# data[].attributes.body
1. "Genetic engineering can be done with plants, animals, or bacteria, and other very small organisms. Genetic engineering allows scientists to move desired genes from one plant or animal into another."

2." Genes can also be moved from an animal to a plant or vice versa. Another name for this is genetically modified organisms or GMOs."

# data[].attributes.body
1. Enhancing food production, reducing environmental impact

2. Optimizing animal health and welfare and production of cutting-edge industrial applications.

# data[].attributes.body
Benefit #1 : Papaya has Anticancer Properties. It can reduce cancer risks and beneficial for ones who are being treated with cancer.

Benefit #2 : Loaded with Nutrients & Delicious. Papaya has an enzyme that is called papain, this breaks down tough protein chains in muscle meat.

# data[].attributes.body
1. Studies have shown that genetically modified corn and soy fed to rats led to a higher risk of them developing liver and kidney problems. ..

2. Transgenic modification produces organism types which would never occur naturally, making them highly unpredictable.

3. GMOs are not always tested thoroughly.

# data[].attributes.body
Fact number one: Researchers injected jellyfish DNA into a mother rabbit's embryos.

Fact number two: Moisyadi told Hawaii local news channel KHON, "These rabbits are like a light bulb glowing, like an LED light all over their body.

# data[].attributes.body
First disadvantage : There is a lack of Cross-Pollination which leads to less production of food.

Second disadvantage : The tree it grows on is hard to transplant because of it's wide root system.

# data[].attributes.body
1. "Humans were created and placed on top to rule the Earth. As a species, there is only one race, which is the human race."

2.  . "Even though.  humans display a cruel and destructive nature, humans have the potential to transform into."

3. 'The most important word for the human race to remember is “humanity”.

# data[].attributes.body
Based on the pros and cons, here is an opinionated statement about this GMO animal.

In my option, I think this GMO animal is really cool because of how it was made by a jellyfish's DNA. I think we should have more of these as pets! This kind of GMO rabbit shows a lot of unique styles. The rabbits are expected to have the same life span as their non-glowing counterparts, but Moisyadi said he understands people can object to this kind of experimentation involving live animals. This is so fascinating to me. This is a good GMO animal to me and I'm sure little kids or kids my age (13) would love to see this in person and automatically fall in love with this creature, Hopefully, one day I can see this GMO rabbit in person and see how it glows in the dark.

# data[].attributes.body
https://www.theguardian.com/world/2013/aug/13/glow-in-dark-rabbits-scientists

# data[].attributes.attachment_link.readable_text
As part of an effort to improve treatments for life-threatening illnesses, a team of scientists have created rabbits that glow in the dark.

Their efforts produced two rabbits out of a litter of eight that went from being a normal, fluffy white to glowing green in the dark. The rabbits were born at the University of Istanbul as part of a collaboration between scientists from universities in Turkey and Hawaii.

The rabbits glow to show that a genetic manipulation technique can work efficiently, though the specific color is more cosmetic than scientific. "The green is not important at all - it's just a marker to show the experiment can be done successfully," said University of Hawaii associate professor Stefan Moisyadi.

To produce the glowing effect, researchers injected jellyfish DNA into a mother rabbit's embryos. Those altered embryos were then inserted back into the mother. Similar experiments have resulted in glowing cockroaches and cats.

Eventually, the researchers hope the technique can lead to new ways to produce medicines, Moisyadi said.

"The final goal is to develop animals that act as barrier reactives to produce beneficial molecules in their milk that can be cheaply extracted, especially in countries that can not afford big pharma plants that make drugs, that usually cost $1bn to build, and be able to produce their own protein-based medication in animals," Moisyadi said.

The rabbits are expected to have the same life span as their non-glowing counterparts, but Moisyadi said he understands people can object to this kind of experimentation involving live animals.

"To the people against, I say: think about, what are the benefits and what are the injuries?" Moisyadi said. "And if the benefits outweigh the injuries, let's go with the benefits."

Moisyadi, a native of Turkey who is now with the University of Hawaii, started developing the project in 2006, and researchers are now waiting to see if pregnant sheep produce similar results.

# data[].attributes.attachment_link.description
As part of an effort to improve treatments for life-threatening illnesses, a team of scientists have created rabbits that glow in the dark. Their efforts produced two rabbits out of a litter of eight that went from being a normal, fluffy white to glowing green in the dark.

