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GMO animal process - Ian I

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Margaret FitzSimons _ cows modified to lack lactose intolerance protein 

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Margaret FitzSimons _ cows modified to lack lactose intolerance protein 

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https://www.pnas.org/content/109/42/16811

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Edited by R. Michael Roberts, University of Missouri, Columbia, MO, and approved August 28, 2012 (received for review June 22, 2012)

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Abstract

Milk from dairy cows contains the protein β-lactoglobulin (BLG), which is not present in human milk. As it is a major milk allergen, we wished to decrease BLG levels in milk by RNAi. In vitro screening of 10 microRNAs (miRNAs), either individually or in tandem combinations, identified several that achieved as much as a 98% knockdown of BLG. One tandem construct was expressed in the mammary gland of an ovine BLG-expressing mouse model, resulting in 96% knockdown of ovine BLG in milk. Following this in vivo validation, we produced a transgenic calf, engineered to express these tandem miRNAs. Analysis of hormonally induced milk from this calf demonstrated absence of BLG and a concurrent increase of all casein milk proteins. The findings demonstrate miRNA-mediated depletion of an allergenic milk protein in cattle and validate targeted miRNA expression as an effective strategy to alter milk composition and other livestock traits.

Milk is the primary source of nutrition for young in mammals and contains all the essential nutrients for mammalian growth. In humans, the incidence of breastfeeding is decreasing and cows' milk has become a substitute for human milk. The protein composition of cows' milk is, however, different from that of human milk, and this may cause allergic reactions. In developed countries, 2% to 3% of infants are allergic to cows' milk proteins in the first year of life ( 1). The milk whey protein β-lactoglobulin (BLG) is produced in cows and other ruminants but is not found in human milk, so it is not surprising that it constitutes a major milk allergen ( 2). It naturally occurs in a number of genetic variants, and the most prevalent bovine variants are known as BLG A and BLG B ( 3). BLG has been associated with the transport of small hydrophobic molecules ( 4), linked to hypocholesterolemia ( 5) and has been ascribed antioxidant properties ( 6), but its exact biological role remains unknown. BLG may primarily constitute another source of protein for the young, and it is generally believed that it is dispensable from milk without greatly altering its properties.

To reduce the allergenic potential of milk, hypoallergenic formulas based on enzymatically hydrolyzed whey proteins have been developed by the dairy industry. However, hydrolysis is not a perfect solution because the resulting peptides can still possess residual antigenicity, it may cause milk to taste bitter as a result of the exposure of hydrophobic amino acids ( 7, 8), and the processing technique is costly. Alternatives to postharvest processing include more direct approaches to reduce BLG, such as knockdown by RNAi or KO of the BLG gene ( LGB) by using homologous recombination ( 9). Gene KOs in livestock species have proven to be notoriously difficult to achieve ( 10), and, despite being an attractive target, no BLG KO animals have yet been reported to our knowledge. Applying zinc-finger nuclease technology, Yu et al. recently demonstrated successful biallelic modification of the LGB gene in bovine cells ( 11). The targeted modifications of the one live LGB mutant calf consisted of small in-frame deletions and did not create the LGB KO alleles required for the production of BLG-free milk.

In comparison with a KO strategy, the use of RNAi is a less complex approach to down-regulate BLG and reduce the allergenicity of cows' milk ( 9). Moreover, RNAi could allow fine-tuning of BLG expression, which may be advantageous if some BLG is required for normal milk physiology. Artificial RNAi molecules that enable the knockdown of target transcripts, either by mRNA degradation or a block of translation, have been used in different forms such as siRNAs, shRNAs, or miRNAs ( 12, 13).

A recent in vitro study demonstrated the effectiveness of several shRNAs and miRNAs directed against the porcine variant of BLG ( 14). We used artificial miRNAs based on the murine miRNA-155 to knock down BLG. miRNAs can be driven by Pol II promoters, which enable spatiotemporally restricted expression and greatly limit off-target effects that may arise from constitutive miRNA expression. Indeed, constitutive expression of BLG-specific RNAi constructs negatively affects primary cell growth, indicating that abundant interfering RNAs aimed at BLG may be toxic ( 14). When the same RNAi constructs were controlled by a lactation-specific promoter, they showed no adverse effects and were compatible with early pig development ( 14). However, the feasibility of reducing BLG in milk by an RNAi-mediated approach remains untested.

Here, we present data on the in vitro screening for efficient BLG-specific miRNAs. Considering the substantial costs involved in generating transgenic cows, the most effective miRNAs were tested in a mouse BLG knockdown model engineered for the mammary gland-specific expression of miRNAs and BLG, which rodents do not normally express. The in vivo validated miRNAs were used to generate a transgenic calf whose milk contained no detectable BLG and more than twice the amount of casein milk proteins.

Results

BLG Knockdown in Cultured Cells.

The whey protein BLG is highly conserved between sheep and cattle. This suggests that miRNAs can be designed that possess knockdown activity against BLG variants of both species. Because dairy cattle will be the ultimate target of this approach, we have used the common bovine variant BLG B for designing effective miRNAs against ovine and bovine BLG. The designs suggested by the RNAi designer tool were screened against the mouse, human, and bovine genomes to exclude miRNAs with strong homology to any off-target sequences. We then selected 10 of the most highly ranked miRNAs targeting homologous sequences within the coding regions of ovine and bovine BLG, avoiding the signal peptide encoding sequence and regions with multiple mismatches between the ovine and bovine sequences ( Fig. 1). By using these design criteria, we expected to identify miRNAs that are effective against ovine and bovine BLG.

BLG expression is limited to mammary tissue during lactation and is difficult to emulate in cultured cells. This made it necessary to first engineer a cell culture system for BLG expression to evaluate the 10 BLG-specific miRNAs for their knockdown potential. Thus, COS-7 cells were cotransfected with an expression plasmid for BLG and a plasmid bicistronically encoding GFP and one of the BLG-specific miRNAs. Knockdown of BLG was determined by Western blot analysis ( Fig. 2). BLG expression for cells cotransfected with ovine BLG and a scrambled control miRNA was set as 0% knockdown. To account for differences in transfection efficiency, BLG expression was normalized against GFP expression encoded on the miRNA construct. Most miRNAs down-regulated ovine BLG expression, with the exception of miRNA 7 (−46 ± 49%) and miRNA 10 (−9 ± 45%), which appeared to have no or possibly a slight enhancing effect. The knockdown activities of the effective miRNAs varied and were classified as low (12 ± 19% for miRNA 1), medium (33 ± 14% for miRNA 3 and 31 ± 16% for miRNA 5), and high (94 ± 2% for miRNA 2, 78 ± 9% for miRNA 4, 93 ± 1% for miRNA 6, 66 ± 7% for miRNA 8, and 83 ± 10% for miRNA 9).

By using an equivalent cotransfection approach, replacing ovine BLG with a bovine BLG expression plasmid, the 10 miRNAs were also analyzed for their potential to knock down bovine BLG. Compared with ovine BLG, a greater number of the miRNAs showed high knockdown activity. miRNA 1 (81 ± 9%), miRNA 2 (92 ± 7%), miRNA 3 (78 ± 17%), miRNA 4 (94 ± 2%), miRNA 5 (80 ± 13%), miRNA 6 (97 ± 3%), miRNA 8 (78 ± 7%), and miRNA 10 (70 ± 28%) reduced bovine BLG levels by 70% or more. Thus, some of the miRNAs ( 1, 3, 5, 10) were more specific for bovine BLG and less active against ovine BLG. miRNA 9 showed the reversed specificity and was more active against ovine than bovine BLG, whereas miRNA 7 slightly enhanced bovine BLG expression (−72 ± 42%) and was the only miRNA that had no knockdown activity against either target.

Cumulative action of multiple miRNAs targeting different regions of the same transcript often yields a higher knockdown than the individual miRNAs on their own. We therefore tested tandem miRNA constructs and compared their activity vs. that of their respective single miRNAs. We generated three tandem constructs with different combinations of three miRNAs that showed high knockdown efficiency for ovine and bovine BLG (miRNAs 2, 4, and 6). All tandem constructs showed high BLG knockdown activity in the range of 90% to 98% with very little difference between ovine and bovine BLG ( Fig. S1). Consistent with the selection of single miRNAs possessing high knockdown efficiency against ovine and bovine BLG, we observed no significant differences between the in vitro activity of single and tandem miRNA constructs ( Fig. S1).

Having established that miRNAs can effectively knock down ovine and bovine BLG in vitro, we selected miRNA 6-4, one of the most efficient tandem constructs, for subsequent in vivo evaluation.

BLG Knockdown in Mice.

To limit miRNA expression to the lactating mammary gland, the constitutive promoter used in vitro was replaced with a murine whey acidic protein (WAP) promoter. In addition, the GFP expression cassette was deleted to avoid any effects the reporter gene may have on endogenous or transgene function in transgenic animals. WAP-miRNA 6-4 transgene fragments were coinjected into zygotes with chicken β-globin insulator sequences to minimize the likelihood of position effects exerted by the chromosomal context on the expression of the miRNAs. Four founders that carried the miRNA 6-4 transgene were produced, but only one transmitted the transgene through its germline and was used for all subsequent experiments.

To determine the effect of the tandem miRNA construct on transcript levels and the production of BLG in milk, we crossed miRNA 6-4 mice with an existing transgenic mouse line ( 15) expressing ovine BLG under the control of a minimal ovine BLG promoter. Single and double transgenic offspring were identified by PCR, and females were bred to induce a natural lactation. Real-time PCR quantification of the BLG transcript levels in lactating mammary gland tissue revealed that expression of the miRNA construct had a strong effect on the level of the ovine BLG mRNA. Relative to the housekeeping gene for GAPDH, ovine BLG transcript levels in BLG mice were 1,460 ± 290 and 0.34 ± 0.02 in BLG/miRNA 6-4 mice. Thus, the level of the mRNA for BLG is down-regulated by more than four orders of magnitude (4,294 fold) in BLG mice expressing miRNA 6-4 compared with BLG mice without the miRNA transgene.

Next, we analyzed the milk proteins by SDS/PAGE and Western blotting and directly compared milk from WT mice, double transgenic mice, and those expressing either transgene ( Fig. 3). As expected, BLG is absent in the milk of WT mice and transgenic mice containing only the miRNA 6-4 transgene, but is the most highly expressed milk protein in the milk produced by the transgenic ovine BLG mice. Double transgenic mice with transgenes for ovine BLG and miRNA 6-4, however, show a 96 ± 2% reduction in BLG expression level compared with milk from transgenic mice expressing BLG but no miRNAs. We also addressed the question whether the miRNA-induced knockdown will persist as lactation progresses. Milk was collected 5, 10, and 15 d after the onset of lactation and was analyzed for BLG levels ( Fig. 3 and Fig. S2). Knockdown of ovine BLG induced by miRNA 6-4 remained at the same high level, which indicates persistent expression of the miRNAs and knockdown activity against BLG. Thus, the results demonstrate the utility of the miRNA approach to reduce the level of BLG in milk.

BLG Knockdown in Cattle.

We next attempted to knock down BLG in cattle. The donor cell line (bovine fetal fibroblasts), previously validated for nuclear transfer (NT) ( 16), was transfected with an expression cassette for miRNA 6-4. Two expanded cell clones, 312/3 and 313/4, were karyotyped and found to have normal chromosome numbers ( Fig. S3). Transgene copy numbers for the two transgenic cell lines were determined from Southern blot analyses to be approximately 30 for 312/3 ( Fig. S4) and more than 200 for 313/4.

Initial attempts to generate live calves from cell line 313/4 were unsuccessful. NT with cell line 312/3 and the transfer of 57 cloned embryos to recipients resulted in five pregnancies at approximately day 65 of gestation ( Table S1). Because most of the available 312/3 cells were used for NT, one of the pregnancies was terminated to recover the fetus and derive rejuvenated cells to preserve the unique genetics and provide future NT options. Of the remaining four pregnancies, one resulted in the production of a live female calf ( Fig. S5). Unexpectedly, the miRNA 6-4 calf was born without a tail.

To obtain milk and evaluate miRNA-mediated knockdown of BLG expression, we hormonally induced the transgenic calf into lactation. Analysis of the milk samples by SDS-gel electrophoresis and Coomassie blue staining revealed that none of the milk samples from the miRNA 6-4 calf contained detectable levels of BLG. By contrast, BLG was readily detectable as a major milk protein in all WT controls, including colostrum and natural and induced milk samples, with the latter showing the highest levels of BLG ( Fig. 4A). A more sensitive analysis of BLG levels by Western blot confirmed the highly effective knockdown, as all milk samples from the transgenic calf were devoid of any detectable BLG ( Fig. 4B). To further quantify BLG levels and determine any effects the knockdown of BLG may have on milk protein composition, the milk samples were quantified for all major milk proteins by HPLC. Consistent with the Coomassie blue staining and Western results, HPLC analysis could not detect any BLG in the transgenic calf milk samples and confirmed comprehensive knockdown of BLG ( Table 1). The absence of BLG had a strong, compensatory effect on the levels of other milk proteins. In comparison with natural and induced WT samples, all other major milk proteins were greatly increased in the milk produced by the transgenic calf, in particular the caseins (α- and β-casein more than twofold and κ-casein approximately fourfold). This had a strong effect on the κ-casein-to-total casein ratio, which increased from an average of 0.21 for the induced WT milks to 0.30 for the transgenic calf samples. Although the concentration of whey protein α-lactalbumin was also more than twofold higher, the strong increase of all caseins and lack of BLG decreased the whey-to-casein ratio from an average of 21:79 for induced WT samples to a 4:96 average ratio for the transgenic calf samples.

Discussion

BLG has been suspected as a major milk allergen for decades, but its biological function(s) and impact on milk composition or nutritional and processing properties remain elusive. This is primarily because of the lack of a mouse model. Rodents do not express the protein, and the development of a mouse KO model-as demonstrated for all other major milk proteins ( 17⇓ ⇓ ⇓ - 21)-is therefore not feasible. Second, KOs in ruminants are much more difficult to achieve than in mice, and, although a first attempt at knocking out BLG in cattle was made recently, the one resulting animal does not exhibit a BLG KO phenotype ( 11). RNAi knockdown technology based on a more basic but versatile overexpression strategy is an attractive alternative, as it was shown in mice to be capable of producing phenotypes similar to a KO ( 22).

Most of the 10 candidate miRNAs we tested in vitro were effective against ovine and bovine BLG, which recapitulates the successful design of effective RNAi molecules against porcine BLG in a previous study ( 14). Although a few of our miRNAs showed some target specificity for either of the two tested forms of BLG, this was not correlated with sequence homology between the miRNA and target. Indeed, miRNA 6, with the most numbers of mismatches for the target sequence, was one of the best-performing miRNAs. Most likely, these 3′ mismatches (one and two for ovine and bovine, respectively) do not impede knockdown because they are outside the "seed region," which determines the specificity of the target recognition in translational repression ( 23).

To date, only a few studies have applied RNAi to modify important traits in livestock animals, with relatively mixed success. Knockdown of the prion protein (PrP) to make cattle resistant to the prion disease bovine spongiform encephalopathy only resulted in knockdown of the PrP encoding mRNA and had no significant effect at the protein level of PrP ( 24). A similar study in goats proved to be more successful, and demonstrated the successful reduction of PrP levels, at least in fetal brain ( 25). Five calves were produced in an attempt to increase muscle mass in cattle by knocking down myostatin. However, the calves showed no correlation between the expression levels of the interfering RNA and myostatin mRNA and, in only one of the calves were the myostatin mRNA levels reduced ( 26). These studies included GFP reporters and expressed a single interfering RNA from strong constitutive promoters. By contrast, we avoided a GFP reporter, relying instead on a tightly spatiotemporally regulated promoter and a tandem array of two miRNAs. This excludes potential interference from the reporter gene, minimizing off-target effects from uncontrolled expression, while targeting two separate regions of the BLG mRNA. Expression of a tandem miRNA construct achieved strong knockdown of ovine BLG in our mouse model and knocked down BLG expression in cattle to levels that could no longer be detected by protein analysis. Although this difference in the degree of knockdown might be a reflection of the strong overexpression of ovine BLG in the transgenic mice, it nevertheless demonstrates that the miRNAs are highly effective against different forms of BLG and are likely to be effective against other natural variants of bovine BLG.

mRNA degradation and the repression of translation have been implicated in the mechanism of miRNA-mediated gene silencing. However, the intricate interconnectivity between these two processes has made it difficult so far to fully understand the molecular mechanism underpinning miRNA-mediated silencing ( 12, 27). Analysis of BLG transcript levels in our mouse model demonstrated that strong mRNA decay is involved in the observed knockdown of BLG. Still, repression of translation may be the initial trigger for the mRNA instability, or, alternatively, may further contribute and enhance the knockdown.

In contrast to our mouse model in which we did not observe any obvious phenotypic anomalies, a phenotype was observed in the transgenic calf, which was born without a tail. Taillessness in cattle is a rare congenital abnormality ( 28), and has been previously reported as a phenotype in a transgenic calf produced from a microinjected, cloned embryo ( 29). A similar tail phenotype was observed in a cloned mouse and was shown to be caused by a mutation in this individual donor cell that was below the detection limit for chromosomal abnormalities and unmasked by cloning ( 30). Thus, it appears unlikely that the tailless phenotype of the calf is correlated with the transgene insertion, but rather with an unrelated mutation present in the donor cell used for NT. Further characterization of this transgenic cattle line will be required to clarify the cause for the taillessness in the miRNA 6-4 calf.

The intended aim of expressing the miRNA 6-4 construct has been to direct production of milk depleted of BLG, which was achieved in transgenic mice and cattle. A more detailed analysis of the milk produced by the transgenic calf demonstrated that this change in BLG also affected the levels of all other milk proteins, revealing an intricate balance of milk proteins synthesis. The increase in all other milk proteins as a result of BLG depletion is consistent with previous observations of a decrease in endogenous milk proteins in transgenic mouse models overexpressing BLG ( 31). Moreover, it is known that low BLG levels are associated with high casein levels in cows' milk ( 32, 33). Astonishingly, the BLG knockdown approach greatly surpasses the efforts to increase β-casein (>200% vs. 20%) and κ-casein (∼400% vs. ∼200%) in cows' milk through overexpression of these two milk proteins ( 16).

Induced-lactation milk has higher levels of BLG than natural lactation milk ( 34) (Fig. 4 A), which is advantageous for a vigorous assessment of BLG knockdown and further strengthens the finding of the high efficiency for the knockdown of BLG. In addition, an induced lactation is a reliable predictor of major changes, such as the observed increase in casein that can be expected in a natural lactation ( 35, 36). Thus, the BLG-depleted milk not only has potentially hypoallergenic properties, its high total casein content, particularly high κ-casein correlated with smaller casein micelle sizes, should provide for increased calcium levels and high cheese yields ( 37, 38). Ultimately, natural-lactation milk will be required to confirm the changes to the milk composition and fully assess the functional properties of this milk.

In conclusion, our results demonstrate that targeted expression of miRNAs is an approach that is capable of abolishing a protein's production in livestock. Thus, it validates miRNAs as an efficient tool to change livestock traits. Importantly, this can be achieved with animals possessing a hemizygous transgenic locus. Considering long gestation times and potentially large herd sizes, this offers a clear advantage for disseminating such a trait over KO technology that is dependent on homozygous KO animals to achieve a similar phenotype. The production of dairy cows with a comprehensive knockdown of BLG, will allow direct testing of the longstanding conjecture that milk with greatly reduced BLG level will be less allergenic than normal cows' milk. Furthermore, the BLG knockdown cattle line will provide the opportunity to fully investigate the impact of the associated milk composition changes on nutritional and processing properties and will provide new insights into the still obscure biological function of BLG.

Materials and Methods

The applied methods are briefly summarized here, and a more detailed description in provided in SI Materials and Methods.

Animal Studies.

All animal studies were undertaken in compliance with New Zealand laws, and were approved by the Environmental Protection Agency of New Zealand and the Ruakura Animal Ethics Committee.

DNA Constructs.

In COS-7 cells, expression of artificial miRNAs was based on the BLOCK-iT Pol II miR RNAi system (Invitrogen) using the pcDNA6.2-GW/EmGFP-miR vector. BLG was constitutively expressed from cDNAs [bovine BLG A (GenBank accession no. X14712) and ovine BLG B (GenBank accession no. NM_001009366)]. In mice, miRNA expression was driven by the WAP promoter without a GFP reporter. For expression of the miRNAs in cattle, tandem chicken β-globin insulators were inserted upstream of the WAP promoter.

Southern Blot.

Genomic DNA from cultured bovine fetal fibroblasts was extracted with a DNeasy kit (Qiagen) and isolated from blood of the transgenic calf using the Illustra DNA extraction kit BACC 2 kit (GE Healthcare). EcoRV/HindIII restricted genomic DNA was hybridized with a 0.7-kb XhoI fragment comprising the proximal WAP promoter and entire miRNAs 6 and 4 that was labeled by using the Gene Images AlkPhos Direct Labeling and Detection System (Amersham).

COS-7 Cells: Transfection and BLG Knockdown Analysis.

COS-7 cells were transiently cotransfected with miRNA and BLG expression constructs at a molar ratio of 1:1 by using Lipofectamine 2000 (Invitrogen). Total cell lysates were prepared in the presence of protein inhibitors 48 h after transfection. Ovine and bovine BLG and GFP were detected on Western blots with rabbit anti-bovine BLG and rabbit anti-GFP primary antibodies, respectively, in combination with goat anti rabbit-HRP conjugate as secondary antibody.

Generation of Transgenic miRNA Mice.

The miRNA transgene fragment was comicroinjected with tandem chicken β-globin insulators ( 39, 40) into the pronuclei of C57/BL6 mouse zygotes and embryos transferred into pseudopregnant recipients according to standard protocols ( 41).

Real-Time PCR Analysis of BLG mRNA Levels.

Total RNA was isolated from lactating mammary tissue of ovine BLG and ovine BLG/miR6-4 mice and converted into cDNA. Transcript copies of ovine BLG were calculated relative to those of the housekeeping gene GAPDH following amplification of respective gene-specific sequences in triplicate by real-time PCR ( 42). Results are presented as mean ± SD.

Knockdown Analysis of BLG Levels in Mouse Milk.

Milk was collected manually into capillary tubes by gentle massage of teats following oxytocin administration. Milk proteins were separated by SDS gel electrophoresis and quantified following Coomassie blue staining. Western blots of milk proteins were performed as described for COS-7 cell extracts.

Generation of Transgenic miRNA Cattle.

Bovine fetal fibroblasts ( 16) homozygous for the BLG variant A were cotransfected at a molar ratio of 5:1 (Lipofectamine 2000; Invitrogen) with tandem chicken β-globin insulators and the miRNA 6-4 transgene, including a puromycin selection cassette. Stable cell lines were isolated following puromycin selection. Zona-free NT with two confirmed cell clones (312/3 and 313/4) was performed essentially as described ( 43). Embryos were nonsurgically transferred singularly to synchronized recipient cows. One live transgenic calf was delivered by Caesarian section on day 255 of gestation after being diagnosed with hydroallantois.

Induction of Lactation and Analysis of Induced Bovine Milk.

Lactation of the transgenic calf was hormonally induced at the age of 7 mo, essentially as described ( 44), except that hormones were injected s.c. WT samples of colostrum (provided by Jessica van Leeuwen, AgResearch, Hamilton, New Zealand), milk generated by hormonal induction ( 35), and natural milk were derived from cows of similar breed. The Western analysis was performed as described for mouse milk with a detection limit of 0.04 μg for bovine BLG. The concentrations of the main milk proteins were determined by reversed-phase HPLC against purified milk protein standards with a detection limit for BLG-A and BLG-B of 0.14 μg and 0.08 μg, respectively ( 45).

Acknowledgments

We thank R. Martinus for his support as university doctorate supervisor for A.J.; R. Broadhurst for pronuclear microinjection; members of the reproductive technologies group for in vitro bovine embryo production and transfers; Ali Cullum and animal facility staff for inducing lactation and dedicated animal husbandry; H. Henderson and P. Hunt for assistance with statistical analyses and the artwork, respectively; and R. Thresher and B. Oback for critical reading of the manuscript. This work was funded by the Ministry of Science and Innovation and AgResearch.

Footnotes

Author contributions: A.J., S.W., and G.L. designed research; A.J., S.W., J.M., and D.N.W. performed research; A.J., S.W., and G.L. analyzed data; and A.J., S.W., and G.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1210057109/-/DCSupplemental.

# data[].attributes.attachment_link.title
Targeted microRNA expression in dairy cattle directs production of β-lactoglobulin-free, high-casein milk

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Milk from dairy cows contains the protein β-lactoglobulin (BLG), which is not present in human milk. As it is a major milk allergen, we wished to decrease BLG levels in milk by RNAi. In vitro screening of 10 microRNAs (miRNAs), either individually or in tandem combinations, identified several that achieved as much as a 98% knockdown of BLG.

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- needs to be a primary research article
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- should have graphs, data tables, or images (at least 2) that you can make sense of and talk about
- you should be able to get the main idea of the article, if you don't, please pick a different one :)

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riku kobayashi

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- cows (margaret)
- apples (lucas)
- pigs (ian)
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- papaya (karina)
- tomato (riku)
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- Ian Irving

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- Ian Irving

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Application of Genetically Engineered Pigs in Biomedical Research - PubMed

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Progress in genetic engineering over the past few decades has made it possible to develop methods that have led to the production of transgenic animals. The development of transgenesis has created new directions in research and possibilities for its practical application. Generating transgenic anima…

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-Ana Lugo

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The GMO Potato: What Consumers Need to Know | Living Non-GMO : The Non-GMO Project

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You’re probably aware of the best-known GMO crops such as corn, soy, and canola. Did you know that there are also GMO potatoes?

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- Ian Irving

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https://pubmed.ncbi.nlm.nih.gov/27457558/

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Genetically modified laboratory mice with sebaceous glands abnormalities - PubMed

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Sebaceous glands (SG) are exocrine glands that release their product by holocrine secretion, meaning that the whole cell becomes a secretion following disruption of the membrane. SG may be found in association with a hair follicle, forming the pilosebaceous unit, or as modified SG at different body…

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Alex - Using Genetically Modified Pigs for Xenotransplantation

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Alex - Using Genetically Modified Pigs for Xenotransplantation

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This article discusses editing the genes of pigs to use as organ donors for humans and the complications that can come with

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With developments in medical science, transplantation of cells, tissues and organs has become a safe and effective method of treating end-stage organ failure or hematopoietic system cancers. The primary problem of transplantology is the disproportion between the number of organs available and the number of patients with indications for transplantation. This disproportion is constantly growing due to the shortage of transplantable organs, combined with the increasing number of indications for this treatment. On average, 18 patients die each day while waiting for a transplant in Europe. Every 10 min, someone is added to the national transplant waiting list. At present, more than 14,500 people are on active organ waiting list ( https://www.eurotransplant.org/cms/). The growing shortage of available organs is very difficult to overcome. Currently, two solutions are being considered: extending the life span of patients awaiting transplants and developing new, alternative methods for obtaining transplantable organs. These methods mainly include tissue engineering and the use of stem cells, artificial organs or bioreactors. One especially interesting possibility is xenotransplantation. The use of xenotransplantation in treatment typically involves the transplantation of animal cells, tissues or organs to replace an injured part of the human recipient. At present, the domestic pig ( Sus scrofa domestica) is considered the best donor of biological material for xenotransplantation. Its anatomical and physiological parameters are similar to humans. The pig breeding is well developed and cost-effective, and the large variety of breeds allows the size of the organs harvested to be matched to the recipient [ 1]. Unfortunately, the molecular incompatibility between the donor and the recipient, resulting from the large phylogenetic distance between pigs and humans, entails a range of immune complications following transplantation, leading to xenograft rejection. Advances in genetic engineering have made it possible to modify the genome of donor animals in a way preventing the recognition of their organs by the human recipient's immune system and inhibiting the processes leading to xenograft rejection. Several techniques for obtaining genetically modified animals are available: pronuclear and cytoplasmic microinjection, somatic cell nuclear transfer (SCNT) and viral transduction of DNA. Moreover, transposon-based technologies can be exploited for transgenesis. Both the Sleeping Beauty and the piggyBac systems offer an easy and efficient method for stable genomic insertion. Integration of the exogenous DNA with the host genome may occur randomly-through non-homologous recombination-or through directed transgenesis. Precise genomic modifications are possible with the use of site-specific nucleases, the most recent advancement in biotechnology. These include: zinc finger nucleases (ZFN), transcription activator-like effector (TALE) nucleases and modifications of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats, CRISPR; CRISPR-associated proteins, Cas) bacterial immune system. The first step leading to the introduction of modification in a specific locus in the genome is connected with the induction of double-strand breaks (DSB) in this region. DSB are repaired typically by non-homologous end joining (NHEJ), as a result of which changes may occur in the original nucleotide sequence in DNA or the formation of an indel (insertion/deletion) mutation. If indel mutations appear in the protein-coding sequence, this may lead to the gene frameshift and appearance of premature STOP codons, ending translation. Although transcription progress may be undisturbed, functional protein is not formed. Apart from NHEJ, DSB may be corrected via homologous recombination (HDR). This mechanism may be used to introduce specific changes in a given locus: point mutations, insertion (e.g., transgene insertion) or specific deletions. ZFNs are generated by fusing a DNA-binding domain with the zinc finger motif and a non-specific DNA-cleavage domain comprised of the I endonuclease. The I domain responsible for DNA hydrolysis requires dimerization of a monomer pair. When two FokI monomers are bound, DNA is cleaved and a DNA double-strand break occurs. Individual zinc finger is responsible for the recognition of three neighboring nucleotides. The DNA-binding domain typically contains between three and six individual zinc finger repeats. In order to generate breaks in the DNA, a ZFN pair is required, in which each DNA-binding domain recognizes a sequence of 9-18 bp. TALENs are similar to ZFNs; however, in this case the zinc finger domains were replaced with DNA-binding domains from proteins of bacteria from the genus Xanthomonas. The DNA-binding domain of TALE protein consists of series of 33-35 amino acid repeats that each is responsible for the recognition of a single base pair in DNA. Generally it is difficult, time-consuming and costly to produce proteins recognizing a specific sequence due to the need to produce a separate pair of proteins for each targeted sequence. An alternative to the above-mentioned methods is provided by the CRISPR/Cas system, in which the specificity of DNA recognition is provided not by a protein (as in the case of ZFN or TALEN), but by a short, complementary RNA molecule. CRISPR/Cas9 system is adaptable immune mechanism used by many bacteria to protect themselves from foreign nucleic acids, such as viruses or plasmids. An advantage of the CRISPR/Cas9 system is connected with its high specificity and easy construct design, dependent mainly on the 20 base pairs comprising guide RNA. Apart from the above, multiplexing and expression of several synthetic RNA obtained within one experiment makes it possible to reduce costs and duration of the experiment [ 2]. The fast development of genetic engineering techniques in recent years has made it possible to perform virtually any kind of genetic manipulation in vitro. Therefore, the prospect of producing multitransgenic pigs whose organs would resist rejection after transplantation is becoming increasingly realistic.

After the Rejection Response

Due to genetic differences between the donor and recipient, transplantation of xenogenic organs triggers immune response in the recipient, leading to complete failure of the transplanted organ. Depending on the mechanism and timing of xenotransplant rejection, the following classification was adopted: hyperacute rejection (HAR), delayed xenograft rejection (DXR), acute cellular rejection (ACR) and chronic rejection (CR).

Risk of Viral Infection with Xenotransplantation

The use of porcine cells, tissues or organs for xenotransplantation raises concerns about the risk of infection with viruses present in the animals. Most pathogens that could potentially be harmful to humans can be eliminated by careful donor selection, breeding in sterile and isolated conditions and screening of each donor candidate. Porcine endogenous retroviruses (PERV) remain a problem, as they constitute an integral part of the porcine genome [ 52]. Endogenous retroviruses are dormant (inactive) in the host, causing no disease symptoms. They may, however, be activated by certain factors and thus become infectious. If an endogenous virus is present in the xenograft, it could potentially become activated and pathogenic in the recipient. Based on nucleotide sequence differences within the env gene, encoding viral envelope glycoproteins, porcine endogenous retroviruses can be divided into three subtypes: PERV-A, PERV-B and PERV-C [ 53]. Research conducted so far indicates that the PERV-A and PERV-B subtypes are present in various pig breeds in all the populations studied, while PERV-C is not found in all animals [ 54]. Both PERV-A and PERV-B can infect various human cell types in vitro [ 55]. The situation is different with regard to the PERV-C subtype, which is practically incapable of infecting human cells by itself, but can, through recombination with the PERV-A subtype, exhibit increased infectivity toward human cells compared with the PERV-A subtype alone [ 56].

PERVs are very difficult to eliminate, as they are encoded in multiple locations in pig genome [ 57]. To reduce the risk of PERV infection in humans, it has been proposed that xenograft donor candidate animals should be tested for retrovirus levels, so that organs can be harvested only from those with low values, while carriers of the PERV-C subtype should be eliminated altogether. Another suggested solution involves the use of small interfering RNA (siRNA) [ 58, 59] and other genome editing techniques (ZFN, TALEN and CRISPR/Cas) to remove PERV-encoding sequences from the animals' genome. For this strategy to succeed, the technique used must deactivate dozens of very similar genes at once. This is why the CRISPR/Cas method is the most promising, as it allows for simultaneous modification of multiple parts of the genome. Using this technology, Yang et al. [ 60] designed two RNA molecules to inactivate 62 copies of the pol gene required for PERV activity. The study on a porcine kidney epithelial cell line demonstrated that the modification produced a 1000-fold reduction in PERV transmission to human cells, compared to non-transgenic control cells, giving rise to great hopes for the complete elimination of these viruses from pigs used as xenograft donors.

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Genetically Modified Pigs as Organ Donors for Xenotransplantation

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The growing shortage of available organs is a major problem in transplantology. Thus, new and alternative sources of organs need to be found. One promising solution could be xenotransplantation, i.e., the use of animal cells, tissues and organs. The domestic ...

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-Ana L

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https://www-liebertpub-com.ezproxy1.lib.asu.edu/doi/10.1089/crispr.2021.0091

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doi: 10.1097/mot.0b013e3283292549.

Affiliation

¹ Massachusetts General Hospital, Transplantation Biology Research Center, Boston, Massachusetts 02129, USA. david.sachs@tbrc.mgh.harvard.edu

Free PMC article

Free PMC article

doi: 10.1097/mot.0b013e3283292549.

Affiliation

¹ Massachusetts General Hospital, Transplantation Biology Research Center, Boston, Massachusetts 02129, USA. david.sachs@tbrc.mgh.harvard.edu

Abstract

Purpose of review: Recent developments in the field of genetic engineering have made it possible to add, delete or exchange genes from one species to another. This technology has special relevance to the field of xenotransplantation, in which the elimination of a species-specific disparity could make the difference between success and failure of an organ transplant. This review focuses on developments in both the techniques and applications of genetically modified animals.

Recent findings: Advances have been made using existing techniques for genetic modifications of swine and in the development of new, emerging technologies, including enzymatic engineering and the use of small interfering RNA. Applications of the modified animals have provided evidence that genetically modified swine have the potential to overcome both physiologic and immunologic barriers that have previously impeded this field. The use of alpha-1,3-galactosyltransferase gene-knockout animals as donors have shown marked improvements in xenograft survivals.

Summary: Techniques for genetic engineering of swine have been directed toward avoiding naturally existing cellular and antibody responses to species-specific antigens. Organs from genetically engineered animals have enjoyed markedly improved survivals in nonhuman primates, especially in protocols directed toward the induction of tolerance, presumably by avoiding immunization to new antigens.

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national center for biotechnology information

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doi: 10.1097/mot.0b013e3283292549. 1 Massachusetts General Hospital, Transplantation Biology Research Center, Boston, Massachusetts 02129, USA. david.sachs@tbrc.mgh.harvard.edu Free PMC article Free PMC article doi: 10.1097/mot.0b013e3283292549. 1 Massachusetts General Hospital, Transplantation Biology Research Center, Boston, Massachusetts 02129, USA. david.sachs@tbrc.mgh.harvard.edu Purpose of review: Recent developments in the field of genetic engineering have made it possible to add, delete or exchange genes from one species to another.

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Genomic Estimates of Inbreeding.

Tigers exist as several population genetic clusters within India with the total population size of these clusters ranging from 60 to 1,000 individuals. Each genetic cluster consists of several protected areas, with varying connectivity with other such protected areas and clusters ( 35, 36, 38). For the purpose of this paper, we defined populations and whether they are "isolated" or "connected" based on population genetic and gene flow analyses using markers across the genome ( 34, 35, 39). Here, the "large" populations currently have hundreds of tigers (minimum 300 tigers), while "small" populations currently have fewer than 100 tigers ( 36). While both large populations are part of connected landscapes, they do not have identical histories of connectivity. The southern large-connected population is disconnected from other tiger population genetic clusters in India (e.g., from central India and northwestern India), while the central large-connected population (central Indian landscape) was connected by gene flow to other tiger genetic clusters until recently ( 35, 39). Site-frequency spectra (SFS)-based demographic history models in Armstrong et al. ( 34) suggest that most Indian tigers diverged from each other recently. Increased agriculture, bounty hunting and illegal poaching, habitat loss, and fragmentation together have led to local extinctions of tiger populations ( 40, 41). The small-isolated population we studied here has fewer than 100 individuals, but was connected (until very recently) to the central large-connected population ( 42) and potentially to the now extinct tiger population in Afghanistan ( 43).

We quantify inbreeding by identifying long stretches of the genome that are homozygous and identical by descent (i.e., runs of homozygosity [ROH]). The summed length of ROH divided by total autosomal size is an accurate genomic measure of inbreeding ( F ROH) ( 44). We found that the mean F ROH, measured using ROH longer than 100 kb ( F ROH>100kb), 1 Mb ( F ROH>1Mb), and 5 Mb ( F ROH>5Mb) of all individuals in our dataset (regardless of population) was above 0.48, 0.31, and 0.23, respectively. Tigers from the small-isolated population were most inbred with an average F ROH>100kb of 0.57, while average inbreeding due to recent ancestors (on average up to five generations ago, contingent on assumed recombination rate from domestic cat) ( 45) was above F ROH>5Mb = 0.37 ( Fig. 1A). The F ROH was proportional to effective population size ( N e), with the small-isolated population having lower N e at all time intervals compared to both large and connected populations. This level of inbreeding is empirically associated with high risk of extinction ( 46⇓ - 48). However, shorter ROH could be due to shared demographic history of bottlenecks (e.g., F ROH = 100 kb) and inbreeding depression may be observed at higher F values for large carnivores ( 49). Pedigree-based studies ( 50) have reported that an inbreeding coefficient of 0.47 is lethal for tigers.

We show that inbreeding was low in the large-connected populations compared to the small-isolated population ( Fig. 1 A and B). We compared our estimates for zoo individuals with their known pedigree values to authenticate these results and found that estimates of recent inbreeding indeed correspond with pedigree values ( 51) (SI Appendix, Table 1). Overall, the long ROH of ≥ 10 Mb, signifying inbreeding due to ancestors as recent as three generations ago, are rare in large populations but frequent in the small-isolated population. The mean inbreeding due to recent ancestors in the small-isolated population ( F ROH>10Mb) was above 0.28, which could lead to several negative effects ( 46, 48). We found that the large-connected populations showed inbreeding due to recent ancestors (on average up to five generations ago) of less than F ROH>5Mb = 0.1. The large southern population has more inbred individuals than the large central population, potentially due to its geographical placement, historic size ( Fig. 1D), and connectivity ( 35, 36, 39). Overall, although the theoretical expectation is that populations with N e < 1,000 are unlikely to persist ( 7, 30, 52, 53), this result indicates that a population with a census size of ∼600 tigers may be sufficiently large to buffer short-term inbreeding effects given some gene flow. The long-term viability of these populations remains to be tested. While there are instances of small tiger (and other carnivore) populations persisting ( 36, 37), this could possibly be due to their ability to purge some deleterious alleles. This implies that other large felid species like lions ( 54), snow leopards ( 55, 56), and jaguars ( 57) that often persist in population sizes close to 600 may not necessarily experience the detrimental effects of inbreeding depression over the short term. We caution that this is likely to depend on founder effects and variation in the mutation load among populations of a species (but see ref. 30) and does not address the likelihood for fitness decline over the long term ( 7).

In addition to having the highest F ROH, we found that pairs of individuals from the small-isolated population also shared large tracts of genome that were IBD ( Fig. 1C). This could be a worrying sign for its long-term future as the pairwise IBD sharing translates into inbreeding in the offspring. On average, pairs of individuals from the small-isolated population shared about 40% of their genome (as more than 1-Mb-long IBD stretches), while pairs from the southern large-connected and the central large-connected populations share about 25 and 15%, respectively, slightly higher than the corresponding mean F ROH values ( Fig. 1A). As with inbreeding in offspring, pairwise IBD sharing is expected to be influenced by founding bottlenecks in addition to recent small N e ( Fig. 1D). The small-isolated population had the lowest mitochondrial haplotype diversity and the lowest number of mitochondrial haplotypes (SI Appendix, Fig. 1). This lack of diverse lineages, large tracts of shared IBD genome between individuals, and the recent small population size may therefore require continued immigration of individuals to sustain genetic variation ( 58) or, in the absence of natural immigration, may require genetic rescue.

Mutation Loads in Small-Isolated and Large-Connected Tiger Populations.

We evaluated the relationships between F ROH and the frequency and genomic distribution of putatively deleterious genotypes identified using the Ensembl Variant Effect Predictor (VEP) ( 59). Under the assumption that most deleterious alleles are partially recessive ( 28, 60, 61), the number of homozygous damaging alleles is likely to be informative of the fitness cost of individual inbreeding. First, we found that the number of homozygous putatively damaging alleles (loss-of-function [LOF] and missense alleles together) was proportional to F ROH in all populations. The number of putatively damaging homozygotes increased from <1,500 for individuals with F ROH ≈0.3 to ∼2,200 for individuals with F≈0.6 ( ROH P < 0.01 for each population, linear regressions; Fig. 2). This genomic evidence suggests a fitness cost associated with increased inbreeding. Additionally, the burden of homozygous putatively damaging mutations was substantially larger on average in the small-isolated population (mean ≈ 2,000 per individual) than in the central large-connected population (mean < 1,600 per individual, P < 0.0001, randomization test) or the southern large-connected population (mean > 1,800 per individual, P = 0.0025, randomization test; Fig. 2). This is likely due to higher N e in the central large-connected population compared to the southern large-connected population ( Fig. 1D).

We further investigated differential mutation load between populations for LOF and missense mutations identified with VEP and used the R XY method ( 28, 62) to estimate relative excess of number of putatively deleterious alleles in one population compared to another. R XY compares the number of derived alleles of a particular category (LOF, missense, or putatively neutral) found in one population relative to the other. If R XY = 1, both populations X and Y have the same load of derived alleles, whereas if R XY > 1, then population X has more derived alleles than Y and vice versa if R XY < 1 ( 28, 62). As above, we found a higher load associated with missense mutations in the small-isolated population ( Fig. 2C). This is likely explained by more efficient purifying selection against missense mutations of relatively small effect, which are typically only slightly recessive ( 61), due to larger N e in the large populations than in the small population. We also found a lower load of LOF mutations in the small-isolated population compared to the large populations ( Fig. 2C). This suggests that higher inbreeding in the small-isolated population facilitated purging of relatively large-effect LOF mutations-which are more likely to be highly recessive than small-effect deleterious mutations ( 61, 63). Together, these different approaches suggest a higher putatively damaging allele load in the small-isolated population coupled with purging of loss-of-function alleles in the small-isolated (and inbred) tiger population.

Despite this evidence for purging of LOF alleles in the small-isolated population, it appears that high inbreeding led to individuals in this population having a higher number of homozygous LOF alleles on average ( n = 250) compared to the southern large-connected ( n = 238) and central large-connected ( n = 218) populations ( Fig. 2B). High homozygosity for remaining LOF alleles suggests that the fitness cost due to LOF mutations may be higher in the small-isolated population compared to the large populations despite partial purging. Importantly, while it is impossible to know the realized fitness effects of homozygosity for LOF and missense mutations, this analysis suggests that purging was not sufficient to result in a smaller fitness cost of inbreeding in the small-isolated population compared to the larger populations. We observe similar results when we used a Genomic Evolutionary Rate Profiling (GERP) ( 64, 65) analysis to identify potentially highly deleterious alleles by keeping loci with GERP scores in the top 0.1% (SI Appendix, Fig. 3). For this analysis and those below, it important to recognize that our measure of mutation load is not a direct measure of fitness. Rather, our inferences rest on the assumption that missense and LOF mutations and mutations at conserved sites are deleterious on average, which is well supported empirically ( 65, 66). Definitively determining whether the bottlenecks faced by these populations ( 40, 41) and the additional bottleneck faced by northwestern Indian population three generations ago in 2004 ( 67) purged part of the mutation load would require historical genetic data to quantify pre- versus postbottleneck mutation loads. Additionally, better estimates of very recent demographic history would help to predict the potential for purging during the bottlenecks.

Annotated sets of deleterious variants are rarely available for endangered species. We compared our missense mutations to annotated sets of domestic cat mutations (SI Appendix, SI Methods and Table 2). These missense mutations mapped to genes responsible for disease states such as hypertrophic cardiomyopathy, progressive retinal atrophy, polycystic kidney disease, cystinuria, gangliosidosis, hyperoxaluria, hypothyroidism, mucolipidosis, and mucopolysaccharidosis. Further studies are needed to functionally validate the effect of these mutations in tigers. Studies on inbred/small populations of Asiatic lions revealed several abnormalities ( 68), and genomic studies here might provide further insights on deleterious mutations in large cats. In the small-isolated population at least one tiger has a potential eye condition (SI Appendix, Fig. 2), although no skeletal defects were observed in an earlier study ( 69). Additional quantitative genetic studies on inbred zoo individuals can aid in understanding the possible phenotypic effects of inbreeding and inbreeding depression for endangered species.

SFS revealed that nearly 14% of both putatively damaging and neutral derived alleles were fixed in the small-isolated population ( Fig. 3). A smaller proportion of derived alleles (both damaging and neutral) were fixed in the large populations (<0.06). However, there were slightly more fixed derived alleles in the southern large-connected population than in the central large-connected population. The higher abundance of fixed derived alleles (both damaging and neutral) in the small-isolated population, and to a lesser extent in the southern large-connected population, suggests that genetic drift (e.g., due to historical population bottlenecks and founder effects) drove previously rare alleles to fixation in these populations ( Fig. 3) ( 70). The fixation of deleterious alleles is consistent with theoretical predictions for very small populations ( 4, 7) and empirical observations of wolves ( 32), Apennine brown bears ( 71), and other species ( 31). In the Isle Royale wolves, high-frequency deleterious alleles were consistent with frequent bone deformities in the population ( 32, 72). The large fraction of fixed putatively damaging mutations in the small-isolated population suggests that partial purging of the mutation load due to LOF mutations ( Fig. 2C) was not sufficient to erase a substantial fraction of the mutation load in this population. While the SFS was essentially flat for polymorphic loci (i.e., excluding loci with derived allele frequencies of 0 or 1) in the small-isolated population (as expected following a population bottleneck), there was a larger fraction of rare derived alleles in the large populations, as expected for large populations at mutation drift equilibrium ( 70). A larger proportion of putatively damaging than neutral alleles were at or near fixation in each of the large populations and across all of India ( Fig. 3), possibly due to positive selection driving selective sweeps on a small fraction of nonsynonymous mutations. The hump-shaped SFS are likely the result of a complex demographic history for Indian tigers, including strong population structure and historical population bottlenecks ( 70, 73, 74).

Finally, the frequency distributions of putatively deleterious and neutral derived alleles reveal signatures of purging via natural selection. Some previously rare, deleterious alleles are expected to be removed from a population by random genetic drift alone, during periods of small N e (e.g., population bottlenecks or founding events). Effective natural selection through purging is expected to eliminate deleterious derived alleles more frequently than neutral alleles or to keep them at lower frequencies than neutral derived alleles. We expected putatively deleterious derived alleles to either be absent or be less frequent (on average) than putatively neutral derived alleles if purging has occurred in our study populations. This prediction was supported via a randomization test in each population and for all populations combined ( P < 2e-4, two-sided randomization test). This suggests that purifying selection has successfully removed a small fraction of the mutation load in each of the three study populations and across India. The higher proportion of fixed and very high-frequency derived putatively damaging alleles in the large populations and across India may be caused by historical selective sweeps on a small fraction of positively selected nonsynonymous mutations, the details of which we leave to future research.

Interestingly, both inbreeding and mutation load for the southern large-connected population (from Western Ghats in India) are higher than for the central large-connected population (from central India, Figs. 1 and 2). This could reflect the central location of this population ( 35, 38, 39). Additionally, the central large-connected population may have had higher historical connectivity (with other population clusters), a two-dimensional network of local populations (versus a linear array in southern large-connected populations), and other ecological factors that allow higher population densities to be achieved in all habitats of central large-connected populations compared to southern large-connected populations.

Genetic Rescue Strategies.

Our data suggest that genomic inbreeding ( F ROH up to 0.6 in the small-isolated population) is comparable to populations experiencing inbreeding depression [e.g., Florida puma, F ROH up to 0.6 ( 75), and Isle Royale wolves, F ROH between 0.1 and 0.5 ( 32)]. While we also demonstrate the effects of purifying selection, homozygous LOF alleles are more frequent and mildly deleterious missense mutations are in excess in the small-isolated population.

Gene flow is the proposed strategy to sustain small and isolated populations ( 18). Gene flow that results in masking of deleterious alleles responsible for genetic load leading to increased population growth rate is genetic rescue. Evolutionary rescue is gene flow that increases genetic variation ( 76) and thus adaptive potential. High F ST at loci with high-frequency damaging alleles in the receiving population (frequency >0.9) should be indicative of good source populations for genetic rescue. In practice, for both evolutionary rescue and genetic rescue, source populations are identified as those that have high genome-wide genetic differentiation with the receiving population (to maximize the heterozygosity of admixed offspring) (reviewed in ref. 19). Gene flow from populations with high genome-wide F ST in theory should result in masking deleterious alleles, allowing both genetic rescue and evolutionary rescue. However, the smaller number of loci with high-frequency damaging alleles compared to all polymorphic loci could lead to discordance between these two proxies for genetic and evolutionary rescue (i.e., genetic differentiation at loci with putatively damaging alleles may not be a good predictor of genome-wide genetic differentiation).