# data[].attributes.body
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7885269/

# data[].attributes.attachment_link.readable_text
Abstract

Rabbits are one of the most used experimental animals for investigating the mechanisms of human cardiovascular disease and lipid metabolism because they are phylogenetically closer to human than rodents (mice and rats). Cholesterol-fed wild-type rabbits were first used to study human atherosclerosis more than 100 years ago and are still playing an important role in cardiovascular research. Furthermore, transgenic rabbits generated by pronuclear microinjection provided another means to investigate many gene functions associated with human disease. Because of the lack of both rabbit embryonic stem cells and the genome information, for a long time, it has been a dream for scientists to obtain knockout rabbits generated by homologous recombination-based genomic manipulation as in mice. This obstacle has greatly hampered using genetically modified rabbits to disclose the molecular mechanisms of many human diseases. The advent of genome editing technologies has dramatically extended the applications of experimental animals including rabbits. In this review, we will update genetically modified rabbits, including transgenic, knock-out, and knock-in rabbits during the past decades regarding their use in cardiovascular research and point out the perspectives in future.

Keywords: atherosclerosis, CRISPR-Cas9, hypercholesterolemia, knock-out rabbits, transgenic rabbits

Introduction

Rabbits were first used for disclosing the pathogenesis of human atherosclerosis a century ago. In 1908, a Russian physician, Alexander I. Ignatowski (1875-1955) fed rabbits with a diet supplemented with animal proteins (milk, meat, and eggs) and found that these rabbits developed pronounced aortic atherosclerosis.

Later, a Russian experimental pathologist, Nikolai N. Anichkov (or Anitschkow) (1885-1964) further demonstrated that it was dietary cholesterol rather than proteins that play the critical role in the pathogenesis of atherosclerosis in rabbits and proposed a causal role of cholesterol in the development of atherosclerosis (Fan et al., 2015). Now, a consensus has been widely hold in this field that, in both humans and experimental animals, high levels of plasma cholesterol carried by apolipoprotein (apo)-B-containing particles such as low density lipoproteins (LDL) initiate the development of atherosclerosis (Steinberg, 2004). These pioneering studies derived from rabbit experiments not only provided the first evidence but also established a theory basis of the "lipid hypothesis" of atherosclerosis (Steinberg, 2004). Since then, cholesterol-fed rabbits along with Watanabe heritable hyperlipidemic (WHHL) rabbits, a mutant rabbit with genetic deficiency of LDL receptor functions, have been extensively used to elucidate multiple facets of the pathophysiology of human atherosclerosis, leading to the discovery of the LDL receptor functions in familial hypercholesterolemia (Goldstein et al., 1983) and the development of the most-prescribed lipid-lowering drug, statin (Brown and Goldstein, 2004). On the other hand, transgenic rabbits with overexpression of various genes were generated from early 90's and served as an alternative tool for investigating the gene functions in cardiovascular disease. Moreover, recent genome editing technology has provided enormous opportunities to create knock-out (KO) and knock-in (KI) rabbits. Important roles of rabbits in studying human atherosclerosis have been extensively reviewed in the previous reviews (Fan et al., 1999a, 2015, 2018; Fan and Watanabe, 2000, 2003). In this review, we will focus on genetically modified rabbits for their applications in cardiovascular research.