We investigated whether evolutionary rescue is a good proxy for genetic rescue. We propose that certain source populations may be ideal if they result in masking of damaging alleles and increased genome-wide heterozygosity ( Fig. 4 A, Top Right). Alternatively, other source populations ( Fig. 4 A, Bottom Right) will increase heterozygosity but not mask damaging alleles, while those in Fig. 4 A, Top Left mask damaging alleles but may not increase heterozygosity substantially genome-wide. Our data and estimates reveal a strong and positive correlation between the two proxies ( Fig. 4B, adjusted r ² = 0.91), reinforcing that selecting source populations based on high genome-wide F ST would result in both genetic and evolutionary rescue. For the small-isolated population, our data suggest that Kaziranga (proportion of damaging loci rescued = 0.74, genome-wide F ST = 0.26, damaging loci F ST = 0.26) is the best source population, while ecologically more similar populations like Kanha (proportion of damaging loci rescued = 0.69, genome-wide F ST = 0.22, damaging loci F ST = 0.23) and Corbett (proportion of damaging loci rescued = 0.65, genome-wide F st = 0.26, damaging loci F ST = 0.28) may be more practical and almost as effective at genetic rescue. For the large-isolated population in southern India, both proxies are lower on average (average proportion of damaging loci rescued = 0.65, average genome-wide F ST = 0.195, average damaging allele F ST = 0.21, across source populations). Note that high genome-wide F ST between a donor and a recipient population could also be due to intense bottlenecks and resulting drift in endangered species, and such population pairs may not result in effective genetic rescue. We caution that these inferences are subject to assumed fitness effects of damaging alleles. Most genome-wide predictions of deleterious alleles in wild species await annotation with phenotypic data ( 77); such a strategy could be implemented when the loci affecting fitness are identified as in the case of Tasmanian devils ( 78) and Soay sheep ( 79).

Whether there is a substantial advantage to selecting source populations for genetic rescue based on differentiation for putatively damaging loci (versus across the whole genome) remains to be tested empirically. Genome-wide surveys would help in identifying source populations that best mask damaging alleles and increase genome-wide heterozygosity; however, there are other considerations (as highlighted in ref. 33) that could impact selection of source populations. A denser sampling of tigers across the Indian tiger range is necessary to predict best rescue individuals for particular target populations. Such dense sampling of genomes can benefit from sequencing of noninvasive samples since acquiring blood samples from endangered species can be difficult ( 80). Methods involving sequencing genomes from shed hair ( 81) or fecal samples ( 82) will be useful in creating such translocation networks and monitoring the populations for gene flow.

Implications for Conservation of Endangered Species.

We find that small and isolated populations, in this case tigers of Ranthambore Tiger Reserve, have high inbreeding arising from both recent ancestors and ancestors in deep history. Our analyses revealed a signature of purging for putatively large effect (LOF) deleterious alleles in this population. However, this small and isolated population also had the highest average number of homozygous damaging alleles of the three study populations, suggesting a potential fitness cost of inbreeding despite purging of some large effect mutations and compared to the large and connected populations. Together, these results demonstrate that purging (as inferred from genomic signatures) does not eliminate all damaging alleles and hence does not contradict the small population paradigm of conservation biology ( 19). Careful predictions of putative phenotypic effects of the existing deleterious alleles at high frequencies might be instructive to understand future inbreeding depression, and these alleles could potentially be used as early warning signs for population decline. However, caution is warranted in this regard because it is still unclear how effectively molecular predictions (i.e., identification of loss-of-function or derived alleles at conserved sites) can translate into predictions of fitness differences among extant individuals and populations.

Historical isolation and bottlenecks due to overharvest appear to have a significant effect on missense (potentially mild effect) mutation load. Our analyses suggest that purging can also occur in large-connected populations in the context of endangered species ( Fig. 3). This could be because all tiger populations are in general small, and the range of population sizes is not very wide ( 36). Continued isolation of these populations will increase mutation load in the future. Thus, maximizing connectivity might be the best strategy to minimize extinction (from an ecological perspective), increase genome-wide genetic variation, and also minimize mutation load in the future. Maximizing connectivity in the wild would require increasing population abundance since large populations are sources of dispersing individuals, but also planning for habitat corridors, since demographics of tiger landscapes are dominated by extinction and recolonization dynamics ( 83, 84). Such strategies may be relevant to several endangered species with wide ranges but fragmented populations, like elephants, lions, and wild dogs.

Critical to providing recommendations for management is understanding ongoing evolutionary trajectories for endangered species. Given the large number of frequent deleterious alleles in the small-isolated population, management could aim to decrease the number of close to fixation damaging alleles, potentially maximizing mean absolute fitness. Our data reveal that source populations with high genome-wide differentiation (with the target population) would adequately mask deleterious alleles and increase heterozygosity. Gene flow from even the relatively proximate large-connected population in central India would decrease the frequency of damaging alleles and allow genetic rescue. We caution that such gene flow could also introduce other damaging alleles (e.g., in wolves) ( 85).

Overall, genomic data and analyses provide richer, more nuanced ways to address inbreeding and genetic rescue of small and isolated populations of endangered species (i.e., the small population paradigm in conservation). We demonstrate effective purging of some loss-of-function alleles, but not mutations with smaller effects, highlighting the need to characterize detrimental mutations and how they will be addressed through genetic rescue in future conservation efforts. Conservation management based on predictive models for fitness effects in the context of observed mutations will aid long-term persistence of populations in the wild.

Sample Collection.

We collected tissue from tranquilized or dead tigers from several tiger reserves (14 protected areas across India); zoo individuals from Sagar et al. ( 51) were also analyzed ( Fig. 5 and SI Appendix, Table 1). Individuals belonged to one of five population clusters: 1) From the small-isolated population in northwestern India (NW) ( n = 19), we obtained tissues from 15 NW individuals and used data from 2 NW individuals in Armstrong et al. ( 34) and 2 NW individuals in Khan et al. ( 81). Our NW samples consist only of individuals that were born in Ranthambore Tiger Reserve (RTR) but could have been translocated to other protected areas (for example, Sariska Tiger Reserve) according to forest department records. 2) From the central large-connected population in central India (C) ( n = 9), we obtained tissue from four Kanha Tiger Reserve (KTR) individuals and used data from Armstrong et al. ( 34) for five other KTR individuals. 3) From the southern large-connected population in southern India ( n = 11), we obtained tissue from two Wayanad Wildlife Sanctuary (WWS) individuals and two Bandipur Tiger Reserve (BTR) individuals. We additionally used data from Armstrong et al. ( 34) for five WWS individuals and Liu et al. ( 86) for two Nagarhole Tiger Reserve (NTR) individuals. Since WWS, BTR, and NTR populations are effectively contiguous, we refer to them together as Wayanad (WAY) tigers. 4) From Nandankanan Zoo, Odisha, India, Sagar et al. ( 51) obtained five blood samples from inbred individuals, and these genomes were used as a control to test our methods. The pedigree-based inbreeding coefficients of these individuals vary from 0.21 ( n = 1) to 0.26 ( n = 1) and 0.28 ( n = 3) (SI Appendix, Table 1). Such pedigree-based estimates are expected to be an underestimate of the actual inbreeding due to the low depth of pedigrees ( 44). 5) From other protected areas, we also sampled one individual tiger from Lalgarh Division Forest, two individuals from Corbett Tiger Reserve (COR), one from Chitwan National Park, four from Kaziranga Tiger Reserve, one from Periyar Tiger Reserve, one from Bor Tiger Reserve, one from Chandrapur district in Maharashtra, and two from Sundarban Tiger Reserve. These individuals combined with others are used for drawing pan-India inferences. The sampling locations are depicted in Fig. 5.

DNA extraction and sequencing were done using the tissue library preparation protocol described in Khan et al. ( 81) (SI Appendix, SI Methods).

Whole-Genome Resequencing and Variant Discovery.

We trimmed the raw reads using TRIMMOMATIC ( 87) to have a mean phred-scaled quality of 30 in a sliding window of 15 bp and removed any read that was shorter than 36 bp after trimming from further analysis. We aligned these reads to a Bengal tiger reference genome (JAHFZI000000000) using BOWTIE2 ( 88). The alignments were then saved in a binary format (BAM) using SAMTOOLS1.9 ( 89). We marked duplicate reads with the Picard Tools "MarkDuplicates" command ( http://broadinstitute.github.io/picard). We called variants from the BAM files using Strelka with default options ( 90). The variants were filtered with VCFtools ( 91) to retain biallelic sites with a minimum minor allele count of 3 and remove indels and loci with mean depth across individuals below the 2.5th percentile and above the 97.5th percentile across all loci. We removed all sites with missingness >20% after removing genotypes with genotype quality (GQ) less than 30. We removed the X chromosome from the analysis. A total of 1,601,148 single-nucleotide polymorphisms (SNPs) with mean read depth = 17.2 (± 1.8) remained for analyses after these filtering steps.

Runs of Homozygosity.

We identified ROH using a sliding-window, likelihood-ratio method ( 92, 93). For this analysis we used SNP data without the minimum genotype quality filter described above and then used genotype likelihoods, rather than called genotypes, as input to maximize the genomic information available for detecting ROH. We split each chromosome into sliding windows of 200 SNPs and step size of 10 SNPs. For each individual i and window j, we calculated the probability of the observed genotype likelihood at each SNP k ( G k ) assuming the two alleles were IBD and also assuming they were non-IBD ( LOD(j,i F ROH>100 kb=∑ ​ R O H >100 kb Total autosomal size. )=∑k=1 k i l o g 10 (P r(G k|IBD)P r(G k|non-B D) ), where LOD scores across all windows and individuals form a bimodal distribution. Windows with LOD score ≥0 were called IBD, while windows in the left-hand side of the distribution were called non-IBD as in Kardos et al. ( 92). We calculated the logarithm of odds (LOD) score for each window as 44, 94). Consecutive IBD windows were joined to map the starting and stop positions of ROH for each individual. F ROH was calculated as the fraction of the autosomal genome in ROH; for example,

We calculated the percentage of the genome in ROH above a particular size class to estimate inbreeding arising from ancestors in different historical time periods. The coalescent times of ROH were estimated as g = 100/(2 rL), where g is the expected time (in generations) back to the parental common ancestor where the IBD haplotypes forming an ROH coalesce ( 95), r is the recombination rate (here, 1.9 cM/Mb based on domestic cat) ( 45), and L is the length of the ROH in megabases. For example, a 100-kb ROH is estimated to arise from a single haplotype in an ancestor 263 generations ago on average. An individual's genomic inbreeding arising from ancestors up to 263 generations ago is then estimated using only ROH longer than 100 kb. Similar calculations were performed for other ROH lengths. Table 1 lists the lengths of ROH and corresponding average estimated coalescent times for the IBD haplotypes that form the ROH.

We constructed percentile bootstrap confidence intervals for mean F ROH in each population ( 96). We randomly resampled individuals within a population with replacement 10,000 times, each time calculating the mean F ROH as described above. The 0.025 and 0.975 quantiles defined the 95% confidence interval for mean F ROH in each population. We used a randomization procedure to test for a difference in the mean F ROH for each pair of populations as follows. First, we randomized the population affiliation for each individual 10,000 times. For each randomization replicate, we calculated the mean F ROH for each of the two populations. We calculated the P value as the proportion of 10,000 randomization replicates where the difference in F ROH was at least as large as the empirical difference in F ROH.

IBD Stretches of Genome Shared between Pairs of Individuals.

We estimated stretches of genome IBD between pairs of individuals using IBDseq default parameters ( 97). We filtered for shared stretches longer than 1 Mb and estimated the sum of shared lengths of more than 1 Mb per autosomal genome size. We estimated this for all pairs of individuals within a population and all pairs across populations.

Estimating Effective Population Size from ROH.

We used the estimates of F ROH using ROH arising from ancestors in four different estimated time frames ( Fig. 1) to estimate N e over each respective time frame. If the estimated coalescent times for ROH are unbiased, then the average F ROH based on ROH with estimated maximum coalescent times less than t generations back in time is an estimator of the inbreeding accumulated in the population from the time of sampling back to t generations ago. We estimated N e from the following expression of mean expected individual inbreeding as a function of N e over t generations:F ROH,t=1−(1−1 2 N e) t.

This approach assumes that the probability of inferring an ROH segment due to several smaller homozygous segments is very low. We calculated 95% confidence intervals for N e by bootstrapping over individuals within each population as described above for our estimates of mean F ROH.

Mitochondrial Haplotype Diversity.

We used the reads aligned to the mitochondrial scaffold of BenTig1.0. We called consensus fasta files for each individual from this as described in Khan et al. ( 81). These multiple consensus fasta files were aligned using clustal omega ( https://www.ebi.ac.uk/Tools/msa/clustalo/) and written to nexus output files. We then used DnaSP 6 ( 98) to estimate haplotype diversity.

Identifying Effect of Mutations.

We refiltered the SNPs as described above with VCFtools ( 91), except here we removed genotypes with phred-scaled genotype quality <30 and then removed loci that were monomorphic, had >20% missing data, or mean read depth in the bottom and top 2.5% quantiles across individuals. We also removed loci with extreme values of F IS ( F IS < −0.5 or F IS > 0.95) estimated across all Indian tigers to remove loci that likely have frequent genotyping or read mapping errors; this expectedly resulted in mean F IS > 0 (0.14).

We used Ensembl Variant Effect Predictor ( 59) on SNPs to identify the consequence of a mutation and classify them as missense, LOF, or intergenic as described by Xue et al. ( 28). We used annotation files generated for the Bengal tiger assembly using RNA seq.

Mutation Load.

We used the R XY method described by Do et al. ( 62) and implemented by Xue et al. ( 28) to estimate relative excess of mutation loads in a population X with respect to another population Y. We randomly selected seven individuals from each population and estimated the relative mutation load for each of the three possible pairwise combinations of the SI, s-LC, and c-LC populations. SDs were obtained by 100 rounds of jackknife. For each round of jackknife for missense and LOF mutations, we randomly excluded 10 to 15% of loci and repeated the estimation. For intergenic variants, 100,000 loci were randomly excluded for each round.

To infer the ancestral state of each site in the tiger reference we converted the references for the domestic cat (GenBank: GCA_000181335.4), cheetah (GCA_003709585.1), and lion (GCA_008795835.1) into FASTQ reads by sliding across the genome in nonoverlapping windows of 100 bp and transforming each window into a separate FASTQ read. The resulting FASTQ reads were then mapped to the tiger reference genome with bwa mem v0.7.17, slightly lowering the mismatch penalty (− B 3) and removing reads that mapped to multiple regions. Mapped reads were realigned around indels using GATK (https://gatk.broadinstitute.org/hc/en-us) IndelRealigner. Next, we converted the mapped reads into a haploid FASTA consensus sequence, excluding all sites with depth above one (as such sites contain at least one mismapped read) using ANGSD (http://www.popgen.dk/angsd/index.php/ANGSD) - dofasta. The ancestral allele at a locus was then determined as the majority allele found in genomic alignment of the domestic cat, cheetah, and lion. Sites where the ancestral allele could not be identified (e.g., where the ancestral allele identified in the cat-cheetah-lion alignment was not present in our sample of tigers) were excluded.

We counted the number of homozygous deleterious alleles per individual as number of derived missense and LOF mutations. We quantified uncertainty in the mean number of homozygous deleterious alleles for each population using percentile bootstrap confidence intervals as described above. We used randomization tests as described above to test for differences in the mean number of homozygous damaging and LOF alleles per individual for each pair of populations.

Site-Frequency Spectrum.

We equalized the sample sizes across the populations and across loci within each population by randomly subsampling 16 nonmissing alleles from each locus in each population before estimating the derived neutral and derived damaging allele frequencies. For the SFS analysis of all populations combined, we subsampled 88 nonmissing alleles from each locus. Repeating the subsampling process and analysis had no effect on the results.

We used randomization tests to determine whether the mean putatively damaging derived allele frequency was statistically significantly different from the mean putatively neutral derived allele frequency in each population and for all populations combined ( Fig. 3). We randomly reassigned the neutral versus damaging status for each allele frequency 5,000 times. For each of the 5,000 replicates, we recalculated the randomized mean derived neutral and damaging allele frequency. A P value was calculated as the proportion of the 5,000 permutations that resulted in a difference in the mean neutral versus damaging derived allele frequency that was larger than observed in the nonrandomized data.

Genetic Rescue and F ST.

We estimated genome-wide genetic differentiation as F ST with VCFtools. For loci with high-frequency (>0.85, closer to fixation) deleterious (missense and LOF) alleles in a population, we listed all the loci in each target population (population in need of rescue). For every possible source population, we estimated the proportion of these loci with frequency <0.85. We then calculated F ST at only these loci to estimate damaging loci F ST. The proportion of loci rescued was calculated as the fraction of loci that had <0.85 frequency in the possible source population.

We investigate this for five possible source populations, including tigers from KTR, WAY, RTR, COR, and Kaziranga Tiger Reserve (KAZ).

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Genomic evidence for inbreeding depression and purging of deleterious genetic variation in Indian tigers

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Habitat fragmentation is sequestering species into small and isolated populations, with high chances of extinction. Are small and isolated populations at risk for extinction from inbreeding depression? Or does inbreeding and purging of deleterious alleles reduce such threat? Using whole genomes from several wild Indian tiger populations, we provide evidence supporting purging of highly deleterious variants in a small-isolated population.

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Plant Biotechnology Journal - 2020 - Blennow - Non‐GMO potato lines  synthesizing increased amylose and resistant starch 

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Process of trans mice - Ian I

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Alex Thoman - GMO Primates to help better understand neurological functions

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Alex Thoman - GMO Primates to help better understand neurological functions

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Since pigs have taken, I switched my topic to GMO primates

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ScienceDirect.com | Science, health and medical journals, full text articles and books.

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ScienceDirect is the world's leading source for scientific, technical, and medical research. Explore journals, books and articles.

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Abstract

Transgenic coho salmon strains containing an all-salmon growth hormone (GH) gene construct (OnMTGH1) have been examined. The transgene utilized is comprised of the metallothionein-B promoter driving the expression of the type-I growth hormone gene from sockeye salmon. Transgene DNA is integrated into the salmon genome, but is transmitted at low frequency from founder transgenic animals consistent with late integration following microinjection resulting in mosaic animals, whereas subsequent generations transmit transgene DNA as a stable Mendelian trait. Different families established from separate founder animals yield lines with unique growth characteristics suggesting important site-of-integration effects on transgene expression. Growth enhancement of transgenic salmon is initiated early, with advanced hatch timing but occurring also throughout the life history, particularly during the early phase in fresh water. GH transgenic fish showed precocious smoltification and onset of sexual maturation, but approximately normal adult body size, indicating that compression of the normal coho salmon life history is occurring. The viability of diploid GH transgenic salmon ranges from reductions to greater than that of controls among strains, and triploid transgenic animals had normal viability relative to diploid transgenic salmon. Triploid transgenic salmon display a reduction in growth rate relative to transgenic diploids, but are still significantly growth enhanced compared with nontransgenic controls. The distinct phenotypic characteristics of GH transgenic families suggest that evaluation for aquaculture and for risk assessments requires examination of strains on a case-by-case basis. Furthermore, strong effects of size at maturity in culture conditions were observed for nontransgenic wild strain coho salmon which were not apparent in GH transgenic salmon, indicating that strong genotype by environment effects strongly influence phenotype and complicate risk assessments.

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Growth, viability and genetic characteristics of GH transgenic coho salmon strains

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Transgenic coho salmon strains containing an all-salmon growth hormone (GH) gene construct (OnMTGH1) have been examined. The transgene utilized is comprised of the metallothionein-B promoter driving the expression of the type-I growth hormone gene from sockeye salmon.

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Transgenic Tobacco Plants Overexpressing a Grass PpEXP1 Gene Exhibit Enhanced Tolerance to Heat Stress

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Heat stress is a detrimental abiotic stress limiting the growth of many plant species and is associated with various cellular and physiological damages. Expansins are a family of proteins which are known to play roles in regulating cell wall elongation and expansion, as well as other growth and deve…

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https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-014-0141-0

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The pioneering work of Stanley Cohen and Herbert Boyer, who invented the technique of DNA cloning, signaled the birth of genetic engineering, which allowed genes to transfer among different biological species with ease [ 1]. Their discovery led to the development of several recombinant proteins with therapeutic applications such as insulin and growth hormone. Genes encoding human insulin and growth hormone were cloned and expressed in E. coli in 1978 and 1979 respectively. The first licensed drug produced using recombinant DNA technology was human insulin, which was developed by Genentech and licensed as well as marketed by Eli Lilly in 1982.

There are more than 300 biopharmaceutical products including therapeutic proteins and antibodies in the market with sales exceeding USD100 billion [ 2],[ 3]. Therapeutic monoclonal antibodies have captured the major market share (>USD18 billion) followed by the hormones (>USD11 billion) and growth factors (>USD10 billion) [ 4]. Biopharmaceuticals approved by the US Food and Drug Administration (FDA) and European Medicines Agency (EMA) from 2004 to 2013 are largely derived from mammalian cell (56%); E. Coli (24%); S. Cerevisiae (13%); Transgenic animals & plants (3%) and insect cells (4%) as shown in Figure 1[ 5]-[ 13]. At present, insulin is being produced predominantly in E. coli and Saccharomyces cerevisiae for treatment of diabetic patients.

Percentage of biopharmaceuticals produced in different expression systems[ 5]-[ 13].

Since the early 1920s, diabetic patients were treated with insulin, which was purified from bovine or porcine pancreas. The development in the field of genetic engineering allowed the production of insulin in E. coli and yeast, which have been approved for therapeutic applications in human by FDA [ 14],[ 15].

Nowadays, recombinant human insulin is mainly produced either in E. coli or Saccharomyces cerevisiae. Using E. coli expression system, the insulin precursors (IP) are produced as inclusion bodies and fully functional polypeptides are obtained finally by solubilization and refolding procedures [ 16]. Yeast based expression system yield soluble IP which is secreted into the culture supernatant [ 17]-[ 19]. Saccharomyces cerevisiae is the most preferred and predominant yeast for large scale commercial production of insulin, however several other alternate yeast strains have been explored for insulin production [ 20]-[ 24]. Besides, E.coli and yeast, mammalian cells, transgenic animals and plant expression systems are also employed as a host for large-scale production of recombinant insulin [ 25]-[ 28].

The incidence of diabetes is increasing at an alarming rate and it has been speculated that the number of diabetic patients worldwide would increase to approximately 300 million by the year 2025 [ 29]. Consequently, the requirement for insulin will increase manifold (approximately more than 16000 kg/ year) and the productivity of current insulin expression system would not be sufficient to meet the future market demands. Efficient expression systems for insulin production are also needed and novel route for insulin administration such as oral or inhalation are to be developed.

Several recombinant protein based drugs, produced by various expression systems are approved by FDA. Among prokaryotes, Escherichia coli has always been preferred for production of recombinant proteins as it offered several advantages including high growth rate, simple media requirement, easy to handle, high yield and very cost effective. However, there are some disadvantages using E. coli expression system, such as loss of plasmid and antibiotic property, unsolicited inducers for gene expression, intracellular accumulation of heterologous proteins as inclusion bodies, improper protein refolding, lack of post- translational modifications (including unable to form disulphide bonds), protein-mediated metabolic burden and stress, endotoxin contamination, poor secretion, proteolytic digestion and complexity in downstream process [ 30]-[ 32].

Among yeast strains, Saccharomyces cerevisiae, Hansenulla polymorpha and Pichia pastoris are very commonly used for production of recombinant proteins [ 21],[ 24],[ 33]-[ 35]. Like E. coli, they grow rapidly and are very easy to handle and amenable to various genetic manipulations. Recombinant proteins produced in yeast are properly folded and glycosylated to a certain extent similar to the one expressed in mammalian cells. Various human therapeutic proteins, including therapeutic monoclonal antibodies are being produced in mammalian cell lines such as Chinese hamster ovary (CHO) and Baby hamster kidney (BHK) cells. Recombinant proteins expressed in mammalian cells are properly folded, glycosylated and generally yield a functionally active protein [ 36]. However, the production cost of biopharmaceuticals using mammalian expression system is very high due to expensive culture media. However, when we look at the number of approved biopharmaceuticals by United States and/or the European Union for 2013 recorded above average as compared to past five years. The mean rate of approval is 13 as shown in the Figure 2[ 5]-[ 13]. Remarkably, the number is same for both the years 2009 and 2013.

Approval numbers of biopharmaceuticals in United States and/or European Union over the past six years with a trend line showing the mean approval rate[ 5]-[ 13].

Structure and function of insulin

The human insulin is comprised of 51 amino acids and has a molecular weight of 5808 Da. It is produced by beta cells of the pancreas and plays a key role in regulating carbohydrate and fat metabolism in the body. Insulin is synthesized as a single polypeptide known as preproinsulin in pancreatic beta cells. Preproinsulin harbours a 24-residue signal peptide, which directs the nascent polypeptide to the endoplasmic reticulum. The signal peptide is cleaved as the polypeptide is translocated into the human of the endoplasmic reticulum resulting in the formation of proinsulin. In the Endoplasmic reticulum, the proinsulin is folded in proper confirmation with the formation of 3 disulphide bonds. Folded proinsulin is then transported to the trans-Golgi network, where it is converted into active insulin by cellular endopeptidases called as prohormone convertases (PC1 and PC2) and exoprotease carboxypeptidase E. The endopeptidases cleaves at two positions, resulting in the release of a fragment termed as C-peptide. The mature insulin, thus formed consists of an A-chain with 21 aminoacids and a B-chain containing 30 aminoacids and both polypeptides linked together by two disulphide bonds. Besides, the A-chain has an intrachain disulphide bond [ 37],[ 38].

E. coli expression system for production of insulin

E. coli is a preferred microorganism for large-scale production of recombinant proteins. However, several disadvantages limit its use for production of recombinant biopharmaceuticals. Various post-translational modifications (PTMs) such as glycosylation, phosphorylation, proteolytic processing and formations of disulfide bonds which are very crucial for biological activity, do not occur in E. coli[ 39],[ 40]. N-linked glycosylation is the most common posttranslational modification of proteins in eukaryotes. It has been discovered that the bacterium Campylobacter jejuni possess the capability to glycosylate the proteins and it was also shown that a functionally active N-glycosylation pathway could be transferred to E. coli[ 41]. Although the structure of bacterial N-glycan is different from that observed in eukaryotes, engineering of Campylobacter N-linked glycosylation pathway into E. coli, provides an opportunity to express heterologous proteins in glycosylated form in E. coli. Expression of Pglb oligosaccharyltransferase or (OTase) from C. jejuni in E. coli showed a significant increase in glycopepetide yield [ 42],[ 43]. Recently efforts has been made to produce glycosylated proteins with substrates other than native and non-native to E. coli and C.jejuni[ 44]-[ 48].

The codon usage of the heterologous protein also plays a major role in determining the expression level of recombinant protein. If the codon usage of heterologous protein differs significantly from the average codon usage of the E. coli host, it could result in very low expression. Usually, the frequency of the codon usage reflects the abundance of their corresponding tRNA. Therefore, significant differences in codon usage could result in premature termination of translation, misincorporation of aminoacids and inhibition of protein synthesis [ 49]. Expression of heterologous proteins in E. coli can be improved by replacing codons that are rarely found in highly expressed E. coli genes with more favorable major codons. Similarly, co-expression of the genes encoding for a number of the tRNA for rare codon, may enhance the expression of heterologous proteins in E. coli. There are some commercial E. coli strains available that encodes for tRNA for rare codons such as BL21 (DE3) CodonPlus-RIL, BL21 (DE3) CodonPlus-RP (Stratagene, USA) and Rosetta (DE3). BL21 (DE3) CodonPlus-RIL harbors tRNA genes for rare codons like AGG, AGA (arginine), AUA (isoleucine) and CUA (leucine). Similarly, Rosetta (DE3) strain harbors tRNA genes for rare codons like AGG, AGA (arginine), CGG (arginine), AUA (isoleucine), CUA (leucine), CCC (proline) and GGA (glycine). These rare codons have been associated with low expression of proteins in E. coli, hence application of these genetically engineered E. coli host strains may improve the expression level of heterologous proteins and thus might result in higher yield of desired protein [ 50]-[ 52]. The use of protease-deficient E. coli strains, which carry mutations that eliminate the production of proteases may also improve the yield of recombinant protein by reducing proteolytic degradation. E. coli strain BL-21, is deficient in two proteases encoded by the lon (cytoplasmic) and ompT (periplasmic) genes. Rather than the external parameters, targeted methods such as modifications in protease or secretion pathways can provide the insight into biology of recombinant proteins [ 53]. In E. coli, complex and large therapeutic proteins can be secreted in periplasm as it provides an oxidizing environment and help in forming disulphide bonds, which facilitate the proper folding of recombinant proteins and likely to yield reliable N- terminus of expressed protein [ 54]. Periplasm has advantages over cytoplasm in less protein concentration and proteolytic activity, improve the production titer [ 55], and enhance the solubility of recombinant protein. Altogether, with these advanced modifications and developments ease the process of target protein production thus accelerating the drug development [ 56].

Heterologous proteins generally accumulate in E. coli as inclusion bodies, which comprise of insoluble misfolded aggregates of proteins. Use of molecular chaperones may increase the protein solubility and assist in proper folding of recombinant protein. Some of the chaperones prevent aggregation of protein and some assist in refolding and solubilization of misfolded proteins. The most important chaperones in E. coli are GroEL, GroES, DnaK, DnaJ, GrpE and Trigger factor. These chaperones may be used singly, or in combination to enhance the protein solubility in E. coli[ 57],[ 58].

Recombinant human insulin was first produced in E. coli by Genentech in 1978, using a approach that required the expression of chemically synthesized cDNA encoding for the insulin A and B chains separately in E. coli[ 59]. After expressing independently, the two chains are purified and co-incubated under optimum reaction conditions that promoted the generation of intact and bioactive insulin by disulphide bond formation. The first commercial recombinant insulin was developed for therapeutic use in human by this two-chain combination procedure [ 60]. Another approach involves the expression of a single chemically synthesized cDNA encoding for human proinsulin in E. coli followed by purification and subsequent excision of C-peptide by proteolytic digestion. This approach was more efficient and convenient for large scale production of therapeutic insulin as compared to the two chain combination approach and has been used commercially since 1986 [ 60]. Eli Lilly followed this technology to produce Humulin, the first recombinant insulin approved in 1982, for the treatment of diabetic patients. These first generation recombinant insulins have an amino acid sequence identical to native human insulin and are preferred over animal derived insulin products [ 14]. However, advancement in the field of genetic engineering and development of technology to chemically synthesize genes with altered nucleotide sequence, facilitated the development of insulin analogues with altered amino acid sequence. It had been observed that native insulin in commercial preparations usually exist in oligomeric form, as zinc-containing hexamer due to very high concentration, but in blood, biologically active insulin is in monomeric form [ 61]. Hence, this oligomeric complex should dissociate so that insulin can be absorbed from the site of injection into the blood. Due to this, subcutaneously injected recombinant insulin usually have a slow onset with peak plasma concentration after 2 hours of injection and longer duration of action that last for 6-8 hours [ 62]. Hence, in order to develop a fast- acting insulin analogue, it was required to modify the amino acids residues whose side chains are involved in dimer or oligomer formation. It has been shown that amino acids residues in insulin B-chain particularly B8, 9,12, 13, 16 and 23-28 play critical role in oligomerization [ 63],[ 64]. Lispro, developed by Eli Lilly, was the first fast acting insulin analogue to obtain regulatory approval in 1996, for therapeutic use [ 60]. Insulin Lispro is engineered in such a way that it has similar amino acid sequence as the native insulin but has an inversion of proline-lysine sequence at position 28 and 29 of the B-chain, which resulted in reduced hydrophobic interactions and thus prevented dimer formations. For commercial production of insulin Lispro, a synthetic cDNA encoding for Lys B28- Pro B29 human proinsulin was expressed in E. coli and insulin Lispro was excised proteolytically from the proinsulin by treating with trypsin and carboxypeptidase. Another rapid-acting insulin analogue, produced in E. coli is Glulisine (Apidra) which was developed by Aventis Pharmaceuticals and approved by US regulatory authorities in 2004. Insulin Glulisine have been generated by replacing B3 asparagine by a lysine and B29 lysine replaced by glutamic acid [ 14].

To avoid multiple injection, long-acting insulin analogues with prolonged duration of actions have also generated. Insulin Glargine is one of such long-acting insulin analogues, which was developed by Aventis Pharmaceuticals and approved by regulatory authorities of USA and EU in 2000. Insulin Glargine was generated by replacing the C-terminal asparagine of the A-chain with a glycine residue and the C-terminal of the B- chain was modified by adding two arginine residues. These modifications resulted in increase of the isoelectric point (pI) from 5.4 to neutral values. Glargine was produced as proinsulin and expressed in E. coli and was finally formulated at pH 4 in soluble form. However, after subcutaneous administration, it precipitated due to neutral pH in the subcutaneous tissue. Resolubilization of insulin occur slowly, resulting in longer duration for its release in the blood [ 14].

Yeast expression system for the production of insulin

Yeast is a preferred host for expression of various heterologous proteins that require post-translational modifications for its biological activity. Yeast cell has the ability to carry out numerous post-translational modifications such as phosphorylation, O-linked glycosylation, N-linked glycosylation, acetylation and acylation. Recombinant proteins are expressed in soluble form in yeast and properly folded in functionally active form. Production of biopharmaceuticals using yeast expression system is also very cost effective and is amenable to scale up using large bioreactors. However, one major concern for producing therapeutic glycoprotein for human application is that yeast N-glycosylation is of the high-mannose type, which confers a short half-life in vivo and hyper-immunogenicity and thus render the therapeutic glycoprotein less effective. Various attempts have been made to humanize yeast N-glycosylation pathways in order to produce therapeutic glycoproteins with humanized N-glycosylation structure [ 65].

The therapeutic proteins produced in yeast are specifically from Saccharomyces cerevisiae and include hormones (insulin, insulin analogues, non-glycosylated human growth hormone somatotropin, glucagon), vaccines (hepatitis B virus surface antigen), uprate oxidase from Aspergillus flavus, granulocyte-macrophage colony stimulating factor, albumin, hirudin of Hirudo medicinalis and human platelets derived growth factor [ 34]. Like E. coli, yeast derived recombinant biopharmaceuticals majorly intended as therapeutics for infectious diseases or endocrine, metabolic disorders. Alternate yeast strains, besides S. cerevisiae, are being explored for large-scale production of biopharmaceuticals. Specifically, Pichia pastoris has the ability to attain high cell densities by its robust methanol-inducible alcohol oxidase 1 (AOX1) promoter and simple developmental approaches contribute to high quality and quantity of recombinant proteins production. In comparison to Saccharomyces cerevisiae, Pichia pastoris provides a major advantage in the glycosylation of secreted proteins because it does not hyperglycosylate the heterologous proteins. Both yeast strains have a majority of N-linked glycosylation of the high-mannose type, but the length of the oligosaccharides chain added to proteins in Pichia (around 8-14 mannose residues per side chain) is much shorter than those expressed in Saccharomyces cerevisiae (approximately 50-150 mannose residues per side chain), suggesting that glycoproteins produced in Pichia pastoris may be more suitable for therapeutic use in humans [ 66],[ 67]. Moreover, very high level of expression of heterologous proteins can be attained in Pichia pastoris, that might constitute about 30% of total cellular protein which is very high as compared to S. cerevisiae[ 68],[ 69]. Therefore, Pichia pastoris can be an attractive alternate for large-scale production of recombinant insulin and insulin analogues. Comparing the different insulin production systems where the bacterial expression systems show higher average specific productivity and maximum biomass concentrations are higher in yeast, the overall production space-time yield remains similar as shown in Table 1[ 70].

Saccharomyces cerevisiae has been extensively used to produce recombinant human insulin since early 1980s [ 17],[ 18] and a large proportion of recombinant commercial insulins are produced by this yeast expression system [ 19],[ 74]. For efficient expression and secretion of recombinant proinsulin in yeast, insulin construct was engineered to contain the native A-chain and a B-chain lacking the C-terminal B30 threonine, either directly fused or linked via a short synthetic C peptide (like AAK). The cDNA sequence encoding for this construct was fused with α-factor signal sequence of Saccharomyces cerevisiae for secreted expression of proinsulin which gave yield upto 80 mg/ml of insulin. The single chain proinsulin was purified and converted to active insulin by a trypsin-mediated transpeptidation reaction in presence of threonine ester [ 19]. Besides native recombinant insulin, various insulin analogues are also being produced in S. cerevisiae. Insulin Aspart is another fast-acting insulin analogue, which was produced in S. cerevisiae, developed by Novo Nordisk and approved by US FDA in 2001 for therapeutic use in human. Insulin Aspart was generated by replacing proline residue at position 28 with aspartic acid in the B-chain. This genetic modification resulted in an increase in inter-chain charge repulsion, decrease in self-association and thus causing rapid entry into the blood from the site of subcutaneous injection [ 63],[ 75].

Insulin Detemir is another recombinant long-acting insulin analogue that was commercially produced in S. cerevisiae, developed by Novo Nordisk and approved for therapeutic use in human in 2004 by European regulatory authorities. Recombinant Detemir have been generated by removing the threonine residue at the 30 position of the B-chain, and a C14 fatty acid chain covalently attached to the lysine residue at the 29 position of the B-chain. These genetic alterations resulted in the binding of insulin to albumin in plasma, which ensured the slow and constant release of insulin and thus prolonging its duration of action up to 24 hours [ 76]-[ 78].

Sacharomyces cerevisiae has been reported for the production of more than 40 different recombinant proteins [ 79]. A few of which related to diabetes are illustrated in Table 2, along with different characteristics. Few proteins secreted extracellularly by Sacharomyces cerevisiae with α-factor leader sequence being repeatedly used for adequate production of recombinant proteins. Furthermore, a synthetic leader sequence had been developed by Kjeldsen and associates at Novo Nordisk for more efficient protein secretion in yeast [ 79],[ 80].

Transgenic plants as host for insulin production

Transgenic plants have been utilized to produce recombinant proteins because of their advantage of cost effectiveness, high quality protein processing, absence of human pathogens, ease of production and presence of eukaryotic machinery for posttranslational modifications. Initially, the human growth hormone was the recombinant protein product extracted from transgenic tobacco plant [ 81]. After that, numerous different products have developed from plants such as Hepatitis-B-Virus surface antigen, antibodies, industrial proteins and milk proteins.

Recombinant human insulin has been successfully expressed and produced in oilseeds of plant Arabidopsis thaliana[ 27]. This technology involved the targeted expression of insulin in subcellular organelles known as oilbodies that allowed very high level of expression with easy recovery of recombinant insulin. Oilbodies are storage organelles inside the oilseeds, which comprises of hydrophobic triacylglycerol core encapsulated by phospholipid membrane and an outer wall of proteins known as oleosins. Genetically engineered oil seeds have been generated with recombinant protein specifically targeted to oilbodies as oleosin fusion [ 27],[ 82],[ 83]. Then the oilbodies are easily separated from other seed components by liquid-liquid phase separation, which reduced the number of chromatography steps required to obtain purified insulin. It has been observed that insulin accumulated to high level in transgenic seed (0.13% of total seed protein). Recombinant insulin was cleaved from the oleosin fusion partner and matured with trypsin digestion following oil body purification to yield a biologically active insulin. This study clearly demonstrated that expression of insulin as oleosin fusion protein in plant allow accumulation of large amount of recombinant insulin within the seed and also provide simple downstream purification by centrifugation i.e. oilbody purification. Subsequent maturation to obtain biologically active insulin can be accomplished using standard enzymatic methods currently used for commercial production of insulin from E. coli and yeast. Oilseeds also act as a natural cellular warehouse, where recombinant insulin can be stockpiled until required [ 27].

In another approach, transgenic plants have been generated, in which, tobacco and lettuce chloroplasts were transformed with human proinsulin comprised of A, B and C-chains fused with the cholera toxin B subunit [ 28]. It has been observed that, old tobacco leaves accumulated proinsulin upto 47% of total leaf protein and similarly, old lettuce leaves amassed proinsulin up to 53% of total leaf protein. Proinsulin stored in leaves of lettuce was found to be very stable as up to 40% of proinsulin was detected even in senescent and dried leaves as shown in Table 3. Proinsulin from tobacco leaves was extracted with 98% purity and cleaved by Furin protease to release insulin peptides. Oral delivery of unprocessed proinsulin encapsulated in plant cell or by injection into mice revealed lowering of blood glucose levels similar to commercially available insulins. Based on the yield (3 mg of proinsulin/gm of leaves), it was estimated that one acre of tobacco plantation could yield upto 20 million daily doses of insulin per year [ 28]. C-peptide of proinsulin, which is not present in current commercially available insulin and insulin analogues derived from E. coli and S. cereviciae, would be a great advantage in long-term treatment of diabetic complications such as stimulation of nerve and renal functions. Very high level of expression of biologically active proinsulin in tobacco and lettuce leaves and long-term stability in dried leaves offers a reliable low-cost technology for both injectable as well as oral delivery of proinsulin.

Table 3 CTB cholera toxin B subunit proinsulin expression in tobacco and lettuce chloroplasts[28]

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Cell factories for insulin production - Microbial Cell Factories

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The rapid increase in the number of diabetic patients globally and exploration of alternate insulin delivery methods such as inhalation or oral route that rely on higher doses, is bound to escalate the demand for recombinant insulin in near future.

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https://watermark-silverchair-com.ezproxy1.lib.asu.edu/erp284.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAAtkwggLVBgkqhkiG9w0BBwagggLGMIICwgIBADCCArsGCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMdrbvVz9gcHE-8C5DAgEQgIICjL45HKiONGm4kcPDzpVH2pb7IW9rQR8gf8ftlH7aYMr_Ifc7RNKnnBVDU0NKJWrbq2RHnz1rl3EkAu1iW_1Rrr3ugByiVdhDiiA-Ea7CBDimUJQeTxgO4R15DIDwEKulUAPDDjs7qHCTi7lGYiE5_jTwNjFNoeXL_NV-utBwXenPpbUqF4jvtrcpEGKjFwuP-nabmu60VgFbq1rdGCxHDAic8vlIJLM_ZeWec7UPUwwylgBTjK3ylU4T1x_l71ju8KHJHm43xjVo4EUsPceDpGm7je2GVCi1bQqkHFHM7VgYyJUccYApJnIQeSkx-C9dJXz0ktwc40HPt9kof5iFtr1b-iQqgX1JRQxf1TKiuckkb7JZTVIQiBhXp41AazBrDBsZ15iSpbHxuJIb_5G3DzhdCH134BX-bNcEXtoY3pexjyk4JKZ8VvMpHu9YWrMVWWrv4GNcLdRuI-MjL2gueXxMofhcSHshdq47hyx0ImOos7BelJPQyek2K6rTUxeBsNycbShGRtqedRsGfM-MTsevDiQZ3xGDosYVMHiQ6oVu-_KRvaz0VfmRCsng6iltnou7xeNfw-axUuEVuRcn4x0VUS0V57Hpg8bjWbJmUDJXZkB6PWBoTFMBApFzr9W4_ONCv_LBI97al29CjYeq_2bv33e3tbcTCZAdaVFQ91cSjuwk_uxAHR-WEqEE6MkSBLqPg4Fgb1zDltrnfwKZyX9Zucv5_4jwXfZ7ZXaplE5qdKdQtALe9PfBs1fh1E-hLYE9joQKM2--gIcw4mSNR_y2GDZxVc6mRjF9ePKQaRJhtkxXWpiID0iNF966qE7g043nt_fLUQzyR7GlXNG1PYPiqTlHbqfuQgKRt4o

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https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108622#s3

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Rolf B. Edvardsen,

Sven Leininger,

Lene Kleppe,

Kai Ove Skaftnesmo,

Anna Wargelius

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Abstract

Understanding the biological function behind key proteins is of great concern in Atlantic salmon, both due to a high commercial importance and an interesting life history. Until recently, functional studies in salmonids appeared to be difficult. However, the recent discovery of targeted mutagenesis using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system enables performing functional studies in Atlantic salmon to a great extent. We used the CRISPR/Cas9 system to target two genes involved in pigmentation, tyrosinase ( tyr) and solute carrier family 45, member 2 ( slc45a2). Embryos were assayed for mutation rates at the 17 somite stage, where 40 and 22% of all injected embryos showed a high degree of mutation induction for slc45a2 and tyr, respectively. At hatching this mutation frequency was also visible for both targeted genes, displaying a graded phenotype ranging from complete lack of pigmentation to partial loss and normal pigmentation. CRISPR slc45a2/Cas9 injected embryos showing a complete lack of pigmentation or just a few spots of pigments also lacked wild type sequences when assaying more than 80 ( slc45a2) sequence clones from whole embryos. This indicates that CRISPR/Cas9 can induce double-allelic knockout in the F0 generation. However, types and frequency of indels might affect the phenotype. Therefore, the variation of indels was assayed in the graded pigmentation phenotypes produced by CRISPR/Cas9- slc45a2. The results show a tendency for fewer types of indels formed in juveniles completely lacking pigmentation compared to juveniles displaying partial pigmentation. Another interesting observation was a high degree of the same indel type in different juveniles. This study shows for the first time successful use of the CRISPR/Cas9 technology in a marine cold water species. Targeted double-allelic mutations were obtained and, though the level of mosaicism has to be considered, we demonstrate that F0 fish can be used for functional studies in Atlantic salmon.

Citation: Edvardsen RB, Leininger S, Kleppe L, Skaftnesmo KO, Wargelius A (2014) Targeted Mutagenesis in Atlantic Salmon ( Salmo salar L.) Using the CRISPR/Cas9 System Induces Complete Knockout Individuals in the F0 Generation. PLoS ONE 9(9): e108622. https://doi.org/10.1371/journal.pone.0108622

Editor: Diego Fraidenraich, Rutgers University -New Jersey Medical School, United States of America

Received: June 26, 2014; Accepted: August 23, 2014; Published: September 25, 2014

Copyright: © 2014 Edvardsen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

Funding: The project was supported by the Norwegian Research Council ( http://www.forskningsradet.no), project number 221648/O30. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Atlantic salmon is an important commercial aquaculture species. Likewise, the growing salmon industry displays a demand for healthy, fast growing and tasty fish. To improve knowledge and to solve bottlenecks of salmon aquaculture, the salmon genome is currently being sequenced [1]. To further implement findings gained from the salmon genome, functional studies of genes linked to health, growth, welfare and filet quality will be necessary. This will increase our knowledge and possibly explain causative relations to some of the challenges present in the aquaculture industry such as egg/sperm quality, reproductive mechanisms, growth rate, disease resistance and immunological mechanisms. In the era of genome wide association studies many genes will be directly linked to a trait and further functional characterization of these genes will significantly improve selective breeding of associated traits.

Functional studies in salmon embryos have so far been difficult to implement, but a few studies have employed knockdown techniques in salmonids using morpholinos [2]-[4]. However, current approaches have not proven to be very efficient. This can partly be ascribed to the characteristics of the egg being difficult to precisely inject due to its large and opaque features and almost invisible first cell stage [5]. This together with the variable efficiencies using morpholinos makes it even more difficult in this non-model species [6].

In recent years the ability to specifically create targeted mutations by gene editing techniques has steadily improved. The first gene editing experiments in teleosts were conducted in zebrafish, starting with targeted mutagenesis using zinc finger nucleases (ZFN, [7], [8]) followed by the more efficient transcription activator-like effectors (TALENS, [9]). A study in rainbow trout used ZFN to induce targeted mutagenesis in the sdy gene, with the purpose to confirm its function in sex determination [10]. The ZFN technology has also been applied to a catfish species, with the aim to elucidate muscle growth mechanisms [11]. The problem using this methodology in salmonids is their long generation time (2-4 years) as previously used targeted mutagenesis protocols do not efficiently induce bi-allelic mutations and therefore require breeding for two more generations to induce a homozygous mutation in targeted genes.

Recently, with the CRISPR (clustered regularly interspaced palindromic repeats)/Cas9 (CRISPR-associated) system, a highly efficient technology has been developed, which has shown to specifically induce mutations in many model and non-model animals including zebrafish and tilapia [12], [13]. This technology is so efficient in zebrafish and Xenopus that it can induce gene specific bi-allelic mutations in the first (F0) generation [14], [15]. This feature makes this new technology very promising for use in non-model organisms with longer life spans such as the Atlantic salmon.

The CRISPR/Cas9 system was identified in bacteria and archaea, serving as an immune system against bacteriophages and foreign DNA [16]-[19]. In these organisms the CRISPR loci incorporates foreign DNA, this DNA is then synthesized into short CRISPR RNAs (crRNA) which bind to a transactivating RNA (tracrRNA) and to foreign DNA catalyzing cleavage and destruction by recruiting the RNA-guided DNA nuclease Cas9 [20]. A construct which has been tailored for targeted mutations is a fusion of crRNA and tracrRNA functioning as a guide RNA (gRNA) which in combination with the Cas9 nuclease can then induce double DNA strand breaks [21]. The mutation is introduced when the doublestrand break is repaired by either high-fidelity homologous recombination or error prone non-homologous end-joining (NHEJ).

The aim of the present study was to attempt creating specifically induced bi-allelic mutations in target genes in Atlantic salmon using the CRISPR/Cas9 system. For this purpose we selected genes producing pronounced pigmentation phenotypes in zebrafish enabling a direct visual enumeration [22]-[23]. Specific target oligonucleotides corresponding to two genes ( tyrosinase and slc45a2) known to be involved in pigmentation in zebrafish and other species were cloned into a CRISPR vector developed for zebrafish [14]. In vitro transcribed RNAs from the cloned constructs were injected into one-cell stage Atlantic salmon embryos together with Cas9 mRNA. Mutation rates were measured in whole embryos prior to hatch and in DNA prepared from finclip versus whole juvenile fish showing lack of pigmentation. Here, we present the first targeted genome editing employing the CRISPR/Cas9 technology on salmonid fish and further extend the list of successfully tested non-model species where functional studies and induction of mutations have previously been problematic.

Materials and Methods

Cloning of target sites for gRNAs

Using a draft salmon genome sequence (AGKD00000000.1) both tyrosinase ( tyr) and slc45a2 exon sequences were predicted by comparing to published sequences in salmon for tyr (Accession: NM_001123643) and in zebrafish for slc45a2 (NP_001103847). From the predicted sequences in the salmon genome the slc45a2 gene was found split into two contigs (Accession: AGKD01080285, AGKD01048537). The salmon tyrosinase ( tyr) coding sequence was found split into four genomic contigs (Accession: AGKD01095509, AGKD01379509, AGKD01094529, AGKD01346345). Suitable oligonucleotides for CRISPR targeting were selected using an online tool provided by http://zifit.partners.org/ZiFiT/ [24]. Candidate target sequences were compared to the current salmon genome draft to exclude unwanted off-target cleavage. Target site and oligonucleotide sequences for salmon slc45a2 and tyr are listed in Table S1. Oligonucletides were annealed and cloned into pT7-gRNA [14], Addgene ID# 46759 as described below: 2 µM of each forward and reverse oligonucleotide was annealed in annealing buffer (0.4 M Tris pH 8, 0.2 M MgCl ₂, 0.5 M NaCl, 10 mM EDTA pH 8) by incubating at 95°C for 5 min, followed by ramping down to 4°C at −2°C/min. 3 µl of annealed oligonucleotides were ligated into 5 ng of BsmBI, BglII, SalI digested pT7-gRNA and gel-extracted using Quick T4 DNA Ligase (NEB) and subsequently cloned using XL1-blue competent cells (Stratagene). Plasmids were prepared using a QIAprep Spin Miniprep Kit (Qiagen).