"Naturally" Genetically Modified Rabbits

Spontaneous mutations in rabbits can be found accidentally and they can be used in controlling the coat color for commercial purposes such as tyrosinase and the melanocortin 1 receptor (Aigner et al., 2000; Xiao et al., 2019). However, some spontaneous mutations in rabbits can cause a pronounced phenotype that can mimic human diseases, such as Watanabe heritable hyperlipidemic (WHHL) rabbits (Watanabe, 1980), St. Thomas hyperlipidemic rabbits (Laville et al., 1987; Seddon et al., 1987) and complement 6 deficient rabbits (Rother, 1986; Liu et al., 2007a). WHHL rabbits were originally established by Dr. Yoshio Watanabe (1927-2008) at Kobe University, Japan, through serial inbreeding (Watanabe, 1980). Homozygous WHHL rabbits exhibit spontaneous hypercholesterolemia characterized by high levels of LDLs and severe atherosclerosis and often serve as a human familial hypercholesterolemia model (Watanabe et al., 1985). Genetic analysis revealed that WHHL rabbits have defective LDL receptor functions due to a deletion of 12 nucleotides in exon 4 of the LDL receptor gene, which leads to a 4-amino acid deletion in the cysteine-rich ligand-binding domain of the LDL receptor protein (Yamamoto et al., 1986). LDL receptor mutations can be easily detected by PCR analysis (Sun et al., 2002a); however, high levels of plasma LDL-cholesterol are the major manifestation observed in homozygous WHHL rabbits. In addition to hyperlipidemia and aortic atherosclerosis, some WHHL rabbits (later designated as WHHL-MI) show coronary atherosclerosis and myocardial infarction (Shiomi et al., 2003; Shiomi and Fan, 2008). Using WHHL rabbits, Tomoike et al. further developed a subline of WHHL designated a hereditary hypertriglyceridemic rabbit after selected in-breeding. This model exhibited postprandial hypertriglyceridemia along with insulin resistance and visceral obesity although polygenetic loci for these pathophysiogical changes have not been determined (Kawai et al., 2006). In addition to WHHL rabbits, the St. Thomas hospital hyperlipidemic rabbits were developed by La Ville et al. in London (Laville et al., 1987; Seddon et al., 1987). Different from WHHL rabbits which have high plasma LDL levels due to LDL receptor dysfunctions, the St. Thomas hospital hyperlipidemic rabbits showed high levels of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL), and LDL, thus this rabbit model resembles human familial combined hyperlipidemia. Elevated plasma cholesterol levels in these rabbits were caused by overproduction of these apo-B-containing lipoproteins in the liver although the genetic mutations responsible for hyperlipidemia have not been examined in details. There is a complement-6 (C6) deficient rabbit originally reported by Rother in 1986 (Rother, 1986). C6 deficiency in these rabbits arises from a single gene defect and is not known to be associated with other genetic abnormalities. In spite of this, C6 deficient rabbits are protective against cholesterol diet-induced atherosclerosis (Schmiedt et al., 1998).

Transgenic Rabbits

Because spontaneous mutant rabbits with obvious phenotypes resembling human disease phenotypes are rare and accidently discovered by experimental animal staff, it is necessary to make genetically modified rabbits according to one's own research purposes. The technology for producing transgenic (Tg) rabbits was almost concurrently reported by German (Brem et al., 1985) and US (Hammer et al., 1985) groups in 1985, but the actual use of Tg rabbit technology as an experimental tool in the field of cardiovascular diseases was not realized until 1994 when John Taylor's laboratory at the Gladstone Institute of Cardiovascular Disease in San Francisco created the first Tg rabbit expressing human hepatic lipase (Fan et al., 1994). Later on, they also produced Tg rabbits expressing human apoB-100 (Fan et al., 1995), apoE (Huang et al., 1997; Fan et al., 1998), and apoB mRNA editing protein (Yamanaka et al., 1995). Until now, more than 20 kinds of Tg rabbits expressing different genes that are involved in lipid metabolism and atherosclerosis have been reported and studies using these Tg rabbits have provided considerable insights into the molecular mechanisms of these gene functions in lipoprotein metabolism and atherosclerosis (Fan and Watanabe, 2003; Peng, 2012; Fan et al., 2015). The transgenes expressed in Tg rabbits for the study of lipoprotein metabolism and atherosclerosis can generally be classified into three categories: (1) those proteins that constitute lipoprotein structures such as apo(a) (Rouy et al., 1998; Fan et al., 1999b), apoAI (Duverger et al., 1996a), apoAII (Koike et al., 2009a; Wang et al., 2013), apoB-100 (Fan et al., 1995), apoCIII (Ding et al., 2011), and apoE (Huang et al., 1997; Fan et al., 1998); (2) those enzymes or transfer proteins that participate in the lipid metabolism such as hepatic lipase (Fan et al., 1994), lipoprotein lipase (Fan et al., 2001a), phospholipid transfer protein (Masson et al., 2011), apoB-100 mRNA editing enzyme catalytic polypeptide protein (Yamanaka et al., 1995), lecithin:cholesterol acyltransferase (Hoeg et al., 1996), endothelial lipase (Wang et al., 2017); and (3) those proteins that may exert some functions on the arterial wall cells which participate in the pathogenesis of atherosclerosis including matrix metalloproteinase-1,9,12 (Liang et al., 2006; Niimi et al., 2019; Chen et al., 2020), 15-lipoxygenase (Shen et al., 1996), C-reactive protein (Koike et al., 2009b), and vascular endothelial growth factor (Kitajima et al., 2005) (). In addition, Tg rabbits have also been used for the investigation of human heart diseases, including LQT syndrome (Brunner et al., 2008), hypertrophic cardiomyopathy (Marian et al., 1999) and tachycardia-induced cardiomyopathy (Suzuki et al., 2009). This is because, in comparison with mice and rats, the rabbit heart is similar to that of humans in both structure and function (Bers, 2002; Marian, 2006; Pogwizd and Bers, 2008). For example, like human heart in which β-myosin heavy chain (β-MyHC) accounts for 90% of total myofibrillar myosin, rabbit heart is composed of 80% β-MyCH which is different from the mouse heart predominated by 95% α-MyHC (Marian, 2005; Bosze et al., 2016). Tg rabbits can be generated by microinjecting a transgenic DNA construct into the pronuclei of fertilized embryos (Fan et al., 1999a; Kitajima et al., 2003). The transgenic constructs are typically composed of the transgene (either cDNA or genomic DNA) under the control of a tissue-specific promoter such as liver- and macrophage-specific promoter. In addition to the pronuclear microinjection method, other methods such as sperm vector (Wang et al., 2003; Li et al., 2006, 2010; Shen et al., 2006), ICSI-mediated transgenesis (Li et al., 2010; Zhang et al., 2016), somatic cell nuclear transfer (SCNT) (Li et al., 2009) or chimeric SCNT (Matsuda et al., 2002; Skrzyszowska et al., 2006), lentiviral vectors (Hiripi et al., 2010), transposon-mediated transgenesis (Katter et al., 2013; Ivics et al., 2014), and novel genome editing technology (Song J. et al., 2016; Yang et al., 2016; Li et al., 2019) have been reported to produce Tg rabbits. In spite of this, the pronuclear microinjection is still the most common method even though transgene integration rate is low.