In-vitro transcription of gRNA and Cas9 mRNA

pT7-gRNA was linearized using BamHI-HF (NEB), containing the respective cloned target sites for either slc45a2 or tyr, were prepared using a QIAprep column (Qiagen) and transcribed using the MEGAscript T7 kit (Ambion) according to the manufacturer's protocol. The mir Vana miRNA Isoltation Kit was used to purify gRNAs.

For producing Cas9 nuclease mRNA, we used the pTST3-nCas9n vector optimized for Zebrafish [14] (Addgene ID# 46757). Prior to in-vitro transcription, the plasmid was linearized using XbaI (NEB) and cleaned up via a QIAprep Spin column. Cas9 mRNA was produced using the mMessage mMachine T3 kit (Ambion) and purified using an RNeasy MiniKit spin column (Qiagen).

Injection procedures

Salmon eggs and sperm obtained from Aquagen (Trondheim, Norway) were sent overnight to Matre Aquaculture station at IMR. Eggs were subsequently fertilized with sperm in fresh water (6-8°C) containing 0.5 mM reduced Gluthathione as described earlier for rainbow trout [25]. After fertilization, embryos were incubated 2-3 hours at 6-8°C until the first cell was visible. Eggs were subsequently injected with a mix containing 50 ng/µl gRNA and 150 ng/µl Cas9 mRNA in MilliQ H ₂ O using the picospritzer III (Parker Automation, UK) and needles from Narishige (Japan). After injection, eggs were incubated at 6°C until hatching.

Analysis of mutations

DNA was obtained from embryos, juveniles and fin clips using DNeasy Blood & Tissue kit (Qiagen) or AllPrep DNA/RNA kit (Qiagen) with the following modifications: Eggs were squashed in Buffer ATL and Proteinase K (DNeasy Blood & Tissue kit, Qiagen), incubated at 56°C for 2-4 h and subjected to phenol/chloroform extraction prior to DNA purification. Briefly, 1 vol. of UltraPure Phenol/Chloroform/Isoamylalcohol (25∶24∶1, Lifetechnologies) was added, mixed and phases separated by centrifugation. The upper phase was mixed with 1 vol. of chloroform, centrifuged and 1 vol. of Buffer AL and 100% EtOH (DNeasy Blood & Tissue kit, Qiagen) was added to the DNA containing aqueous phase and subjected to subsequent steps of the DNeasy Blood & Tissue kit protocol. Juveniles (separated from the yolk sac) and fin clips were homogenized using Zirconium oxide beads and a homogenizer (Precellys) in buffer ATL or buffer RLTplus/β-mercaptoethanol (AllPrep kit, Qiagen) prior to DNA extraction. PCR was performed on genomic DNA to obtain a fragment that covered the targeted mutagenesis site (PCR primer sequences are listed in Table S1). Fragments were both directly sequenced and subcloned into pCR4-TOPO using the TOPO TA cloning kit for sequencing (Invitrogen) to either measure the general effect in the target site in the whole preparation or in single sequences from clones to assess the level of mosaic mutation rate in each individual or sample.

Ethics statement

All animal experiments within the study were approved by NARA, the governmental Norwegian Animal Research Authority ( http://www.fdu.no/fdu/). To avoid unnecessary pain all fish larvae were sedated with metomidate prior to sampling.

Results and Discussion

For the establishment of the CRISPR/Cas method in salmon, target genes were selected based on their known role in pigmentation of fish, as a resulting phenotype is clearly visible at early developmental stages and throughout life. Since salmonid genomes are partly tetraploid [26], the two selected pigmentation genes, tyrosinase and slc45a2, were also chosen because both occur only once in the genome making them suitable for gene knockout. Tyrosinase ( tyr) is essential for normal pigmentation both in medaka [27]-[28] and zebrafish [22]. In addition Tyr has previously been shown to affect pigmentation in rainbow trout using morpholinos [29]. Likewise, Slc45a2 is essential for pigmentation in both zebrafish [23] and medaka [30].

Since development is relatively slow in Atlantic salmon, it takes almost three months until a visible pigmentation phenotype can be observed in hatched embryos at about 500 day° (∼84 days at 6°C [5]). In salmon, the developmental stage is calculated as day° by multiplying the incubation temperature (in °C) with the amount of days since fertilization. At stages prior to visual examination, embryos can be screened by PCR for targeted mutations. Consequently, injected embryos were harvested two weeks after fertilization (17 somite stage, 72 day°), DNA was extracted from whole embryos and PCR screenings were performed for both gRNA targets, CRISPR slc45a2/Cas9 (n = 20 embryos) and CRISPR tyr/Cas9 (n = 9 embryos). This analysis revealed that both CRISPR slc45a2/Cas9 and CRISPR tyr/Cas9 resulted in induced mutations in 40% and 22% of the embryos at early developmental stages, respectively (see Table 1). This reflects that in our experiments the overall mutation rate is lower than what has been observed in zebrafish (75-90%) [14] but within a similar range as it has been obtained for tilapia (24-50%) [13]. A possible reason for a lower mutation rate in Atlantic salmon can be the lower incubation temperature (6-8°C) causing the Cas9 enzyme to be less effective. Another more plausible explanation is the injection procedure. Because of the opaque character of newly fertilized salmon eggs and the hardness of the chorion it is difficult to inject directly into the cytoplasm of the first cell. Therefore, the relatively low number of embryos displaying mutations is possible due to failed injections. However, we assume that successful injections result in a high incidence of mutations ( Figure 1 and 2) which implies that the mutation efficiency is as high as previously observed for zebrafish [14]. An increased early mutation frequency could possibly also be obtained by injecting the Cas9 protein directly or by improving the timing of injection after fertilization. It has been shown that the introduction of off-target mutations might constitute a problem using the CRISPR/Cas9 system. In this study we did not measure off-target mutations, however previous studies in zebrafish has shown a low off-target mutation frequency produced by CRISPR [31]. CRISPR sites were also carefully selected in this study to prevent off-target effects.

Mutation rates and number of indel variants found in Atlantic salmon whole juvenile fish extracts at 650 day° (B).

(A) Fish analyzed for indels in fin clip and whole juvenile fish preparations. (B-D) Frequency [%] (x-axis) of a specific indel (y-axis) in fin clip (grey bars) and whole fish (black bars) of Atlantic salmon juveniles at 600 day°.

At 650 day° phenotypes in mutated juvenile groups were visually determined and 8.7% and 5.1% of juveniles displayed a graded loss of pigmentation ( Figure 1A). The similar graded degree of phenotype induction has previously been observed in both zebrafish and Xenopus for targeting the tyr gene in these species [14], [32]. In contrast to these studies a dose dependent severety of phenotype could not be established, since we only used one empirically determined concentration of gRNA and Cas9 mRNA in our work. However, we cannot exclude a dose-dependent effect due to injection procedures as explained above. Fish showing a graded phenotype due to mutations in either slc45a2 or tyr, were selected based on the level of pigmentation loss. Hence, the following juveniles were subjected to PCR screening and sequencing of induced mutations; full ( slc45a2-1), almost full ( tyr-1), medium ( slc45a2-2, tyr-2) and little pigmentation loss ( slc45a2-3, tyr-3, Figure 1, Table S2a-c and S3a-c). Additionally, two fish from each group, targeted for either slc45a2 or tyr, which showed no visual pigmentation loss were analyzed ( neg slc45a2#1, neg slc45a2#2, neg tyr#1 and neg tyr#2, Figure 1, Table S2e-f and S3e-f). To quantify mutation frequencies in each juvenile fish, DNA was extracted from the whole fish, a PCR product covering the CRISPR target site was amplified and the product was subsequently subcloned (50-100 clones per fish). In the mutated slc45a2-1 fish, which displayed a complete loss of pigmentation, no wild type sequences could be detected in any of the clones assayed (n = 88). However, medium pigmentation loss phenotype in slc45a2-2 ( Figure 1, Table S2b) mutated fish also lacked wild type sequences even though 87 clones were sequenced. Simply undersampling might be an explanation for this. Nevertheless, more wild type sequences have been detected in fish with less severe phenotypes, while none or few could be found in strong (no/medium pigmentation) phenotypes. This trend was also observed in two of the five tyr phenotypes ( tyr-1 and tyr-2), where only one wild type sequence of 58 and 54 sequenced clones, respectively, could be detected in each fish ( Figure 1, Table S3a-b). Additionally, we screened 21 and 11 fish targeted for either slc45a2 or tyr, displaying a wild type phenotype at 650 day° (neg slc45a2#1, neg slc45a2#2, neg tyr#1 and neg tyr#2, Figure 1B). In these groups, 9,5% ( slc45a2) and 45% ( tyr) of juveniles were shown to have indel mutations ( Table 1, Table S2d-e and S3d-e). As expected these fish displayed a low frequency of targeted mutations explaining the wild type character of the fish ( Figure 1B).

We further wanted to know if embryo/juvenile stages could be sorted based on mutation frequencies in fin clips only, instead of sacrificing the whole fish. This is important in future studies when targeting genes with unknown function and where mutated embryos/juveniles show no visual phenotype in F0. To investigate if fin clips could mirror the mutation type found in whole fish, we screened fin clip and whole fish DNA preparations from additional juveniles (600 day°) which displayed graded pigmentation phenotypes in the slc45a2 knockout attempts. This included three fish slc45a2-4 which only displayed a few pigmentation spots, slc45a2-5 displayed some more pigmentation spots and slc45a2-6 displayed even more pigmentation spots ( Figure 2A). These fish were screened for indel types and frequencies which are presented in Figure 2 B-D and in Table S4a-c. This comparison revealed similar frequencies for the most common indel variants in all three fish. However, among the less frequently occurring indels we observe more indel variants in the whole fish compared to fin clip. These results show that the fin clip to a certain degree mirrors the mosaicism in the knockout fish and can therefore be used as a tool to evaluate knockout phenotypes.

Another interesting finding was the independent generation of the same mutation types in different fish injected with the same gRNA ( Table 2). By thorough screening of the 6 scl45a2 mutated fish ( slc45a2-1 to slc45a2-6, Figure 1A and 2A), we found 7 of the indel variants to be present in at least three of the seven fish ( Table 2). However, we were not able to link the abundance of certain indel types to specific phenotypes. These mutations were all in proximity of the PAM site (bold and underlined in Table 2), which is similar to what has been observed previously in zebrafish [14], [24]. A previous study using the CRISPR/Cas9 technology in Xenopus also indicated that the same mutations were formed using the same construct in different F0 individuals [15]. This further suggests that individual F0 fish may hold the exact same indel variants and crosses of these can produce a non mosaic homozygous mutant already in the F1 generation. This technology is therefore especially relevant for species with long generation times like Atlantic salmon.

In conclusion, we show here for the first time successful targeted mutagenesis in salmon using the CRISPR/Cas9 technology. We anticipate that this technology will be an important tool for functional studies in this biologically and commercially interesting fish species. In the future the use of this technology will be facilitated since the rainbow trout genome has recently been sequenced [26] and the Atlantic salmon genome has been made available (Acc. No. AGKD00000000.3). In our hands, genome editing using CRISPR/Cas9 in salmon seems to be somewhat less efficient than reported for other fish species [12]-[14], probably due to the features of the egg and hence obstacles during injections procedures. Nevertheless, using knockout of pigmentation genes as shown here, can be a useful marker in co-injections with gRNAs targeting other genes of interest which show no obvious phenotype. This approach could facilitate the selection of putative positive fish for confirming a successful knockout of targeted genes. Previous targeted knock out technologies such as TALENs and ZFN did induce lower mutation frequencies in the F0 compared to CRISPR/Cas9 [7]-[9]. Hence, due to the general high efficiency and biallelic mutation capacity of the CRISPR/Cas9 technology, the F0 generation can already be used for the evaluation of protein functions in salmonids, as has been previously demonstrated for tilapia and Xenopus [13], [15]. Furthermore, this paper substantiates that the stronger the knockout phenotype found in the F0 generation is, the less indel variants are found. We also tested and confirmed that fin clips can be used as an indicator of an efficient knockout. To conclude, the establishment of functional studies through the CRISPR/Cas9 technique in salmonids opens up new doors to understand their basic biology and provides a useful tool for investigating traits important for the aquaculture industry.

Acknowledgments

We would like to thank Lise Dyrhovden for excellent care of embryo/juvenile rearing and Stig Mæhle for expert lab assistance.

Author Contributions

Conceived and designed the experiments: AW RBE. Performed the experiments: AW RBE SL LK KS. Analyzed the data: AW RBE SL. Contributed reagents/materials/analysis tools: SL. Contributed to the writing of the manuscript: AW SL RBE. Designed the CRISPR: RBE SL. Cloned all CRISPR constructs and produced gRNA and Cas9 for injection: SL. Injected embryos with CRISPR/Cas9 and followed up on rearing of embryos after injection: AW RBE SL LK KS. Extracted DNA and cloned and sequenced mutated embryos: SL.

References

1. 1. Davidson WS, Koop BF, Jones SJ, Iturra P, Vidal R, et al. (2010) Sequencing the genome of the Atlantic salmon (Salmo salar). Genome biology 11: 403.

2. 5. Gorodilov YN (1996) Description of the early ontogeny of the Atlantic salmon, Salmo salar, with a novel system of interval (state) identification. Environmental Biology of Fishes 47: 109-127.

3. 30. Fukamachi S, Kinoshita M, Tsujimura T, Shimada A, Oda S, et al. (2008) Rescue from oculocutaneous albinism type 4 using medaka slc45a2 cDNA driven by its own promoter. Genetics 178: 761-769.

# data[].attributes.attachment_link.title
Targeted Mutagenesis in Atlantic Salmon (Salmo salar L.) Using the CRISPR/Cas9 System Induces Complete Knockout Individuals in the F0 Generation

# data[].attributes.attachment_link.description
Understanding the biological function behind key proteins is of great concern in Atlantic salmon, both due to a high commercial importance and an interesting life history. Until recently, functional studies in salmonids appeared to be difficult. However, the recent discovery of targeted mutagenesis using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system enables performing functional studies in Atlantic salmon to a great extent.

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https://www.embopress.org/doi/full/10.1038/sj.embor.7400171

Chicken

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Bashir

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Abstract

A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein ( RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 ± 7.5 per recipient). Three surrogate mothers were successfully impregnated (27.3%) and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline-specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole-body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cat but not in the surrogate mother cat. Red fluorescence was detected in tissue samples, including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue, and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.

Introduction

Domestic cats have been proposed as a biomedical model for the study of several human diseases [ 1]. Domestic cats are phylogenetically close to humans, and the feline gene map displays a higher level of systemic conservation with humans than rodents or other laboratory mammals [ 2]. Genetic manipulation of the cat is hampered by a lack of suitable technologies, such as embryonic stem cells (ESs), which are routinely used to generate targeted mutations in the mouse. Cloning through somatic cell nuclear transfer (SCNT) is a potential alternative approach in species for which ES technologies are unavailable. The SCNT technique has been used to produce cloned and transgenic cattle [ 3], sheep [ 4, 5], goats [ 6], and pigs [ 7, 8]. In order to improve the efficiency of transgenic animal production by SCNT, a method for screening both genetically modified nuclear donor cells and reconstructed embryos before embryo transfer to the surrogate females is critical. Green fluorescent protein ( GFP) selection of donor cells has been used to produce transgenic offspring by SCNT in mice [ 9], pigs [ 10], and cattle [ 11]. A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases [ 1]. As a species, the domestic cat exhibits several distinctive characteristics, one of which is a close genetic relationship to the human genome [ 2, 12]. The feline gene map displays a higher level of systemic conservation with humans than rodents or other laboratory mammals [ 2]. The efficient use of nuclear transfer that is associated with the genetic modification of donor cells in domestic cats would substantially improve the efficacy of the production of genetically identical cats that may carry genes for the study of specific human disorders [ 1]. In addition, these methods could be applied to the production of genetically engineered designer pets with selected favorable attributes, such as an allergen-free cat [ 1]. The current study was designed to evaluate the feasibility of producing transgenic cats expressing RFP as a reporter gene.

In this study we used the SCNT technique to produce transgenic cats that ubiquitously express RFP. We used a Moloney murine leukemia virus-based pseudotyped retroviral vector system in which recombinant viruses are packaged with vesicular stomatitis virus G glycoprotein [ 13] to deliver the RFP transgene to the cat somatic cells. The significance of this work stems from its precedence: it is the first report of the successful production of a cloned transgenic cat expressing an exogenous gene. Our nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.

Materials and Methods

Source of Reagents

All of the chemicals were purchased from the Sigma-Aldrich Chemical Company (St. Louis, MO) unless otherwise stated.

Animal Care and Use

The Stroller female cats that were used as oocyte donors were housed in stainless steel cages measuring 0.9 × 0.7 × 0.65 m and were provided with dry food and water ad libitum. They were maintained in an environmentally controlled room on a 14L:10D photoperiod cycle with light onset at 0600 h. All animal care and use procedures were approved by the Institutional Animal Care and Use Committee of Sunchon National University.

Ovary Recovery and Oocyte Maturation

The ovarian follicular development of mature cats was stimulated by injection with 200 IU eCG (Daesung, Seoul, South Korea), and oocyte maturation was induced 4 days later by treatment with 100 IU hCG (Daesung). Ovaries were removed 24 h after hCG injection by routine ovariohysterectomy and were minced in m-Tyrode medium-Hepes using a scalpel blade to release cumulus oocyte complexes [ 14]. Collected oocytes were matured in TCM199 (M-7528) supplemented with 10% fetal bovine serum (FBS; Gibco/Invitrogen, Carlsbad, CA) and 1% penicillin G/streptomycin (P-4333) for 4 h at 38°C in an atmosphere of 5% CO ₂ in air.

Construction of the Retrovirus Vector and Virus Production

A plasmid (pLHCRW) containing a retrovirus vector sequence was constructed by replacing the TRE sequence of pRevTRE (Clontech, Mountain View, CA) with a fragment containing the CMV promoter, DsRed2 gene, and WPRE (woodchuck hepatitis virus posttrancriptional regulatory element) sequences ( Fig. 1). The CMV promoter and the DsRed2 gene were derived from pLNCX and pDsRed2-C1, respectively (Clontech). The WPRE sequence from woodchuck hepatitis virus 2 genomic DNA (GenBank accession number M11082) was introduced following the strategy of Zufferey et al. [ 15]. Retrovirus-producing cells were constructed following the procedure described by Koo et al. [ 16]. PT67 cells (Clontech) were transiently transfected with pLHCRW; the resultant LHCRW viruses were harvested and applied to the GP2-293 cells (Clontech). PT67 are retrovirus packaging cells that are characterized by the expression of the Gibbon ape leukemia virus envelope gene, whereas GP2-293 cells are specialized to express the gag and pol genes of Moloney murine leukemia virus. LHCRW-infected GP2-293 cells were selected with hygromycin (150 μl/ml) for 2 wk, and the resultant Hyg R (hygromycin-resistant) cells were transfected with pVSV-G (Clontech) to express VSV-G protein. Viruses were harvested 48 h after transfection. All virus-producing cells were grown at 37°C in a 5% CO ₂ incubator in Dulbecco modified Eagle medium (DMEM) with 4.5 g/l glucose (Gibco BRL, Grand Island, NY) supplemented with fetal calf serum (10%), penicillin (100 μg/ml), and streptomycin (100 μg/ml). The virus-containing medium that was harvested from the virus-producing cells was filtered through a 0.45-μm pore size filter and used to infect cat fibroblast cells. LHCRW-infected fibroblasts were selected with hygromycin (150 μl/ml) for 2 wk.

Establishment of Cat Fibroblast Cell Lines

Several fibroblast cell sublines were established from single, 6-mm skin biopsies taken aseptically from a male, odd-eyed, all-white cat. Primary cultures were obtained by mincing dermal tissue with sterile scissors in a 35-mm Petri dish (Nunc, Roskilde, Denmark) and culturing the fragments at 38°C with 5% CO ₂ in air in DMEM containing 10% FBS until 90% confluence was observed. Cells then were trypsinized and reconstituted at 1 × 10 ⁶ cells/ml, grown to confluence, and frozen as zero passage cells (1 vial/35-mm dish) in DMEM (Gibco BRL) containing 10% dimethylsulfoxide and 10% FBS. Cells were thawed and cultured for RFP gene transfection. RFP-transfected cells yielding confluent growth after culture in serum-free medium for 3 days were used for SCNT [ 14]. Single RFP-transfected cells were selected under an inverted microscope equipped with a G-2A filter (EX: 510-560 nm, BA: 590 nm; Fig. 2).

Somatic Cell Nuclear Transfer

The SCNT protocol was described previously [ 14]. Briefly, cumulus cells were removed from oocytes by gentle pipetting in TCM199 supplemented with 0.1% hyaluronidase. The denuded oocytes were cultured in TCM199 supplemented with 0.2 μg/ml demecolcine for 1 h and then placed in TCM199 containing 5 μg/ml cytochalasin B and 0.2 μg/ml demecolcine. The protruding first polar body and chromatin plate were removed with a beveled micropipette mounted on micromanipulators (Narishige, Tokyo, Japan) while being viewed with an inverted microscope (Nikon, Tokyo, Japan) using Hoffman modulation contrast optics. Successful enucleation was confirmed by staining the oocytes with Hoechst 33342 (5 μg/ml) and observing them with a fluorescent microscope under ultraviolet light. Fibroblast donor cells then were dissociated using 1% trypsin-EDTA and placed in Ca ²⁺-free and Mg ²⁺-free D-PBS supplemented with 0.3% BSA (fatty acid free). Micromanipulation was used to place a midsized (20-25 μm) single donor cell nucleus into the perivitelline space of each enucleated oocyte. Cytoplast-cell couplets were equilibrated in 0.3 M mannitol containing 0.1 mM Mg ²⁺ and were transferred to an electrofusion chamber containing the same medium. Cell fusion was induced by applying 2.0 kV/cm in 20-μsec DC pulses (2×) delivered by an electro cell fusion generator (Nepagene, Chiba, Japan). Couplets were removed from the fusion chamber, washed, and incubated in TCM199 supplemented with 0.3% BSA at 38°C in an atmosphere of 5% CO ₂ in air. At 1 h after electrofusion, fused couplets were equilibrated in 0.3 M mannitol containing 0.1 mM Ca ²⁺ and 0.1 mM Mg ²⁺, placed into a fusion chamber containing the same medium, and electropulsed by applying 1.0 kV/cm in 20-μsec DC pulses, 0.1 sec apart (2×). Activated couplets then were washed and incubated for 4 h in TCM199 supplemented with 0.3% BSA and 2 mM 6-DMAP at 38°C in an atmosphere of 5% CO ₂ in air.

In Vitro and In Vivo Development of Cloned Embryos Derived from RFP Transgenic Fibroblasts

To culture the in vitro embryos, activated cytoplast-cell couplets were grown in 50-μl droplets of m-Tyrode medium [ 17] supplemented with 1% MEM nonessential amino acid and 3 mg/ml BSA (Tyrode 1) for 3 days before being changed to m-Tyrode medium with 1% nonessential amino acid, 2% MEM BME amino acids (EAA), and 10% FBS (Tyrode 2) for the remaining 4 days of culture. The frequency of cleavage was assessed on Day 3, and the incidence of blastocyst development was visually determined on Day 7. To culture and collect the in vivo embryos, three female cats were treated with eCG and hCG as described below. At 36 h after treatment with hCG, activated cytoplast-cell couplets were surgically transferred into the oviduct of the recipient. Seven days after transfer, blastocysts were flushed from the excised uterus using TALP-Hepes buffered saline containing 0.3% BSA (A-9647).

Evaluation of Cell Number

Blastocysts that were derived from the in vitro and in vivo development were fixed for 15 min at room temperature in PBS containing 3.7% paraformaldehyde and 0.1% BSA before being stained with Hoechst 33342 (20 μg/ml). Embryos then were placed in a drop of glycerol on a slide, covered with a cover slip, and examined for nuclei staining using a fluorescence microscope.

Cloned Embryo Transfer into Synchronized Recipients

Activated cytoplast-cell couplets were cultured in 50-μl droplets of TCM-199 with 4 mg/ml BSA under mineral oil in a humidified atmosphere of 5% CO ₂ in air for 24 h. The number of embryos at the two- to four-cell stage were counted and transferred into the oviducts of healthy, mature, female domestic cats that had been synchronized by an injection of 100 IU eCG followed by an injection of 100 IU hCG 96 h after ovulation. The cloned embryos were transferred approximately 30 h after hCG injection. Each recipient was anesthetized with acepromazine maleate (0.025 mg/kg; Sedaject; Samwoo) and ketamine (5 mg/kg; Daesung) before laparotomy. Only cats that had fresh ovulation sites in their ovaries were used as recipients. Pregnancies were detected on Day 40 or 45 after transfer using a SONOACE 900 ultrasound scanner (Medison Co. Ltd., Seoul, South Korea) with an attached 7.0-MHz linear probe.

Microsatellite Analysis of Cloned Cats

TG cats obtained by SCNT were subjected to parentage analysis. DNA was extracted from tissue collected from newborn cats, surrogate recipients, and donor cells. Nine feline DNA microsatellite markers (FCA229, FCA290, FCA305, FCA441, FCA078, FCA201, FCA224, FCA170, and FCA304) were used to confirm the genetic identity of the cloned cats and the donor fibroblast cells [ 14]. Genomic DNA was amplified by PCR using nine feline microsatellite primers that were fluorescently tagged with HEX (Applied Biosystems, Foster City, CA). All of the PCR products were run on a 3100 Genetic Analyzer, and the allele sizes were calculated using Genescan software (Applied Biosystems).

Genomic DNA Analyses

Genomic DNA was extracted from cats using the G-DEX Genomic DNA Extraction Kit (iNtRON Biotechnology, Seoul, South Korea). To amplify the RFP gene by PCR, the nucleotide sequence of the pDsRed2-C1 cloning vector (catalogue number 632407; Clontech) was used to design an upstream (5′-GTTCCAGTACGGCTCCAAGGTGTA-3′) and downstream (5′-ATGGTGTAGTCCTCGTTGTGGGAG-3′) primer pair corresponding to the pDsRed2-C1 nucleotide sequence at positions 804-827 and 1218-1241 ( Fig. 1), respectively, yielding a predicted amplification product of 438 base pairs. Each reaction mixture contained 100 ng genomic DNA extract, 10 pmol of each primer, and 10 μl of 2× GoTaq Green Master Mix (Promega, Madison, WI) in a final reaction volume of 20 μl. The initial denaturation was performed at 94°C for 5 min, followed by 35 cycles of PCR amplification. The amplification profile consisted of three steps: 94°C for 30 sec (denaturation), 58°C for 30 sec (annealing), and 72°C for 30 sec (extension). After 35 amplification cycles, the samples were maintained at 72°C for 7 min to ensure that complete strand extension had taken place. For Southern blot analysis, genomic DNA (25 μg) was digested with KpnI, and fragments containing the full sequence of the RFP gene were separated on a 0.8% agarose gel. For blot analysis, primers for the synthesis of the RFP gene probe were 5′-CGCCACCATGGCCTCCTC-3′ and 5′-CAGGAACAGGTGGTGGCG-3′, corresponding to pDsRed2-C1 nucleotide sequence positions 606-623 and 1270-1287, respectively ( Fig. 1). The probe was synthesized using a PCR DIG Probe Synthesis kit (Roche, Mannheim, Germany), purified by agarose gel electrophoresis, and labeled with digoxigenin before hybridization. Detection of labeled DNA on the positively charged nylon membrane was performed using a DIG luminescent detection kit (Roche).

Detection of Red Fluorescence Protein

Red fluorescence of liveborn and stillborn RFP TG cats was detected using excitation-emission filter sets. Light was supplied from a 70-W surgery light equipped with an excitation filter (HQ 540/40; Chroma Technology Corp., Rockingham, VT). Red fluorescence emission from the cat and organs was detected using a Samsung digital camera (GX10) equipped with emission filters (HQ 600/50; Chroma Technology) that were attached proximal to the camera lens. The filter was easily removed for imaging with white light illumination.

Statistical Analysis

Each experiment was repeated at least three times. The effects of different culture media on activated oocyte cleavage and development to the blastocyst stage were analyzed by the chi-square test. The blastocyst cell number and the difference between groups were evaluated by the Student t-test. The level of significance was set at P < 0.05.

Results

In Vivo and In Vitro Developmental Potential of Cloned RFP TG Embryos

We monitored in vitro development and RFP expression in reconstructed embryos. Within 1 h after fusion, strong fluorescence was detected in reconstructed oocytes. This fluorescence was probably not derived from transcription of the donor cell genome; rather, it probably originated from the cytoplasm of the donor cell, which contained RFP mRNA as well as RFP protein. Since the fusion method was employed in this experiment, cytoplasmic material from donor cells was fused into the cytoplasm of oocytes. Fluorescence in NT embryos became weaker at the four-cell and morula stages, similar to results reported for swine [ 18]. At 2 h after fusion, strong fluorescence was sustained in reconstructed oocytes; however, at 15-48 h after fusion, the fluorescence in NT embryos became weaker or disappeared. After Day 3, cleaved embryos at the two- to four-cell stages started to express RFP again. We compared the in vitro and in vivo developmental competence of embryos generated from RFP-transfected cell lines by SCNT. The incidence of cleavage was similar between in vitro- and in vivo-developed embryos (78.3% vs. 69.6%; Table 1); however, development to the blastocyst stage was significantly higher in in vivo development than in vitro cultures (6.7% vs. 3.0%; P < 0.05). The average cell number of in vivo blastocysts was significantly higher than that in in vitro blastocysts (508 ± 477.9 vs. 78.2 ± 45.5; P < 0.05). In our in vitro culture system, the percentage of embryos that developed to the blastocyst stage was only 3%. Eight blastocysts were examined in order to determine the average blastocyst cell number. These data suggest that the in vitro culture media was not appropriate for cat embryo culture. To overcome this problem, we limited the pretransfer culture time to less than 24 h.

Production of RFP TG Kittens and Confirmation of the Genetic Identification of Cloned Cats

In order to produce RFP TG kittens, cytoplasmic/cell couples were cultured in TCM199 for 24 h and were then assessed for positive developmental cleavage. Cleaved embryos were transferred into the oviducts of recipient cats that were synchronized with the embryos' developmental stage. A total of 176 cleaved, reconstructed cat embryos were transferred into 11 surrogate mothers (mean = 16 ± 7.5 per recipient; Table 2). Three surrogate mothers were confirmed pregnant (27.3%) by ultrasound scans at 40-45 days; S-2 delivered one liveborn and one stillborn kitten (TG-A and TG-B) at 65 days of gestation, and S-3 delivered one liveborn kitten (TG-C) at 66 days of gestation. The body weights of TG-A, TG-B, and TG-C kittens at birth were 110, 122, and 136 g, respectively. The placental weights of the liveborn TG-A and TG-C kittens were 20 and 29 g, respectively ( Table 3). The placenta of TG-B was not recovered due to a natural delivery at midnight. Analysis of eight feline specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat ( Table 4).

Analysis of RFP Expression in RFP TG Cloned Embryos and Kittens

All of the fibroblasts that emitted a red color under fluorescent light were used as donor cells to make cloned embryos ( Fig. 2). At each stage, all of the cloned embryos expressed the RFP transgene; RFP transgene expression in in vitro- and in vivo-derived SCNT embryos was confirmed under conventional and fluorescence light sources. Although the number of cloned embryos was not enough for statistical analysis, each one that was examined positively expressed RFP (100% in each group; Table 1). At 60 days after birth, the transgenic cloned cats and control cat were examined under a portable surgery light fitted with excitation and emission filter sets to assess the expression level of RFP. Red fluorescence was observed in TG-A and TG-C cats, but not in the control cat ( Fig. 3). The expression pattern of the RFP transgene was determined in several tissues taken from the TG stillborn kitten (TG-B) and non-TG cat. Red fluorescence was detected in tissues, including the brain, heart, liver, kidney, testis, lung, muscle, intestine, thymus, spleen, seminal vesicle, skin, hair, fat, and cultured blood cells that were isolated from the transgenic cat's umbilical cord blood; however, no red fluorescence was observed in tissues from control cats ( Fig. 4). Under brightfield illumination, the transgenic cats appeared red and were readily distinguished from the wild-type cats; this observation is similar to that described in a previous report [ 19]. The level of red fluorescence was pronounced and easily apparent in most of the tissues examined, but was relatively subtle in heart tissue. These data are comparable with the RFP expression observed in mice [ 19], which exhibited lower levels of RFP expression in adipose, lung, spleen, and testis tissue, and no RFP expression in red blood cells. The presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses ( Fig. 5). RFP gene expression was detected in muscle and skin from the stillborn kitten (TG-B), but not from the placenta of a non-TG cat.

Discussion

The GFP and RFP genes are useful markers for the identification of transgenic nuclear transfer embryos prior to embryo transfer [ 20]. Use of these reporter genes might significantly enhance the efficiency of transgenic animal production, because expression of these genes allows positive embryos to be selected before they are transferred into the surrogate mother [ 21]. In addition, the descendants of a transgenic pig expressing GFP will probably provide a variety of genetically marked tissues, which would be very useful for basic research where such marked cells are required [ 18]. In this study, we achieved the ubiquitous, nontoxic expression of RFP in transgenic cats using the SCNT technique. This method could be offered as an alternative to genetic manipulations, such as lineage tracing in chimeric cats or establishing RFP expression in embryonic stem cell lines. The enhanced green ( EGFP) [ 22], yellow ( EYFP) [ 23], and cyan ( ECFP) [ 24] fluorescent proteins have served as invaluable tools for these purposes for many years; however, the emission spectra of these fluorochromes are relatively close, which makes their visual separation difficult with readily available imaging systems. For these reasons, RFPs such as DsRed [ 25] and its improved mutant variants may offer greater usefulness in certain applications. Red fluorescent protein represents a type of fluorescent protein that was initially isolated as DsRed from the sea anemone relative, Discosoma [ 26].

A desired gene can be transferred into the somatic cell by infection with a viral vector or by electroporation with lipid (liposome) or nonlipid (polymer) reagents carrying DNA. For instance, Park et al. [ 27] used a retroviral vector to produce GFP-cloned piglets, Lai et al. [ 28] used electroporation to produce 1,3-galactosyltransferase knockout cloned piglets, and Hyun et al. [ 29] introduced a GFP gene by liposome-mediated gene transfer to produce transgenic cloned piglets. The rate of infection of the RFP gene using the retrovirus vector was more than 80% (Kong et al., unpublished data), which represents a high level of efficacy for cat fibroblast RFP gene infection. In agreement with our result, several previous reports [ 30- 32] showed that not all of the cells that survived after antibiotic selection were transgenic, suggesting a requirement for prescreening SCNT embryos derived from these donor cells prior to embryo transfer into the recipient females.

Transgenic cats were examined under a portable, long-wave surgical lamp to assess the expression level of RFP, and all three kittens exhibited red fluorescence. The expression pattern of the RFP transgene in tissues and cells that were taken from the stillborn transgenic cat was determined. Despite high levels of RFP expression, including expression in the brain and muscle, RFP transgenic cats were apparently normal and showed gross behavior similar to control cats. These observations are consistent with those for pCXmRFP1 transgenic mice, which were apparently normal and exhibited fertility, gross behavior, and a lifespan similar to that of wild-type FVB/NJ mice. Analysis of body weight and the length and size of organs, including heart, liver, kidney, and spleen, revealed no significant difference between age- and sex-matched pCX-mRFP1 transgenic mice and wild-type mice [ 19].

In conclusion, we report for the first time the production of cloned transgenic cats with systemic expression of red fluorescent protein by somatic cell nuclear transfer. Application of the SCNT procedure to produce genetically modified cats would be valuable for generating biomedical models of human disease, as well as for the production of designer pets.

References

Author notes

# data[].attributes.attachment_link.title
Generation of Cloned Transgenic Cats Expressing Red Fluorescence Protein1

# data[].attributes.attachment_link.description
A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries.

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PAPAYA
https://www.nature.com/articles/nature06856.pdf

# data[].attributes.headline
Giselle Hernandez - Transgenic Mosquitos 

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Giselle Hernandez - Transgenic Mosquitos 

# data[].attributes.body
https://www.pnas.org/content/102/38/13568.full

# data[].attributes.attachment_link.title
Transgenic alteration of Toll immune pathway in the female mosquito Aedes aegypti

# data[].attributes.attachment_link.description
Reverse genetics is a powerful tool for understanding gene functions and their interactions in the mosquito innate immunity. We took the transgenic approach, in combination with the RNA interference (RNAi) technique, to elucidate the role of mosquito REL1, a homolog of Drosophila Dorsal, in regulati…

# data[].attributes.body
Bashir Musse

# data[].attributes.attachment_link.readable_text
Abstract

A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein ( RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 ± 7.5 per recipient). Three surrogate mothers were successfully impregnated (27.3%) and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline-specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole-body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cat but not in the surrogate mother cat. Red fluorescence was detected in tissue samples, including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue, and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.

Introduction

Domestic cats have been proposed as a biomedical model for the study of several human diseases [ 1]. Domestic cats are phylogenetically close to humans, and the feline gene map displays a higher level of systemic conservation with humans than rodents or other laboratory mammals [ 2]. Genetic manipulation of the cat is hampered by a lack of suitable technologies, such as embryonic stem cells (ESs), which are routinely used to generate targeted mutations in the mouse. Cloning through somatic cell nuclear transfer (SCNT) is a potential alternative approach in species for which ES technologies are unavailable. The SCNT technique has been used to produce cloned and transgenic cattle [ 3], sheep [ 4, 5], goats [ 6], and pigs [ 7, 8]. In order to improve the efficiency of transgenic animal production by SCNT, a method for screening both genetically modified nuclear donor cells and reconstructed embryos before embryo transfer to the surrogate females is critical. Green fluorescent protein ( GFP) selection of donor cells has been used to produce transgenic offspring by SCNT in mice [ 9], pigs [ 10], and cattle [ 11]. A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases [ 1]. As a species, the domestic cat exhibits several distinctive characteristics, one of which is a close genetic relationship to the human genome [ 2, 12]. The feline gene map displays a higher level of systemic conservation with humans than rodents or other laboratory mammals [ 2]. The efficient use of nuclear transfer that is associated with the genetic modification of donor cells in domestic cats would substantially improve the efficacy of the production of genetically identical cats that may carry genes for the study of specific human disorders [ 1]. In addition, these methods could be applied to the production of genetically engineered designer pets with selected favorable attributes, such as an allergen-free cat [ 1]. The current study was designed to evaluate the feasibility of producing transgenic cats expressing RFP as a reporter gene.

In this study we used the SCNT technique to produce transgenic cats that ubiquitously express RFP. We used a Moloney murine leukemia virus-based pseudotyped retroviral vector system in which recombinant viruses are packaged with vesicular stomatitis virus G glycoprotein [ 13] to deliver the RFP transgene to the cat somatic cells. The significance of this work stems from its precedence: it is the first report of the successful production of a cloned transgenic cat expressing an exogenous gene. Our nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.

Materials and Methods

Source of Reagents

All of the chemicals were purchased from the Sigma-Aldrich Chemical Company (St. Louis, MO) unless otherwise stated.

Animal Care and Use

The Stroller female cats that were used as oocyte donors were housed in stainless steel cages measuring 0.9 × 0.7 × 0.65 m and were provided with dry food and water ad libitum. They were maintained in an environmentally controlled room on a 14L:10D photoperiod cycle with light onset at 0600 h. All animal care and use procedures were approved by the Institutional Animal Care and Use Committee of Sunchon National University.

Ovary Recovery and Oocyte Maturation

The ovarian follicular development of mature cats was stimulated by injection with 200 IU eCG (Daesung, Seoul, South Korea), and oocyte maturation was induced 4 days later by treatment with 100 IU hCG (Daesung). Ovaries were removed 24 h after hCG injection by routine ovariohysterectomy and were minced in m-Tyrode medium-Hepes using a scalpel blade to release cumulus oocyte complexes [ 14]. Collected oocytes were matured in TCM199 (M-7528) supplemented with 10% fetal bovine serum (FBS; Gibco/Invitrogen, Carlsbad, CA) and 1% penicillin G/streptomycin (P-4333) for 4 h at 38°C in an atmosphere of 5% CO ₂ in air.

Construction of the Retrovirus Vector and Virus Production

A plasmid (pLHCRW) containing a retrovirus vector sequence was constructed by replacing the TRE sequence of pRevTRE (Clontech, Mountain View, CA) with a fragment containing the CMV promoter, DsRed2 gene, and WPRE (woodchuck hepatitis virus posttrancriptional regulatory element) sequences ( Fig. 1). The CMV promoter and the DsRed2 gene were derived from pLNCX and pDsRed2-C1, respectively (Clontech). The WPRE sequence from woodchuck hepatitis virus 2 genomic DNA (GenBank accession number M11082) was introduced following the strategy of Zufferey et al. [ 15]. Retrovirus-producing cells were constructed following the procedure described by Koo et al. [ 16]. PT67 cells (Clontech) were transiently transfected with pLHCRW; the resultant LHCRW viruses were harvested and applied to the GP2-293 cells (Clontech). PT67 are retrovirus packaging cells that are characterized by the expression of the Gibbon ape leukemia virus envelope gene, whereas GP2-293 cells are specialized to express the gag and pol genes of Moloney murine leukemia virus. LHCRW-infected GP2-293 cells were selected with hygromycin (150 μl/ml) for 2 wk, and the resultant Hyg R (hygromycin-resistant) cells were transfected with pVSV-G (Clontech) to express VSV-G protein. Viruses were harvested 48 h after transfection. All virus-producing cells were grown at 37°C in a 5% CO ₂ incubator in Dulbecco modified Eagle medium (DMEM) with 4.5 g/l glucose (Gibco BRL, Grand Island, NY) supplemented with fetal calf serum (10%), penicillin (100 μg/ml), and streptomycin (100 μg/ml). The virus-containing medium that was harvested from the virus-producing cells was filtered through a 0.45-μm pore size filter and used to infect cat fibroblast cells. LHCRW-infected fibroblasts were selected with hygromycin (150 μl/ml) for 2 wk.

Establishment of Cat Fibroblast Cell Lines

Several fibroblast cell sublines were established from single, 6-mm skin biopsies taken aseptically from a male, odd-eyed, all-white cat. Primary cultures were obtained by mincing dermal tissue with sterile scissors in a 35-mm Petri dish (Nunc, Roskilde, Denmark) and culturing the fragments at 38°C with 5% CO ₂ in air in DMEM containing 10% FBS until 90% confluence was observed. Cells then were trypsinized and reconstituted at 1 × 10 ⁶ cells/ml, grown to confluence, and frozen as zero passage cells (1 vial/35-mm dish) in DMEM (Gibco BRL) containing 10% dimethylsulfoxide and 10% FBS. Cells were thawed and cultured for RFP gene transfection. RFP-transfected cells yielding confluent growth after culture in serum-free medium for 3 days were used for SCNT [ 14]. Single RFP-transfected cells were selected under an inverted microscope equipped with a G-2A filter (EX: 510-560 nm, BA: 590 nm; Fig. 2).

Somatic Cell Nuclear Transfer

The SCNT protocol was described previously [ 14]. Briefly, cumulus cells were removed from oocytes by gentle pipetting in TCM199 supplemented with 0.1% hyaluronidase. The denuded oocytes were cultured in TCM199 supplemented with 0.2 μg/ml demecolcine for 1 h and then placed in TCM199 containing 5 μg/ml cytochalasin B and 0.2 μg/ml demecolcine. The protruding first polar body and chromatin plate were removed with a beveled micropipette mounted on micromanipulators (Narishige, Tokyo, Japan) while being viewed with an inverted microscope (Nikon, Tokyo, Japan) using Hoffman modulation contrast optics. Successful enucleation was confirmed by staining the oocytes with Hoechst 33342 (5 μg/ml) and observing them with a fluorescent microscope under ultraviolet light. Fibroblast donor cells then were dissociated using 1% trypsin-EDTA and placed in Ca ²⁺-free and Mg ²⁺-free D-PBS supplemented with 0.3% BSA (fatty acid free). Micromanipulation was used to place a midsized (20-25 μm) single donor cell nucleus into the perivitelline space of each enucleated oocyte. Cytoplast-cell couplets were equilibrated in 0.3 M mannitol containing 0.1 mM Mg ²⁺ and were transferred to an electrofusion chamber containing the same medium. Cell fusion was induced by applying 2.0 kV/cm in 20-μsec DC pulses (2×) delivered by an electro cell fusion generator (Nepagene, Chiba, Japan). Couplets were removed from the fusion chamber, washed, and incubated in TCM199 supplemented with 0.3% BSA at 38°C in an atmosphere of 5% CO ₂ in air. At 1 h after electrofusion, fused couplets were equilibrated in 0.3 M mannitol containing 0.1 mM Ca ²⁺ and 0.1 mM Mg ²⁺, placed into a fusion chamber containing the same medium, and electropulsed by applying 1.0 kV/cm in 20-μsec DC pulses, 0.1 sec apart (2×). Activated couplets then were washed and incubated for 4 h in TCM199 supplemented with 0.3% BSA and 2 mM 6-DMAP at 38°C in an atmosphere of 5% CO ₂ in air.

In Vitro and In Vivo Development of Cloned Embryos Derived from RFP Transgenic Fibroblasts

To culture the in vitro embryos, activated cytoplast-cell couplets were grown in 50-μl droplets of m-Tyrode medium [ 17] supplemented with 1% MEM nonessential amino acid and 3 mg/ml BSA (Tyrode 1) for 3 days before being changed to m-Tyrode medium with 1% nonessential amino acid, 2% MEM BME amino acids (EAA), and 10% FBS (Tyrode 2) for the remaining 4 days of culture. The frequency of cleavage was assessed on Day 3, and the incidence of blastocyst development was visually determined on Day 7. To culture and collect the in vivo embryos, three female cats were treated with eCG and hCG as described below. At 36 h after treatment with hCG, activated cytoplast-cell couplets were surgically transferred into the oviduct of the recipient. Seven days after transfer, blastocysts were flushed from the excised uterus using TALP-Hepes buffered saline containing 0.3% BSA (A-9647).

Evaluation of Cell Number

Blastocysts that were derived from the in vitro and in vivo development were fixed for 15 min at room temperature in PBS containing 3.7% paraformaldehyde and 0.1% BSA before being stained with Hoechst 33342 (20 μg/ml). Embryos then were placed in a drop of glycerol on a slide, covered with a cover slip, and examined for nuclei staining using a fluorescence microscope.

Cloned Embryo Transfer into Synchronized Recipients

Activated cytoplast-cell couplets were cultured in 50-μl droplets of TCM-199 with 4 mg/ml BSA under mineral oil in a humidified atmosphere of 5% CO ₂ in air for 24 h. The number of embryos at the two- to four-cell stage were counted and transferred into the oviducts of healthy, mature, female domestic cats that had been synchronized by an injection of 100 IU eCG followed by an injection of 100 IU hCG 96 h after ovulation. The cloned embryos were transferred approximately 30 h after hCG injection. Each recipient was anesthetized with acepromazine maleate (0.025 mg/kg; Sedaject; Samwoo) and ketamine (5 mg/kg; Daesung) before laparotomy. Only cats that had fresh ovulation sites in their ovaries were used as recipients. Pregnancies were detected on Day 40 or 45 after transfer using a SONOACE 900 ultrasound scanner (Medison Co. Ltd., Seoul, South Korea) with an attached 7.0-MHz linear probe.

Microsatellite Analysis of Cloned Cats

TG cats obtained by SCNT were subjected to parentage analysis. DNA was extracted from tissue collected from newborn cats, surrogate recipients, and donor cells. Nine feline DNA microsatellite markers (FCA229, FCA290, FCA305, FCA441, FCA078, FCA201, FCA224, FCA170, and FCA304) were used to confirm the genetic identity of the cloned cats and the donor fibroblast cells [ 14]. Genomic DNA was amplified by PCR using nine feline microsatellite primers that were fluorescently tagged with HEX (Applied Biosystems, Foster City, CA). All of the PCR products were run on a 3100 Genetic Analyzer, and the allele sizes were calculated using Genescan software (Applied Biosystems).

Genomic DNA Analyses

Genomic DNA was extracted from cats using the G-DEX Genomic DNA Extraction Kit (iNtRON Biotechnology, Seoul, South Korea). To amplify the RFP gene by PCR, the nucleotide sequence of the pDsRed2-C1 cloning vector (catalogue number 632407; Clontech) was used to design an upstream (5′-GTTCCAGTACGGCTCCAAGGTGTA-3′) and downstream (5′-ATGGTGTAGTCCTCGTTGTGGGAG-3′) primer pair corresponding to the pDsRed2-C1 nucleotide sequence at positions 804-827 and 1218-1241 ( Fig. 1), respectively, yielding a predicted amplification product of 438 base pairs. Each reaction mixture contained 100 ng genomic DNA extract, 10 pmol of each primer, and 10 μl of 2× GoTaq Green Master Mix (Promega, Madison, WI) in a final reaction volume of 20 μl. The initial denaturation was performed at 94°C for 5 min, followed by 35 cycles of PCR amplification. The amplification profile consisted of three steps: 94°C for 30 sec (denaturation), 58°C for 30 sec (annealing), and 72°C for 30 sec (extension). After 35 amplification cycles, the samples were maintained at 72°C for 7 min to ensure that complete strand extension had taken place. For Southern blot analysis, genomic DNA (25 μg) was digested with KpnI, and fragments containing the full sequence of the RFP gene were separated on a 0.8% agarose gel. For blot analysis, primers for the synthesis of the RFP gene probe were 5′-CGCCACCATGGCCTCCTC-3′ and 5′-CAGGAACAGGTGGTGGCG-3′, corresponding to pDsRed2-C1 nucleotide sequence positions 606-623 and 1270-1287, respectively ( Fig. 1). The probe was synthesized using a PCR DIG Probe Synthesis kit (Roche, Mannheim, Germany), purified by agarose gel electrophoresis, and labeled with digoxigenin before hybridization. Detection of labeled DNA on the positively charged nylon membrane was performed using a DIG luminescent detection kit (Roche).

Detection of Red Fluorescence Protein

Red fluorescence of liveborn and stillborn RFP TG cats was detected using excitation-emission filter sets. Light was supplied from a 70-W surgery light equipped with an excitation filter (HQ 540/40; Chroma Technology Corp., Rockingham, VT). Red fluorescence emission from the cat and organs was detected using a Samsung digital camera (GX10) equipped with emission filters (HQ 600/50; Chroma Technology) that were attached proximal to the camera lens. The filter was easily removed for imaging with white light illumination.