Table 1

Rabbit Embryonic Stem Cells and Genome Information

Because of the lack of both rabbit embryonic stem (ES) cells and the genome information, it has been considered impossible to create KO rabbits by homologous recombination-based genomic modification as to generate KO mice. Unavailability of KO rabbits also constitutes another obstacle that hampers researchers to study loss-of-functions of genes in rabbits. We strived to use somatic cell nuclear transfer technique to generate KO rabbits after Chesne et al. reported the first cloned rabbit about 17 years ago (Chesne et al., 2002). However, after enormous attempts, we got to the conclusion that the production of KO rabbits by somatic cell nuclear transfer is far remote from reality. As a research tool, nuclear transfer technique is unworkable owing to the extraordinarily low efficiency of gene transfer into somatic cells and the possibility in generating cloned rabbits (Song J. et al., 2020). Many groups reported that they could obtain rabbit ES-like cells, but none of these so-called ES-like cells have been proved to be able to generate chimera rabbits (Fan et al., 2015). Rabbit genome has long been an empty area mainly because of budget insufficiency and narrow research communities. In 2014, Carneiro et al. successfully reported a high-quality reference genome using the European rabbit with references to domestication and speciation (Carneiro et al., 2014a, b). Almost at the same period, we along with researchers from the US, Japan and China organized an International Rabbit Genome Sequencing Project Consortium aiming at implementing more extensive whole-genome sequencing of three kinds of common laboratory rabbits: Japanese white rabbits, New Zealand white rabbits and WHHL rabbits. In addition, we performed deep transcriptome sequencing of the aortas, livers, hearts, and kidneys of cholesterol-fed and WHHL rabbits (Wang et al., 2016). After a 2-year collaborative work, we were able to completed whole-genome sequencing of 10 male rabbits for each line with coverage of 13x for each individual after alignment to the reference genome. With the successful completion of rabbit genome sequencing (Carneiro et al., 2014a, b; Wang et al., 2016), researchers now can easily not only design PCR primers to study gene expression in rabbits but also to generate KO rabbits using genome editing techniques as described below. Rabbit genome information is now available from the NCBI database and a comprehensive rabbit transcriptome information established by the Chinese Academy of Sciences in Shanghai (Zhou et al., 2018) is also available at http://www.picb.ac.cn/RabGTD/.