Statistical Analysis

Each experiment was repeated at least three times. The effects of different culture media on activated oocyte cleavage and development to the blastocyst stage were analyzed by the chi-square test. The blastocyst cell number and the difference between groups were evaluated by the Student t-test. The level of significance was set at P < 0.05.

Results

In Vivo and In Vitro Developmental Potential of Cloned RFP TG Embryos

We monitored in vitro development and RFP expression in reconstructed embryos. Within 1 h after fusion, strong fluorescence was detected in reconstructed oocytes. This fluorescence was probably not derived from transcription of the donor cell genome; rather, it probably originated from the cytoplasm of the donor cell, which contained RFP mRNA as well as RFP protein. Since the fusion method was employed in this experiment, cytoplasmic material from donor cells was fused into the cytoplasm of oocytes. Fluorescence in NT embryos became weaker at the four-cell and morula stages, similar to results reported for swine [ 18]. At 2 h after fusion, strong fluorescence was sustained in reconstructed oocytes; however, at 15-48 h after fusion, the fluorescence in NT embryos became weaker or disappeared. After Day 3, cleaved embryos at the two- to four-cell stages started to express RFP again. We compared the in vitro and in vivo developmental competence of embryos generated from RFP-transfected cell lines by SCNT. The incidence of cleavage was similar between in vitro- and in vivo-developed embryos (78.3% vs. 69.6%; Table 1); however, development to the blastocyst stage was significantly higher in in vivo development than in vitro cultures (6.7% vs. 3.0%; P < 0.05). The average cell number of in vivo blastocysts was significantly higher than that in in vitro blastocysts (508 ± 477.9 vs. 78.2 ± 45.5; P < 0.05). In our in vitro culture system, the percentage of embryos that developed to the blastocyst stage was only 3%. Eight blastocysts were examined in order to determine the average blastocyst cell number. These data suggest that the in vitro culture media was not appropriate for cat embryo culture. To overcome this problem, we limited the pretransfer culture time to less than 24 h.

Production of RFP TG Kittens and Confirmation of the Genetic Identification of Cloned Cats

In order to produce RFP TG kittens, cytoplasmic/cell couples were cultured in TCM199 for 24 h and were then assessed for positive developmental cleavage. Cleaved embryos were transferred into the oviducts of recipient cats that were synchronized with the embryos' developmental stage. A total of 176 cleaved, reconstructed cat embryos were transferred into 11 surrogate mothers (mean = 16 ± 7.5 per recipient; Table 2). Three surrogate mothers were confirmed pregnant (27.3%) by ultrasound scans at 40-45 days; S-2 delivered one liveborn and one stillborn kitten (TG-A and TG-B) at 65 days of gestation, and S-3 delivered one liveborn kitten (TG-C) at 66 days of gestation. The body weights of TG-A, TG-B, and TG-C kittens at birth were 110, 122, and 136 g, respectively. The placental weights of the liveborn TG-A and TG-C kittens were 20 and 29 g, respectively ( Table 3). The placenta of TG-B was not recovered due to a natural delivery at midnight. Analysis of eight feline specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat ( Table 4).

Analysis of RFP Expression in RFP TG Cloned Embryos and Kittens

All of the fibroblasts that emitted a red color under fluorescent light were used as donor cells to make cloned embryos ( Fig. 2). At each stage, all of the cloned embryos expressed the RFP transgene; RFP transgene expression in in vitro- and in vivo-derived SCNT embryos was confirmed under conventional and fluorescence light sources. Although the number of cloned embryos was not enough for statistical analysis, each one that was examined positively expressed RFP (100% in each group; Table 1). At 60 days after birth, the transgenic cloned cats and control cat were examined under a portable surgery light fitted with excitation and emission filter sets to assess the expression level of RFP. Red fluorescence was observed in TG-A and TG-C cats, but not in the control cat ( Fig. 3). The expression pattern of the RFP transgene was determined in several tissues taken from the TG stillborn kitten (TG-B) and non-TG cat. Red fluorescence was detected in tissues, including the brain, heart, liver, kidney, testis, lung, muscle, intestine, thymus, spleen, seminal vesicle, skin, hair, fat, and cultured blood cells that were isolated from the transgenic cat's umbilical cord blood; however, no red fluorescence was observed in tissues from control cats ( Fig. 4). Under brightfield illumination, the transgenic cats appeared red and were readily distinguished from the wild-type cats; this observation is similar to that described in a previous report [ 19]. The level of red fluorescence was pronounced and easily apparent in most of the tissues examined, but was relatively subtle in heart tissue. These data are comparable with the RFP expression observed in mice [ 19], which exhibited lower levels of RFP expression in adipose, lung, spleen, and testis tissue, and no RFP expression in red blood cells. The presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses ( Fig. 5). RFP gene expression was detected in muscle and skin from the stillborn kitten (TG-B), but not from the placenta of a non-TG cat.

Discussion

The GFP and RFP genes are useful markers for the identification of transgenic nuclear transfer embryos prior to embryo transfer [ 20]. Use of these reporter genes might significantly enhance the efficiency of transgenic animal production, because expression of these genes allows positive embryos to be selected before they are transferred into the surrogate mother [ 21]. In addition, the descendants of a transgenic pig expressing GFP will probably provide a variety of genetically marked tissues, which would be very useful for basic research where such marked cells are required [ 18]. In this study, we achieved the ubiquitous, nontoxic expression of RFP in transgenic cats using the SCNT technique. This method could be offered as an alternative to genetic manipulations, such as lineage tracing in chimeric cats or establishing RFP expression in embryonic stem cell lines. The enhanced green ( EGFP) [ 22], yellow ( EYFP) [ 23], and cyan ( ECFP) [ 24] fluorescent proteins have served as invaluable tools for these purposes for many years; however, the emission spectra of these fluorochromes are relatively close, which makes their visual separation difficult with readily available imaging systems. For these reasons, RFPs such as DsRed [ 25] and its improved mutant variants may offer greater usefulness in certain applications. Red fluorescent protein represents a type of fluorescent protein that was initially isolated as DsRed from the sea anemone relative, Discosoma [ 26].

A desired gene can be transferred into the somatic cell by infection with a viral vector or by electroporation with lipid (liposome) or nonlipid (polymer) reagents carrying DNA. For instance, Park et al. [ 27] used a retroviral vector to produce GFP-cloned piglets, Lai et al. [ 28] used electroporation to produce 1,3-galactosyltransferase knockout cloned piglets, and Hyun et al. [ 29] introduced a GFP gene by liposome-mediated gene transfer to produce transgenic cloned piglets. The rate of infection of the RFP gene using the retrovirus vector was more than 80% (Kong et al., unpublished data), which represents a high level of efficacy for cat fibroblast RFP gene infection. In agreement with our result, several previous reports [ 30- 32] showed that not all of the cells that survived after antibiotic selection were transgenic, suggesting a requirement for prescreening SCNT embryos derived from these donor cells prior to embryo transfer into the recipient females.

Transgenic cats were examined under a portable, long-wave surgical lamp to assess the expression level of RFP, and all three kittens exhibited red fluorescence. The expression pattern of the RFP transgene in tissues and cells that were taken from the stillborn transgenic cat was determined. Despite high levels of RFP expression, including expression in the brain and muscle, RFP transgenic cats were apparently normal and showed gross behavior similar to control cats. These observations are consistent with those for pCXmRFP1 transgenic mice, which were apparently normal and exhibited fertility, gross behavior, and a lifespan similar to that of wild-type FVB/NJ mice. Analysis of body weight and the length and size of organs, including heart, liver, kidney, and spleen, revealed no significant difference between age- and sex-matched pCX-mRFP1 transgenic mice and wild-type mice [ 19].

In conclusion, we report for the first time the production of cloned transgenic cats with systemic expression of red fluorescent protein by somatic cell nuclear transfer. Application of the SCNT procedure to produce genetically modified cats would be valuable for generating biomedical models of human disease, as well as for the production of designer pets.

References

Author notes

# data[].attributes.attachment_link.title
Generation of Cloned Transgenic Cats Expressing Red Fluorescence Protein1

# data[].attributes.attachment_link.description
A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries.

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Expression of Cry3Bb1 in transgenic corn MON88017

# data[].attributes.attachment_link.description
To evaluate the effects of transgenic expression of Coleopteran-specific Bt protein Cry3Bb1 on target and nontarget insects in fields with Bt crops, it is necessary to quantify the Cry3Bb1 contents in the plants. Here, we describe the optimization and validation of the quantitative detection of Cry3…

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https://pubs.acs.org/doi/pdf/10.1021/jf050594v

# data[].attributes.attachment_link.title
Transgenic Expression of Bean α-Amylase Inhibitor in Peas Results in Altered Structure and Immunogenicity

# data[].attributes.attachment_link.description
The development of modern gene technologies allows for the expression of recombinant proteins in non-native hosts. Diversity in translational and post-translational modification pathways between species could potentially lead to discrete changes in the molecular architecture of the expressed protei…

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https://besjournals.onlinelibrary.wiley.com/doi/pdfdirect/10.1046/j.1365-2664.1999.00448.x

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Alex Peart - Transgenic Medaka Salmon and Trojan Genes

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Alex Peart - Transgenic Medaka Salmon and Trojan Genes

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https://pubmed.ncbi.nlm.nih.gov/14976259/

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Affiliation

¹ Departments of Biological Sciences, Forestry and Natural Resources, and Animal Sciences, Purdue University, West Lafayette, IN 47907, USA. rhoward@bilbo.bio.purdue.edu

Free PMC article

Abstract

Genetically modified (GM) strains now exist for many organisms, producing significant promise for agricultural production. However, if these organisms have some fitness advantage, they may also pose an environmental harm when released. High mating success of GM males relative to WT males provides such an important fitness advantage. Here, we provide documentation that GM male medaka fish modified with salmon growth hormone possess an overwhelming mating advantage. GM medaka offspring possess a survival disadvantage relative to WT, however. When both of these fitness components are included in our model, the transgene is predicted to spread if GM individuals enter wild populations (because of the mating advantage) and ultimately lead to population extinction (because of the viability disadvantage). Mating trials indicate that WT males use alternative mating tactics in an effort to counter the mating advantage of GM males, and we use genetic markers to ascertain the success of these alternative strategies. Finally, we model the impact of alternative mating tactics by WT males on transgene spread. Such tactics may reduce the rate of transgene spread, but not the outcome.

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Transgenic coho salmon strains containing an all-salmon growth hormone (GH) gene construct (OnMTGH1) have been examined. The transgene utilized is comprised of the metallothionein-B promoter driving the expression of the type-I growth hormone gene from sockeye salmon. Transgene DNA is integrated into the salmon genome, but is transmitted at low frequency from founder transgenic animals consistent with late integration following microinjection resulting in mosaic animals, whereas subsequent generations transmit transgene DNA as a stable Mendelian trait. Different families established from separate founder animals yield lines with unique growth characteristics suggesting important site-of-integration effects on transgene expression. Growth enhancement of transgenic salmon is initiated early, with advanced hatch timing but occurring also throughout the life history, particularly during the early phase in fresh water. GH transgenic fish showed precocious smoltification and onset of sexual maturation, but approximately normal adult body size, indicating that compression of the normal coho salmon life history is occurring. The viability of diploid GH transgenic salmon ranges from reductions to greater than that of controls among strains, and triploid transgenic animals had normal viability relative to diploid transgenic salmon. Triploid transgenic salmon display a reduction in growth rate relative to transgenic diploids, but are still significantly growth enhanced compared with nontransgenic controls. The distinct phenotypic characteristics of GH transgenic families suggest that evaluation for aquaculture and for risk assessments requires examination of strains on a case-by-case basis. Furthermore, strong effects of size at maturity in culture conditions were observed for nontransgenic wild strain coho salmon which were not apparent in GH transgenic salmon, indicating that strong genotype by environment effects strongly influence phenotype and complicate risk assessments.

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Abstract

INTRODUCTION

Expansion in brain size and improvement in cognitive skills are among the most fundamental evolutionary changes that set humans apart from other primates. Comparative genomic analyses between humans and non-human primates suggest that these dramatic phenotypic divergences may be due to several underlying genetic changes: rapid evolution of protein-coding genes [ 1, 2] and non-coding RNA genes [ 3-5], emergence of human-specific segmental duplications [ 6-8], as well as alterations in gene expression [ 9-12] and epigenetic regulation [ 13-15]. Despite a great deal of effort in previous studies, we are still on the way in searching for the responsible genes and dissecting the genetic mechanisms that shape the human brain.

Among the reported genes that play important roles in human brain development, MCPH1 (also known as BRIT1) is one of the strong candidates that may contribute to human brain evolution [ 16]. It is one of the fast-evolving genes in primates [ 17]. In particular, MCPH1 has accumulated seven human-specific amino acid changes that are fixed in modern humans [ 17]. Our previous in vitro experiments showed that these human-specific protein-sequence changes could alter the regulation of MCPH1 on its downstream genes [ 18]. Importantly, at the transcriptional level, MCPH1 also has shown human-specific changes. During postnatal brain development, MCPH1 is abundantly expressed in humans, but less so in non-human primates (macaque and chimpanzee, Supplementary Fig. 1A) [ 11]. In addition, we have shown that the MCPH1 transcriptional activity was significantly higher in human than in rhesus monkey [ 18]. Collectively, current evidence suggests that not only the human-specific protein-sequence changes, but also gene-expression alteration of MCPH1 may contribute to human brain development and function.

MCPH1 encodes a pleiotrophic protein. It functions as a transcription factor by interacting with E2F1 (E2F transcription factor 1) to regulate cell cycle and cell apoptosis [ 19]. It also works as a DNA-damage response protein and is involved in chromatic remodeling to control DNA repair [ 16, 20, 21]. In the central nervous system, as a centrosome protein, MCPH1 plays a conserved role in neurogenesis by regulating the neuronal progenitor divison mode via the Chk1-Cdc25B pathway [ 22]. In humans, truncated mutations of MCPH1 cause primary microcephaly (MCPH, OMIM251200)-a rare human brain-developmental disorder, characterized by significantly reduced brain volume and mental retardation [ 23-25]. Consistently, the MCPH1 knockout animal models (mouse and monkey) reproduced the phenotypes of human microcephaly, notably the reduced brain size [ 22, 26]. During human brain development, MCPH1 has the highest expression at the prenatal stage and the expression reduces after birth and remains at a constant level through adulthood ( Supplementary Fig. 1B). At the prenatal stage, MCPH1 is highly expressed in all cell types in the cortex, including neural progenitor cells, inter-neurons, astrocytes and microglia cells [ 27] ( Supplementary Fig. 1B). In a mouse study, it was demonstrated that MCPH1 controls precise mitotic spindle orientation and regulates the progenitor division mode to maintain brain size [ 22]. However, although MCPH1 loss of function causes abnormal brain development, resulting in a reduced brain size in human and animals, the functional consequence of the human-specific seqence and expression changes remains to be understood.

To interrogate the genetic basis of human brain evolution, the traditional mouse or rat models are less ideal due to the vast dissimilarities in brain size and structure between humans and rodents. Instead, a non-human primate transgenic model would be far more effective. The rhesus monkey ( Macaca mulatta), an Old World Monkey species widely used for biomedical research, is an ideal choice, due to its high sequence similarity with humans (>93% for protein-coding genes) [ 28] and yet relatively large phylogenetic distance (about 25 million years of divergence from humans), which alleviates ethical concerns [ 29].

For MCPH1, the coding sequence similarity is 94.9% between human and rhesus monkey, while it is only 67.5% between human and mouse. Similarly, the 5' non-coding sequence (∼5 kb) of MCPH1 likely contains regulatory elements for gene-expression regulation and it has 88.7% similarity between human and rhesus monkey, while it is only 40.4% between human and mouse. Additionally, we have shown that, during primate evolution, the MCPH1 promoter region has acquired a primate-specific E2F1-binding motif that is absent in rodents and other mammalian species [ 30]. Taken together, a rhesus monkey model is promising to study the functional impact of the human-specific changes (protein sequence and gene expression) on human brain evolution.

In this study, to mimic the human-specific genetic changes, using lentivirus transfection, we introduced the human MCPH1 copies (huMCPH1) into the rhesus monkey genome so that the transgenic (TG) monkeys have an overexpression of human MCPH1. We successfully generated eight first-generation (F0) and three second-generation (F1) TG monkeys carrying human MCPH1 copies. Brain-development tracking via magnetic resonance imaging (MRI) of a tissue section with cellular markers showed that the TG monkeys experienced delayed neuronal maturation and neural-fiber myelination, both of which are human-like features of brain-developmental neoteny. Accordingly, transcriptome analysis of prenatal and postnatal brain development revealed an altered gene-expression profile in neuro-progenitors and neurons with shifted expression time of synapse-related genes in the TG monkeys. Remarkably, our preliminary cognitive test detected an improved short-term memory in the TG monkeys.

RESULTS

Generation of transgenic monkeys carrying human MCPH1 copies

All animal procedures were conducted following the international standards, and were approved in advance by the Institutional Animal Care and Use Committee of Kunming Institute of Zoology, Chinese Academy of Sciences and Yunnan Key Laboratory of Primate Biomedical Research (Approval No: SYDW-2010002 and KBI_K001115033-01,01). Lentivirus delivery was used to introduce the human MCPH1 copy (huMCPH1) into the rhesus monkey genome. A high titer (>1 × 10 ¹⁰ infection particles per ml) simian immunodeficiency virus (SIV) vector containing lentivirus was produced for gene transfer ( Supplementary Fig. 1C; see the 'Methods' section for more details). The human MCPH1 gene was cloned into the SIV vector containing an eGFP (enhanced green fluorescent protein) gene copy and a universal promoter (the CMV-enhanced chicken beta actin (CAG) promoter) ( Supplementary Fig. 1D). The monkey oocytes were obtained by super-ovulation and fertilized in vitro (IVF). The early-cleavage-stage embryos were injected with 50-100 pl lentivirus.

In total, five pregnant surrogates produced eight F0 monkeys (T_01-T_08), among which six are twins (T_01/T_02, T_03/T_04 and T_06/T_07) (Fig. 1 and Table 1). Caesarean section was used to deliver baby monkeys at around 155 days' gestation except for the twins (T_03 and T_04) with premature abortion at embryonic 136 days. Multiple tissues (blood, placenta, umbilical cord endothelial cells and skin) were sampled to test the transgenic status and all monkeys turned out to be positive ( Supplementary Fig. 1E-P). We detected strong GFP signals in the nucleus of the TG monkeys ( Supplementary Fig. 1P). Since MCPH1 is known to work in the nucleus [ 31], this result suggests that the huMCPH1 transgenes were correctly positioned in the cell.

To determine the integrated genomic locations and copy numbers, we conducted captured next-generation sequencing according to the reported method [ 32]. As expected for lentivirus, the huMCPH1 copies were randomly integrated into the monkey genomes. The eight TG monkeys have two to nine huMCPH1 copies (Table 1 and Fig. 1B). Importantly, all integration sites are located in either inter-genic or non-coding regions and presumably will not interfere with the function of the monkey endogenous genes ( Supplementary Table 1).

For comparison, we recruited six wild-type (WT) monkeys (Fig. 1C and Supplementary Table 2) and initially they were divided into two groups. The first contained three age-matched monkeys (WT_01, WT_02 and WT_03) raised by their biological mothers. The second WT group contained three age-matched monkeys (WT_06, WT_07 and WT_08) who were separated from their biological mothers 6-25 days after birth and raised by humans under the same conditions as the TG monkeys ( Supplementary Table 2). Unfortunately, TG_02 died of unknown cause 76 days after birth. A biopsy did not reveal any organ damage. As mentioned above, TG_03 and TG_04 were abortions at embryonic 136 days. For comparison of brain-tissue section and transcriptome analysis, six additional WT monkeys were sacrificed at the corresponding developmental stages (76 days after birth for WT_04 and WT_05, and embryonic 130-145 days for WT_09, WT_10, WT_11 and WT_12) ( Supplementary Table 2). In total, we collected data from 8 TG monkeys and 12 WT monkeys.

Brain-development tracking using structural MRI suggests a delayed neural maturation

To test whether the integrated huMCPH1 copies influenced the brain development of the TG monkeys, we first performed a non-invasive analysis, namely structural MRI (Philips Achieva 3.0T TX). The tested monkeys included five TG monkeys (TG_01, TG_05, TG_06, TG_07 and TG_08) and six WT monkeys (WT_01, WT_02, WT_03, WT_06, WT_07 and WT_08). The MRI scans were conducted at scheduled intervals (once every month while 2-12 months old and then once every 6 months until 2-3 years old) (Fig. 1C). Both T1-weighted image and diffusion tensor imaging (DTI) data were collected. Using the T1 image data, we calculated the volumes of total brain (TB), cerebellum, lobes and subcortical regions based on the published method and monkey atlas [ 33] ( Supplementary Fig. 2). To rule out the potential influence of feeding types, we first compared the two WT monkey groups (monkey feeding vs human feeding) and we did not detect any difference in volumes ( Supplementary Table 3). Therefore, all WT monkeys were grouped together in the following analysis.

In general, the TB volume and body weight of the TG monkeys were smaller than the WT monkeys during early development, likely due to the C-section delivery of the TG monkeys at 155 days of pregnancy-about 1 week earlier than the natural delivery of the WT controls. Also, three of the five TG monkeys were twins, which usually weigh less than single-birth monkeys (all WT monkeys were single-birth). However, this difference became smaller when the monkeys grew older and the TG monkeys eventually caught up with the WT monkeys at about 3 years old ( Supplementary Fig. 3A and B). Of note, the relative brain volume (TB volume adjusted by body weight) of the TG monkeys was larger than the WT monkeys during early postnatal development and this difference became invisible when the monkeys grew older (Fig. 1D, linear mixed model (LMM) model, group effect P = 0.05), while the cortex thickness was similar between them throughout development (Fig. 1E).

As the brain is mainly composed of cortex gray matter (GM, mostly neurons) and subcortical white matter (WM, mostly glial cells) [ 34], we next conducted segmentation analyses. Notably, during brain development, the cortex GM volume of the TG monkeys increased more slowly than that of the WT monkeys, and there was on average 164 days' delay of peak time for the TG monkeys (Fig. 1F). When the cortex was divided into four lobes (frontal, parietal, occipital and temporal lobes), we saw the same pattern in all lobes ( Supplementary Fig. 3C-F). Interestingly, the cortex GM ratios (the proportion of the cortex GM volume vs the TB volume) of the TG monkeys were larger than those of the WT monkeys with a similar ratio peak time delay (Fig. 1F, LMM model, group effect P = 0.06). By contrast, we did not detect such a delay in the cerebellum or subcortical region ( Supplementary Fig. 3G and H).

The developmental pattern of WM was different from that of GM. There were no volume/ratio peaks for the cortex WM and the TG monkeys kept a significantly lower volume and cortex WM ratio than the WT monkeys during development (Fig. 1G, LMM model, group effect P = 0.0006) (Fig. 1G, LMM model, group effect P = 0.01). By contrast, the WM volume of the subcortical region did not show such a difference and an opposite pattern was seen for the cerebellum, although statistically not significant ( Supplementary Fig. 3G and H, group effect P = 0.59). Given the observed patterns, we reasoned that the observed brain-developmental changes of the TG monkeys might reflect a delay in cortex development rather than a developmental impairment. Consistently with this view, the cortex GM volume curves of the TG and WT monkeys started to converge 1 year after birth (Fig. 1F) and the same pattern was observed when looking at the curves of the four lobes ( Supplementary Fig. 3C-F). In particular, TG_01 and three WT monkeys (WT_01, WT_02 and WT_03) had MRI data at later stages (∼3 years old) and the cortex GM and WM volumes of TG_01 had already caught up with the WT monkeys ( Supplementary Fig. 4), supporting the proposed brain-developmental delay.

To further explore the brain-developmental changes of the TG monkeys, we analysed the MRI-DTI data to evaluate the WM properties, growth of brain structures and fiber tracts that connect them [ 35]. The fractional anisotropy (FA) index was used in characterizing the degree of diffusion directionality and is sensitive to the axon size, density as well as the degree of myelination [ 36]. Consistently with the observation of brain-volume change, in three types of WM tracts (projection fibers, association fiber and commissural fibers), the TG monkeys exhibited relatively lower FA values compared with the WT monkeys, although statistically not significant ( Supplementary Fig. 5A-C and Supplementary Fig. 6). The lower FA values suggest lower levels of myelination. A similar pattern was also observed when looking at the MD (mean diffusivity) values [ 37] ( Supplementary Fig. 7). Hence, the MRI-DTI data indicated a lower myelination level in the TG monkeys, implying a delayed neural-fiber myelination and neural-network maturation, which seem to mimic the known brain-developmental delay (neoteny) of humans [ 38].

We also checked whether there was a correlation between the number of carried huMCPH1 copies and the brain structural measurements, and we did not find a significant correlation with any measurements including TB volume, cortex volume and cortex thickness, etc. ( Supplementary Fig. 8), suggesting that a gene-dosage effect is not obvious.