Knock-Out and Knock-In Rabbits by Genome Editing Techniques

In the past decade, the emergence of three powerful genome editing technologies has dramatically enhanced the application of genetically modified rabbits (Song J. et al., 2020). The first one is the zinc finger nuclease (ZFN)-mediated genome editing method by which KO rats were successfully created in 2009 (Geurts et al., 2009). Two years later after the birth of KO rats, Flisikowska et al. generated the first immunoglobulin KO rabbits in an attempt to produce humanized antibodies (Flisikowska et al., 2011). Almost at the same time, we successfully created apoCIII KO rabbits with ZFN-mediated genome editing technology (Yang et al., 2013). ZFNs are engineered DNA-cleaving enzymes made by fusing a tailor-made DNA-binding domain to the DNA cleavage domain of Fok1, a type II restriction enzyme. ZFNs generate site-specific double-strand breaks in the DNA at researcher-assigned sites, thus resulting in targeted modification of the genome. However, while ZFNs were not extensively applied in this field, the second generation of the genome editing tool, transcription activator-like effector nucleases (TALEN) were shown up to make the first KO rats in 2011 (Tesson et al., 2011). TALENs are considered much simpler to design and assemble than ZFNs. The DNA binding domain in TALENs was derived from Xanthomonas spp. Bacteria (Christian et al., 2010; Miller et al., 2011). While TALENs utilize the same Fok I endonuclease domain as ZFNs, its DNA binding domain contains a repeated highly conserved 33-34 amino acid sequence with divergent 12th and 13th amino acids which called Repeat Variable Diresidue (RVD) for recognizing one specific nucleotide, for example, NN for guanine, NI for adenine, HD for cytosine, and NG for thymine. This direct relationship between amino acid sequence and DNA recognition has made engineering sequence specific binding domains much easier than ZFNs (Boch et al., 2009; Moscou and Bogdanove, 2009). Using TALEN technology, Lai's laboratory at GIBH, immediately generated two kinds of KO rabbits: an immunodeficent KO rabbit with deficiency of Rag1 and Rag2 genes (Song et al., 2013) and fumarylacetoacetate hydrolase deficient rabbits (Li et al., 2017), which mimics human genetic disease tyrosinemia type I, an autosomal recessive disorder caused by mutations in the both copies of the gene encoding the enzyme. Although TALENs are considered superior to ZFNs in terms of fewer off-target effects, easy design and production, it was soon replaced by the CRISPR-Cas9 based genome editing technology, which is even more rapid and modular than the TALEN platform. Cas9 is an endonuclease playing a protective role against foreign nucleic acids in the adaptive immune system in bacteria. The feature of bacterial CRISPR immune system is that genetic materials taken up from previous invasive elements are expressed in crRNA, which could direct the Cas9 endonuclease to cut foreign DNA elements containing the same sequences (Jinek et al., 2012). Therefore, the CRISPR/Cas9 system has been remolded from bacterial immune system to the genome editing tool, using a designed RNA to guide Cas9 nuclease to the specific DNA sequence (Hsu et al., 2014). Binding of Cas9 nuclease on a specific protospacer adjacent motif (PAM) sequence on the genome (NGG for spCas9) will unwind the adjacent sequence, allowing the RNA:DNA pairing, which activates the nuclease domains in Cas9 to cut DNA and make double-strand breaks. While ZFNs and TALENs rely on protein-DNA recognition, which is less predictable for design and more labor and time consuming for assembly, the CRISPR/Cas9 system relies on the RNA-DNA recognition, which is much simpler and more predictable.

Because CRISPR-Cas9 technique is so efficient and powerful, it was quickly adopted to generate KO rabbits. In this respect, Chen's laboratory at the University of Michigan first established a number of KO rabbits aiming at studying human cardiovascular disease (Yang et al., 2014) and then KO rabbit boom started. Lai's laboratory at GIBH and Li's laboratory at Jilin University made more than 30 KO rabbits using CRISPR-Cas9 along with base-editing (Liu et al., 2018) and CRISPR/Cpf1 (Wu et al., 2018). Most KO rabbits were created in attempt to recapitulate human genetic or congenital disorders or immunodeficient rabbits (Song J. et al., 2017, 2018) as shown in . In addition, this technique has been tried to target the tyrosinase gene to modify rabbit coat colors (Honda et al., 2015; Song Y. N. et al., 2016, 2017, 2018). The standard protocol for generation of KO rabbits using CRISPR-Cas9 has been recently published (Yang et al., 2019). The techniques have been further refined (Liu et al., 2018, 2020) so we can predict that in the next few years, more and more KO or KI rabbits will be made using this technology. Here we will briefly review some valuable KO rabbits created recently to discuss their usefulness in disclosing the molecular mechanisms of atherosclerosis.

Table 2

ApoCIII KO Rabbits

ApoCIII is a major component of plasma chylomicrons and VLDLs, and is a minor component of high density lipoproteins (HDLs) and was first reported by Brown et al. 50 years ago (Brown et al., 1969). It is generally believed that physiological functions of apoCIII is to mediate the triglyceride(TG)-rich lipoprotein metabolism thereby maintaining the plasma TG homeostasis and high plasma levels of apoCIII are positively associated with plasma TG and increases the risk of ischemic heart disease (Huff and Hegele, 2013; Norata et al., 2015; Ramms and Gordts, 2018). However, for a long time, it is not clear whether apoCIII was directly involved in the pathogenesis of atherosclerosis because mouse models failed to provide a clear answer (Yan et al., 2020a). Yang et al. first generated apoCIII KO rabbits using ZNF (Yang et al., 2013) and after several years efforts to breed enough numbers of homozygous apoCIII KO rabbits, we were able to examine the hypothesis whether apoCIII may participate in atherosclerosis. Recently, we have shown that that genetic deletion of the apoCIII gene in KO rabbits significantly accelerates catabolism of TG-rich lipoproteins in the liver and apoCIII deficiency leads to the resistance of KO rabbits to a cholesterol diet-induced hyperlipidemia and inhibits atherosclerosis (Yan et al., 2020a). These results indicate that therapeutic inhibition of apoCIII expression may become a novel strategy for the treatment of hyperlipidemia and atherosclerosis.

ApoE KO Rabbits

ApoE is a ligand for both LDL receptor and LRP and plays an important role in the catabolism of remnant lipoproteins in the liver and genetic deficiency of apoE is a cause of human type III hyperlipoproteinemia (Mahley, 1988; Mahley et al., 1999). Deletion of apoE in mice even on a normal chow diet exhibited hyperlipidemia along with spontaneous aortic atherosclerosis (Plump et al., 1992; Zhang et al., 1992). ApoE KO rabbits were produced at University of Michigan using CRISPR-Cas9 (Yang et al., 2014) and Sage Company using ZFN (Ji et al., 2015), respectively. Even though different techniques were adopted, apoE KO rabbits generated by these two methods exhibit the same phenotypes (Niimi et al., 2016). Homozygous apoE KO rabbits on a normal diet only showed mild hyperlipidemia and their plasma total cholesterol levels reached ~200 mg/dL, similar to human type III hyperlipoproteinemia patients, whose cholesterol levels are elevated to 300~350 mg/dL (Mahley et al., 1999). Because plasma levels of cholesterol in apoE KO rabbits on a normal diet are not high to be atherogenic, there are not spontaneous atherosclerosis, which is different from apoE KO mice. However, when apoE KO rabbits were fed a cholesterol diet, they developed more prominent hypercholesterolemia than WT rabbits, which is basically caused by the remarkable accumulation of intestinally-derived remnant lipoproteins, β-VLDLs (Niimi et al., 2016). Recently, we found that apoE KO rabbits are highly susceptible to a cholesterol diet-induced atherosclerosis. Therefore, apoE KO rabbits will serve as a new model for human hyperlipidemia.

LDL Receptor KO Rabbits

In humans, genetic deficiency of LDL receptor functions causes severe hypercholesterolemia and atherosclerosis at early ages, called familial hypercholesterolemia (FH). FH is an autosomal dominant genetic disorder characterized by elevated plasma LDL levels due to LDL receptor dysfunctions (Soutar and Naoumova, 2007). Two laboratories have successfully generated LDL receptor KO rabbits using CRISPR-Cas9 (Yang et al., 2014; Lu et al., 2018). Similar to human FH, homozygous LDL receptor KO rabbits develop spontaneous hypercholesterolemia and atherosclerosis (Lu et al., 2018). Therefore, like WHHL rabbits, LDL receptor KO rabbits can be used for the study of human FH.

Cholesteryl Ester Transfer Protein KO Rabbits

Cholesteryl ester transfer protein (CETP) is a glycoprotein that transfers plasma lipids between HDLs and apoB-containing particles therefore plays an important role in lipoprotein metabolism. However, it is not known whether inhibition of CETP activity can prevent cardiovascular disease because four CETP inhibitors (torcetrapib, dalcetrapib, evacetrapib, and anacetrapib) failed to prove their efficacy in terms of reduction of cardiovascular risk by clinical trials( https://en.wikipedia.org/wiki/CETP_inhibitor). Since CETP is genetically absent in rodents (mice and rats) and pigs, rabbits are considered the best model for investigation of CETP functions because rabbits have high levels of CETP in the plasma as humans. Taking this advantage, Zhang et al. created CETP KO rabbits and found that CETP KO rabbits showed higher plasma levels of HDL-cholesterol (Zhang et al., 2017). When fed a cholesterol-rich diet, CETP KO rabbits still exhibited higher HDL-cholesterol levels accompanied by lower total cholesterol levels than wild-type (WT) rabbits (Zhang et al., 2017). CETP KO rabbits had significant less atherosclerosis in both aorta and coronary arteries than WT rabbits (Zhang et al., 2017). These results indicate that genetic ablation of CETP gene inhibits the development of atherosclerosis in cholesterol-fed rabbits.