Brain-tissue-section analysis indicates delayed neuronal differentiation

To detect brain-developmental changes at the cellular level, we conducted brain-tissue-section analysis of the frontal lobe at both the prenatal (two TG and four WT monkeys at embryonic day 136) and postnatal (one TG and two WT monkeys at 76 days after birth) stages (Fig. 2A). Two marker genes were used to examine the status of neural-cell proliferation and differentiation, including NeuN for matured neurons and glial fibrillary acidic protein (GFAP) for matured astrocytes. At the prenatal stage (E136), there were 80% NeuN-positive cells in the WT monkeys, contrasting with only 20% in TG_03 and 60% in TG_04 ( P = 1.25E-08, two-tailed t-test; Fig. 2B). At the postnatal stage (P76), 40-60% of cells were NeuN-positive in the WT monkeys, but only 10% in TG_01 ( P = 6.0E-04, two-tailed t-test; Fig. 2B). We detected similar ratio differences for astrocytes, with the WT monkeys having twice the ratio of GFAP-positive cells compared with that of the TG monkeys ( P < 0.01, two-tailed t-test; Fig. 2C).

The reduced ratios of matured neurons and astrocytes in the TG monkeys would predict elevated ratios of immature cells. To test this, we used two additional makers, namely DCX (doublecortin) for immature neurons and fatty acid binding protein (FABP) for immature astrocytes. As expected, the TG monkeys possessed much higher ratios of immature neurons and glia cells compared with the WT monkeys ( P < 0.001, two-tailed t-test; Fig. 2D and 2E). Of note, the total numbers of cells in the brain were similar between the TG monkeys and the WT monkeys ( Supplementary Fig. 9). Collectively, this cell-level difference is consistent with the observed myelination delay of the MRI data, as the fiber tracts are mostly composed of glial cells and myelinated nerve cells (axons).

Brain-transcriptome analysis using bulk tissue

To gain insight into the molecular mechanism underling the speculated cortex-developmental delay, we conducted RNAseq of the prefrontal cortex of the prenatal (two TG vs four WT at E136) and postnatal monkeys (one TG vs two WT at P76), with liver and muscle as the references. As expected, the overall MCPH1 expression was much higher in the TG monkeys than in the WT monkeys for all tissue types and the integrated huMCPH1 copies had much higher expression than the endogenous monkey MCPH1 ( Supplementary Figs 10A and 11A). In the brain, there was a large number of differentially expressed genes (DEGs) between the TG and the WT monkeys (970 genes at embryonic day 136 and 1933 genes at postnatal day 76) ( Supplementary Figs 10B, C, 11B and C and Supplementary Table 4). The numbers of DEGs were comparable in muscles and much lower in livers. Only a small portion of DEGs overlapped among tissues ( Supplementary Figs 10D and 11D and Supplementary Table 4), implying that the transgene huMCPH1 affects the gene expression of the TG monkeys in a tissue-type-dependent manner.

To see the functional enrichment of the DEGs in the brain, we performed gene ontology (GO) ontology analysis using the ToppGene Suite [ 39]. At the prenatal stage (E136), there were 350 significantly enriched functional categories for the TG-down-regulated genes ( Supplementary Table 5, FDR B&H < 0.05) and the top four categories were all related to synaptic signaling ( Supplementary Fig. 10E, left panel). In contrast, the enriched categories for the TG-up-regulated genes (60 categories, Supplementary Table 6) were mostly basic cellular functions such as translation and protein localization to the endoplasmic reticulum, not closely related to neural function ( Supplementary Fig. 10E, right panel). Similarly, at the postnatal stage (P76), the top 10 enriched categories for the TG-down-regulated genes were all related to neuron differentiation and neuron development, and synaptic-signaling genes were also over-represented, while the top categories for the TG-up-regulated genes were mostly related to neuron projection ( Supplementary Fig. 11E and Supplementary Table 7). The transcriptomic changes in the developing brains suggest that many neuron-maturation and differentiation-related genes were suppressed in the TG monkeys, consistently with the observed delay of neuron differentiation in the tissue-section analysis (Fig. 2).

Importantly, we found 107 brain DEGs shared between the prenatal E136 and postnatal P76 stages (Fig. 3A). GO ontology analysis with these 107 genes indicated that the highest enrichment category was synapse-related function, confirming the observed pattern when using all brain DEGs (Fig. 3B, 6/10). Remarkably, 35 of the 107 genes (32.7%) overlapped with the known synapse genes in the datasets of synaptome [ 40] and synsysnet [ 41] (Fig. 3C). Further analysis showed that about 50% of the 35 genes are either post-synapse- or synapse-related genes, and only 0.9% are pre-synapse-related genes (Fig. 3D). The hierarchical clustering analysis using the shared brain DEGs clearly distinguished the TG and the WT monkeys with the most prominent distinction between brain and muscle/liver (Fig. 3E). For example, NR4A1 is a downstream gene of MEF2A-a gene playing a critical role in activity-induced synaptic modification [ 42]. This gene showed 76% (at E136) and 26% (at P76) expression reduction in the TG monkeys. In the mouse model, overexpression of NR4A1 would eliminate dendritic spines while knock-down of NR4A1 could cause excessive number of spines and major postsynaptic density [ 43]. Together, these data suggest that, at the bulk-tissue level, the transgene huMCPH1 mainly suppresses the expression of neural differentiation and synapse-function-related genes.

Generation of F1 TG monkeys and transcriptome analysis of fetal cortical lamina

To further dissect the impact of the huMCPH1 copies on brain development, we generated three F1 TG monkeys by IVF using the sperms of TG_01 that we proved were carrying the huMCPH1 copies in the germ line ( Supplementary Fig. 12). The three F1 TG monkeys were sacrificed at embryonic day 76 (TG_09 at E76) and embryonic day 92 (TG_10 and TG_11 at E92)-the two developmental time points during the neurogenesis peak in rhesus monkeys [ 44]. Captured sequencing analysis indicated that the three F1 TG monkeys all carried the huMCPH1 copies at the same integrated sites as TG_01 ( Supplementary Table 1). With the use of IVF, we also obtained five WT fetal monkeys at the corresponding developmental points (two WT at E76 and three WT at E92, Supplementary Table 2). To conduct more detailed developmental tracking, we sampled the frontal cortex and dissected (using laser micro-dissection) the brain tissue into four cortical laminae, as they reflect different stages of neural proliferation, differentiation and migration, including the cortical plate (CP), outer sub-ventricular zone (OSVZ), sub-ventricular zone (SVZ) and ventricular zone (VZ) (Fig. 4A). RNAseq was performed for each lamina.

First, we checked MCPH1 expression in WT monkey embryos and we found that, at E76, no difference existed between the CP and the germinal zone (OSVZ, SVZ and VZ), while, at E92, MCPH1 expression was higher in the germinal zone than in the CP, consistently with the reported pattern in mouse [ 24] ( Supplementary Fig. 13A). As expected,

in the TG monkey embryos, the transgene huMCPH1 had a much higher expression than the endogenous MCPH1 in all laminae at both E76 and E92 ( Supplementary Fig. 13B). The fold changes were different among different laminae. SVZ had the highest fold change at E76, while CP had the highest fold change at E92 ( Supplementary Fig. 13B).

The principal component analysis (PCA) showed that the RNA profiles can distinguish different developmental cortical laminae. The VZ and CP were clearly separated, as they represent undifferentiated neuro-progenitors and differentiated neural cells, respectively, while the separation between the SVZ and OSVZ was incomplete because they are the intermediate stages in view of cell proliferation, migration and differentiation (Fig. 4A). Consistently, the marker genes for neuro-progenitors ( SOX2) and neurons ( SYT1) showed the expected expression pattern in these laminae (Fig. 4A). We then analysed DEGs of the four laminae between the TG monkeys and the WT monkeys. As expected, the transgene huMCPH1 caused expression changes of many genes and this pattern was more pronounced at E92 compared with E76 (140-350 genes for E76 and 3000-9000 genes for E92, Supplementary Table 8) ( Supplementary Fig. 13C and D). The GO ontology analysis showed that, at E76, there were 25 enriched categories for CP, 26 for OSVZ, 41 for SVZ and 10 for VZ ( Supplementary Tables 9-12, FDR B&H < 0.05). Among the top 10 categories, the enriched functional terms were cell development (CP, 5/10), synapse signaling (OSVZ, 5/10), cell differentiation and proliferation (SVZ, 5/10) and cilium function (VZ, 3/10) ( Supplementary Fig. 13E, left panel, FDR B&H < 0.05). In contrast, there were a lot more enriched categories at E92 (453 for CP, 109 for OSVZ, 102 for SVZ and 786 for VZ) ( Supplementary Tables 13-16, FDR B&H < 0.05), among which the top categories were mRNA catabolic process (CP, 3/10), neuron differentiation, neurogenesis and cell migration (OSVZ, 5/10), cell cycle and mRNA processing (SVZ, 10/10) and immune response (VZ, 8/10) ( Supplementary Fig. 13E, right panel, FDR B&H < 0.05).

We next conducted lamina-to-lamina pairwise comparisons in the TG and the WT monkeys separately. Markedly, at E76, the TG monkeys exhibited much less between-lamina expression difference than the WT monkeys. In particular, there were no DEGs when comparing SVZ with OSVZ in the TG monkeys, contrasting with 557 DEGs in the WT monkeys (Fig. 4B and Supplementary Table 17). A similar pattern was seen at E92. For example, there were 61 CP vs OSVZ DEGs in the TG monkeys, while there were 9186 DEGs in the WT monkeys (Fig. 4C and Supplementary Table 18). This result suggests that cortical-lamina distinction is much weaker for the TG monkeys compared with the WT monkeys, supporting the proposed delay of neuronal maturation and differentiation. Consistently, we observed delayed expression peaks of four known neuron-differentiation markers, including SYP (Synaptic vesicle protein p38), ENO2 (Cytosolic protein t), GAD1 (Glutamic acid decarboxylase) and MAP2 (Cytoskeletal protein) (Fig. 4D).

Furthermore, in order to see the temporal pattern of gene-expression delay in the TG monkeys, we combined the RNAseq data of all four developmental stages, including E76, E92, E136 and P76 (6 TG vs 11 WT). We classified those genes showing expression delay in the TG monkeys into three types according to their expression peak times in the WT monkeys ( Supplementary Fig. 14A and B). The Type-1 genes are those with an expression peak shift from E92 in the WT monkeys to E136 in the TG monkeys (e.g. the SLC44A2 gene). The Type-2 genes had a peak shift from E136 in the WT monkeys to P76 in the TG monkeys (e.g. the SYP gene), while the Type-3 genes had a peak shift from E92 in the WT monkeys to P76 in the TG monkeys (e.g. the CDK5 gene) ( Supplementary Fig. 14A). In total, we identified 185, 347 and 50 genes for Type-1, Type-2 and Type-3 delays, respectively ( Supplementary Fig. 14B). We then performed GO ontology analysis and only the Type-2 genes showed significant enrichment of functional categories, implying that the developmental stage close to birth was the most affected in the TG monkeys. Consistently with the results of bulk-tissue RNAseq, the Type-2 genes from the cortical laminae containing undifferentiated cells (VZ, SVZ and OSVZ) were mainly enriched for synapse-related functions such as trans-synapse signaling, chemical-synapse transmission and synaptic signaling ( Supplementary Fig. 14C, P < 0.001) and the involved genes showed delayed expression peaks at P76 or later stages in the TG monkeys.

To test whether the observed gene-expression delay in the TG monkeys show human-like features, we obtained data from a previous study in which genes with human-specific expression delay were identified by comparing postnatal brain development in the prefrontal cortex of humans, chimpanzees and rhesus macaques [ 11]. We found that only the Type-2 genes were enriched in the reported Module-I gene set with human-specific expression delay ( P < 0.0001; hypergeometric test; Supplementary Fig. 14D and Supplementary Table 19). For example, MEF2A is a Type-2 gene and also a Module-I gene, which not only plays a role in neuron differentiation [ 45], but also mediates a human-specific time shift of cortex synaptic development [ 11]. Hence, the patterns of gene-expression delay are consistent between the data from the bulk tissue and the data for the laminae, and many neural-differentiation-related genes were suppressed in the TG monkeys with human-like expression delays during brain development.

General behavior analysis and test of short-term memory

To test whether the observed brain-developmental delay at the molecular and cellular levels in the TG monkeys can be transformed into cognitive changes, we first performed an analysis of general behaviors (four TG vs four WT age-matched monkeys 24-36 months old; see the 'Methods' section for details). A total of nine indexes for general behaviors were measured [ 46], including self-injury behavior, stereotypical behavior, feeding, self-grooming, locomotion, resting, bouts of wake, wake and sleep. No difference was detected between the TG and the WT monkeys, suggesting that the transgene huMCPH1 did not cause abnormal behaviors in the TG monkeys ( Supplementary Fig. 15).

Next, we performed a test of short-term memory using the delayed-matching-to-sample (DMS) task, which was known to be correlated with prefrontal-cortex function [ 47-49]. The computerized touch-screen behavioral battery (Cambridge Neuropsychological Test Automated Batteries, CANTAB; Lafayette, USA) was used. The DMS task requires the monkeys to remember the color and the shape of a stimulus on the screen for a specified delayed time. The monkeys were initially habituated in a testing room for 5 days and then subject to touch training. Touch training was divided into two phases. In Phase-1, monkeys were subject to touch training for 15 days continuously. In Phase-2, monkeys were required to have a >85% correction rate for 3 continuous days in the training sessions and then were subject to the DMS task test (Fig. 5A, Supplementary Fig. 16 and Supplementary Video 1; more details about touch training is provided in the 'Methods' section). The results showed that, at 0 ∼ 4-sec delayed times, the TG monkeys performed significantly better than the WT monkeys (Fig. 5B, general linear model (GLM) model, group effect P = 0.0086) and this difference became more pronounced in the sessions with 8 and 16-sec delayed times (Fig. 5B; GLM model, 8 s, group effect P = 0.0022; 16 s, group effect P = 5.5E-04). When the delayed time extended to 32 sec, the difference remained (Fig. 5B, GLM model, group effect P = 0.032). When all sessions with different delayed times were combined together, the TG monkeys had a significantly better performance than the WT monkeys (Fig. 5B, GLM model, group effect P = 7.5E-04). Interestingly, we also observed significantly shorter reaction times (response latency) in the TG monkeys for all categories of delayed times ( P < 0.001, two-tailed t-test, Fig. 5B). Collectively, the TG monkeys exhibited better performance in the DMS task than the WT monkeys, suggesting that the brain-developmental delay caused by the transgene huMCPH1 may have enhanced the short-term memory of the TG monkeys.

DISCUSSION

MCPH1 is one of the strong candidates for human brain evolution, since it has accumulated a human-specific protein-sequence and gene-expression changes [ 17, 18]. Ideally, a gene-replacement model would be preferred so that the influence of the endogenous monkey MCPH1 could be removed. However, due to the long generation time of monkeys (4-5 years), current gene-editing tools are still impractical in generating such a model in monkeys. We argue that a transgenic monkey model is practical and to a large extent can mimic the human-specific status. The transgenic monkeys carry the human MCPH1 copies so that the effect of the human-specific protein-sequence changes can be tested. At the same time, since the transgenic monkeys over-express the huMCPH1 transgene, this can mimic the human-specific increase in gene expression.

Because MCPH1 is a key gene for neurogenesis, one of the expected phenotypic outcomes in the transgenic monkeys would be a larger brain, which was not the case in this study. We showed that the TG monkeys carrying the huMCPH1 transgene did not manifest an enlarged brain size, implying that a single gene likely has a limited effect on neural progenitor pool proliferation during brain development. Alternatively, it is equally possible that the human-specific changes in MCPH1 may not enhance its known function in neuro-progenitor proliferation [ 22]; rather, they work on the unknown function of MCPH1 in neuronal maturation, neural plasticity and synapse signaling, which were supported by multiple lines of evidence presented in this study.

Our analyses found a developmental delay of GM in the brain of the TG monkeys, suggesting that the huMCPH1 transgene may delay neuron differentiation and maturation during brain development. Consistently with the cortex-developmental delay, there were much fewer mature neurons and glia cells in the TG monkeys compared with the WT monkeys during the early period of postnatal development. Consistently, the tissue-level transcriptome comparisons indicated a large amount of neuron differentiation and development, and synapse genes were suppressed in the TG monkeys, providing a possible molecular basis for the observed delay in cell maturation and fiber myelination in the brain. In fact, our previous in vitro experiments demonstrated that MCPH1 can act as a transcription repressor and repress telomerase activity [ 50]. Furthermore, the cortical-lamina transcriptome comparisons showed that the huMCPH1 transgene can influence gene expression at all laminae including the CP, OSVZ, SVZ and VZ as early as prenatal E76, indicating that the huMCPH1 transgene may alter neurogenesis by affecting the neuro-progenitor cell division and differentiation. In fact, previous mouse studies have already shown that MCPH1 is required for precise mitotic spindle orientation during neurogenesis [ 22]. Additionally, as expected, cortical-lamina pairwise comparisons suggested that the between-laminae differences in the TG monkeys were not as obvious as in the WT monkeys, consistently with the proposed delay in neural differentiation. Hence, the huMCPH1 transgene may have contributed to delaying cortical-lamina differentiation in the TG monkeys. Taken together, we propose that overexpression of the huMCPH1 transgene can cause neural-developmental delay due to the down-regulation of many neural-differentiation-related genes. Future experiments are warranted to reveal the detailed molecular pathways.

One hallmark difference between humans and non-human primates is that humans require a much longer time to shape their neuro-networks during development, greatly elongating childhood, namely the so-called 'neoteny'. Myelination is the process of generating myelin sheaths around nerve fibers so that neural signals can be propagated more swiftly with less signal loss. This process is considered a key developmental aspect of the human brain and continues for at least 10-12 years after birth, providing an extended window of neural-network plasticity [ 51]. In fact, human neocortical myelination is developmentally protracted compared with that of chimpanzees [ 52]. We speculate that the observed neural-maturation delay in the TG monkeys may have extended their time window of neural-network plasticity, similar to the brain-developmental neoteny of humans. In support of our speculation, when we combined the RNAseq data of all developmental stages, we found that many of the delay genes were synapse-related genes, which are required for the experience-dependent process of neural-network plasticity [ 53]. More interestingly, most of the delay genes showed human-specific changes in the timing of synaptic development in the previous study [ 11]. For example, MEF2A and SYP were among the key genes showing human-specific delay of youth-like expression compared with chimpanzee and macaque [ 11]. Notably, synapse and spine density in the human-projection neurons is much higher than that in rhesus macaque, which is associated with the higher cognitive performance in humans [ 11, 54].

The speculated extension of neural-network plasticity in the TG monkeys gained further support from our preliminary cognitive data. The TG monkeys showed an improved short-term memory, suggesting that the observed brain-developmental delay in the TG monkeys is beneficial, possibly through extending the time window of neural-network plasticity. More interestingly, the TG monkeys displayed a significantly shorter reaction time than the WT monkeys during the DMS task, which is another hint at cognitive improvement. More sophisticated cognitive tests are needed to understand the long-term effect of the huMCPH1 transgene in the TG monkeys.

Our findings demonstrated that transgenic non-human primates (excluding ape species) have the potential to provide important-and potentially unique-insights into the basic questions of what actually makes humans unique, as well as into disorders and clinically relevant phenotypes, such as neurodegenerative and social-behavior disorders that are difficult to study by other means [ 32, 46, 55]. But such gains must invariably be weighed against potential ethical concerns [ 29, 56, 57]. We noted that the transgenic monkey model also has limitations, including the influence of the endogenous monkey gene copy and the incapability to differentiate the effects of protein-sequence changes from gene-expression changes. Several recent technical improvements (e.g. CRISPR-Cas9) have shown the hope of conducting precision genome editing in monkeys [ 26, 58-61], providing more powerful tools for future studies in understanding the genetic basis of human brain evolution.

METHODS

The detailed methods and materials are available as Supplementary Data at NSR online.

Acknowledgements

The wild-type MCPH1 plasmid was kindly provided by Prof. Xingzhi Xu (Capital Normal University, China). We are grateful to Prof. Xintian Hu and Drs. Dongdong Qin and Shihao Wu for their advice on behavioral and cognitive tests. The monkey icon in Fig. 5 was obtained using Freepick at www.flaticon.com. We thank Hui Zhang and Yan Guo for their technical assistance in this study.

FUNDING

This study was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB13000000 to B.S.), the National Natural Science Foundation of China (31730088 and 31621062 to B.S.) and the Youth Innovation Promotion Association of CAS (to L.S.).

AUTHOR CONTRIBUTIONS

L.S. and B.S. designed the study; L.S., X.L., J.J., T.L., M.L. and Q.L. performed the experiments; H.W. contributed to animal care and management; C.R.L. and J.H.W. assisted with the MRI image data analysis; Y.N., X.H., Y.C. and W.J. assisted in the monkey reproductive technique and virus-gene transfer; L.S., X.L., Y.L., Y.Y.M., X.S. and H.Y. conducted MRI scanning; Y.L. and T.L. assisted with the histological studies; L.S. and B.S. performed data analysis; L.S., X.L., J.J. and B.S. wrote the manuscript.

Conflict of interest statement. None declared.

REFERENCES

Author notes

# data[].attributes.attachment_link.title
Transgenic rhesus monkeys carrying the human MCPH1 gene copies show human-like neoteny of brain development

# data[].attributes.attachment_link.description
Expansion in brain size and improvement in cognitive skills are among the most fundamental evolutionary changes that set humans apart from other primates.

# data[].attributes.body
https://link.springer.com/article/10.1007/s11033-019-05028-7#Abs1

# data[].attributes.attachment_link.readable_text
Molecular Biology Reports 46, 5941-5953 (2019) Cite this article

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Abstract

Sunflower ( Helianthus annuus. L) is one of the principal oil seed crops affected by the salinity stress, which limits the oil content and crop yield of sunflower plants. The acclimatization of plants to abiotic stresses such as salinity tolerance is mainly mediated by the vacuolar Na⁺/H⁺ antiporters ( NHX) by tagging Na⁺ into vacuoles from the cytosol. We show here that the over-expression of wheat TaNHX2 gene in transgenic sunflower conferred improved salinity stress tolerance and growth performance. Transgenic sunflower plants were produced by infecting the embryonic axis ex-plants with Agrobacterium tumefaciens strain EHA105 containing a pBin438- TaNHX2 binary vector that carried a wheat antiporter ( TaNHX2) gene under the control of a double CaMV 35S promoter with NPT II gene as a selectable marker. PCR analysis of T ₀ and T ₁ transgenic plants confirmed the integration of TaNHX2 in sunflower genome. Stable integration and expression of TaNHX2 in sunflower genome was further verified by Southern hybridization and semi-quantitative RT-PCR analyses. As compared to the non-transformed plants, TaNHX2 expressing transgenic plants showed better growth performance and accumulated higher Na⁺, K⁺ contents in leaves and roots under salt stress (200 mM NaCl). Transgenic sunflower plants displayed improved protection against cell damage exhibiting stable relative water content, chlorophyll content, increased proline accumulation and improved reactive oxygen species (ROS) scavenging because of higher activities of the antioxidant enzymes like superoxide dismutase and ascorbate peroxidase, along with decreased production of hydrogen peroxide, free oxygen radical and malondialdehyde (MDA) under salt stress (200 mM NaCl). Taken together, our findings suggest that TaNHX2 expression in sunflower plants contributed towards improving growth performance under sodium chloride stress.

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Abbreviations

APX:

Ascorbate peroxidase

DW:

Dry weight

FW:

Fresh weight

MDA:

Malondialdehyde

RT-PCR:

Reverse transcription polymerase chain reaction

RWC:

Relative water content

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

TW:

Fully turgid weight

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Acknowledgements

The authors acknowledge the Head, Department of Plant Sciences, University of Hyderabad for access to the research facilities provided by DST-FIST, DBT-CREBB, and UGC-SAP in undertaking this investigation. Authors also thank Prof.Souyi Chen and Jinsong Zhang, Institute of Genetics and Developmental Biology, CAS, Beijing for providing the pBin438- TaNHX2 construct. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Author notes

1. P. B. Kirti

Present address: Rajendra Prasad Central Agricultural University, Pusa-Samsthipur, Bihar, India

2. Ramesh Mushke and Rajesh Yarra have contributed equally to this work.

Affiliations

1. Aegis Agro Chemical India Pvt Ltd, Plot No: B 11/1, Industrial Developmental Area, Uppal, Hyderabad, Telangana, 500039, India

Ramesh Mushke

2. Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, 500049, India

Rajesh Yarra & P. B. Kirti

3. Agri Biotech Foundation, Rajendranagar, Hyderabad, Telangana, India

P. B. Kirti

Contributions

RY and PBK conceived the experiments. RM and RY performed the experiments. RM, RY, and PBK analyzed the data. RY and PBK wrote the manuscript. All authors approved the final version of the manuscript.

Corresponding author

Correspondence to Rajesh Yarra.

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Mushke, R., Yarra, R. & Kirti, P.B. Improved salinity tolerance and growth performance in transgenic sunflower plants via ectopic expression of a wheat antiporter gene ( TaNHX2). Mol Biol Rep 46, 5941-5953 (2019). https://doi.org/10.1007/s11033-019-05028-7
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DOI: https://doi.org/10.1007/s11033-019-05028-7

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Keywords

TaNHX2

Sunflower

Helianthus annuus

Salt stress

Antioxidant enzymes

ROS scavenging

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# data[].attributes.attachment_link.title
Improved salinity tolerance and growth performance in transgenic sunflower plants via ectopic expression of a wheat antiporter gene (TaNHX2) - Molecular Biology Reports

# data[].attributes.attachment_link.description
Sunflower (Helianthus annuus. L) is one of the principal oil seed crops affected by the salinity stress, which limits the oil content and crop yield of sunflower plants. The acclimatization of plants to abiotic stresses such as salinity tolerance is mainly mediated by the vacuolar Na+/H+ antiporters (NHX) by tagging Na+ into vacuoles from the cytosol.