ApoAII KI Rabbits

ApoAII is the second major apolipoproteins in HDLs. However, its physiological functions are largely unknown compared with apoAI. Interestingly, WT rabbits are genetically deficient in apoAII so their HDLs only contain apoAI. This unique feature makes WT rabbits as a "natural" apoAII KO model. We first made Tg rabbits expressing human apoAII gene and found that hepatic expression of human apoAII inhibits cholesterol diet-induced atherosclerosis (Wang et al., 2013). To examine the apoAII specific functions in the absence of apoAI, we further replaced the rabbit endogenous apoAI with human apoAII gene through knock-in (KI) using TALEN technology (Koike et al., 2021). In this way, apoAII KI rabbits expressed exclusively human apoAII without apoAI in HDL particles, which enables us to compare the net functions of apoAI-only-HDLs in WT rabbits with apoAII-only-HDL in KI rabbits in terms of HDL metabolism and atherosclerosis. In the latest study, we found that apoAII KI rabbits showed consistently lower TG and higher HDL-cholesterol levels and developed significantly less aortic atherosclerosis on a cholesterol diet (Koike et al., 2021).

Considerations and Future Perspectives

Although genetically modified rabbits are an important experimental model in cardiovascular research, they should not be simply used as a substitute of mice and rats, as discussed above. Because rabbits are more expensive, require larger space, and need more time to breed compared with mice, the generation of genetically modified rabbits should be carefully planned to solve those specific problems that cannot be well-examined in other experimental animal models, such as the development of lipid-lowering drugs (Niimi et al., 2020). However, off-target effects in these animals remain a concern as the genome editing is extremely productive and efficient. So far, almost all studies claimed that off-targets in genetically modified rabbits through genome editing are either none or negligible as comprehensively discussed in the recent review (Song J. et al., 2020); nevertheless, there is a need to performed careful genotyping, including sequencing, and expression validation of genetically modified rabbit models. It can be expected that more and more genetically modified rabbits will be made and used in a variety of medical sciences which will certainly expand our knowledge to explore new mechanisms of human diseases. Genome editing technique may eventually replace the pronuclear microinjection for the generation of Tg rabbits. However, complicated gene manipulation in rabbits, such as conditional KO in an organ- or cell-specific and time-controlled manner using the Cre/LoxP system is still lacking, thus it will be absolutely necessary to build such a platform in future. Finally, the preservation of valuable strains of genetically modified rabbits is an urgent task with increased number of rabbit models produced. In this aspect, various procedures for cryopreservation of rabbit sperm (Vicente and Viudes-de-Castro, 1996; Dalimata and Graham, 1997; Nishijima et al., 2015) and embryos (al-Hasani et al., 1992; Kasai et al., 1992; Marco-Jimenez et al., 2016) have been reported but have not been standardized. In the future, it may be necessary to establish an international rabbit bio-resource center or sperm and embryo bank to stock and share valuable rabbit models worldwide.

Author Contributions

JF, YW, and YC wrote the manuscript. All authors contributed to the article and approved the submitted version.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We would like to thank the following people for their contribution to this project. Jun Song, Jie Xu, Dongshan Yang, and Jifeng Zhang at the University of Michigan, Yajie Chen, Tomonari Koike, and Manabu Niimi at University of Yamanashi, and Shuji Kitajima and Fumikazu Matsuhisa at Saga University.

Glossary

Footnotes

Funding. This work was supported in part by the Research grant from Ono Medical Foundation, JSPS KAKENHI (JP15H04718), the National Natural Science Foundation of China (Nos. 81941001 and 81770457), the JSPS-CAS Bilateral Joint Research Program (JPJSBP 120187204), and NIH grant (HL117491, HL147527, and HL129778).

References

al-Hasani S., Hepnar C., Diedrich K., van der Ven H., Krebs D. (1992). Cryopreservation of rabbit zygotes. Human Reprod. 7(Suppl. 1), 81-83. 10.1093/humrep/7.suppl_1.81 [[PubMed] [CrossRef] Google Scholar]

Kitajima S., Morimoto M., Watanabe T., Fan J. (2003). Transgenic rabbits: production and applications for human disease models. Res. Dev. Biophy. Biochem. 3, 179-194. [Google Scholar]

Peng X. (2012). Transgenic rabbit models for studying human cardiovascular diseases. Comp. Med. 62, 472-9. [[PubMed] PMC free article] [Google Scholar]

# data[].attributes.attachment_link.title
Genetically Modified Rabbits for Cardiovascular Research

# data[].attributes.attachment_link.description
Rabbits are one of the most used experimental animals for investigating the mechanisms of human cardiovascular disease and lipid metabolism because they are phylogenetically closer to human than rodents (mice and rats). Cholesterol-fed wild-type rabbits were first used to study human atherosclerosis more than 100 years ago and are still playing an important role in cardiovascular research.

# data[].attributes.body
I used https://www.bartleby.com/essay/Pros-And-Cons-Of-Humanity-PJ4WEFKBR6. And I used google.

# data[].attributes.attachment_link.readable_text
Humans were created and placed on top to rule the Earth. As a species, there is only one race, which is the human race. Even through humans display a cruel and destructive nature, humans have the potential to transform into. The most important word for the human race to remember is "humanity". The word "humanity" defined in context to this essay is humaneness; benevolence. "Humanity" enables people to be compassionate, allows individualism, and incorporate ethnicity.
Humans are like coins, having two faces. On one side is the positive potential, but on the other side of is an abyss of darkness. Throughout history humans have been portrayed as creature of mass destruction, leaving their trail of despair as they pave their path to the future. The human race is plagued by sin: acts of transgression against divine law. In particular the seven deadly sins: Lust, Gluttony, Sloth, Wrath, Envy, and Pride. Humans are susceptible to the teachings of the evil. Humans lust for immoral sexual acts. The impurity of lust mutates one into "a servant of the devil". Humans overindulge in matters to the point of ruin. The amount of waste that humans conceive is heaping. Humans pollute themselves with the things they create. Greed is the desire of material possessions, which is more than ones needs. Humans strive to have everything money, clothes, cars, and etc. Sloth is a lack of interest in exertion comprised of states (primarily mentally and physically). Humans are lazy creatures that don't

# data[].attributes.attachment_link.description
Humans were created and placed on top to rule the Earth. As a species, there is only one race, which is the human race. Even through humans display a cruel and destructive nature, humans have the potential to transform into. The most important word for the human race to remember is "humanity".

# data[].attributes.body
First Fact : Papaya is also known as "papaw" in Australia and other parts of the world, but in the US, the papaw is a mango shaped fruit.

Second Fact : A papaya is brotonically, a berry. It grows from a tree, but is a fruit of a herb.

# data[].attributes.body
In my opinion, I think Papaya is a great GMO plant. It has more advantages than disadvantages. They reduce the cancer risks, which saves some lives. They're also very tasty & filled with nutrients. They can actually help with skin too, their are some downfalls to it, but it still a great GMO plant. I recommend having papaya. They're hard to transplant because of their width and length, but they're still very beneficial in multiple ways.

# data[].attributes.body
1. "There are things that go on in our bodies, things we have no idea about as we go on about our day. Be it your new found height in the morning or how many times your heart beats per day, here are 15 fascinating facts about your body."

2.  " The human body is a beautiful, wacky, and mysterious machine."

# data[].attributes.body
I used: https://www.nectarsleep.com/posts/fun-facts-about-the-human-body/

# data[].attributes.attachment_link.readable_text
There are things that go on in our bodies, things we have no idea about as we go on about our day. Be it your new found height in the morning or how many times your heart beats per day, here are 15 fascinating facts about your body.

Fun Facts About The Human Body - The human body is a beautiful, wacky, and mysterious machine. Here's the thing: we live with it, so sometimes it's easy to forget how amazing things like internal organs, eyes, or even the basic building blocks of life (cells) are, right? Here are 15 facts about the human body that are sure to get your brain going.

15. You're going to be taller in the morning

Measure yourself in the morning, then again at night. You're going to be taller in the morning because of how the cartilage in your bones compresses during the day. Thanks a lot, gravity!

Share

14. your heart beats about 100,000 times a day

Depending on your beats per minute, your heart beats about 100,000 times a day. That means it sends 2,000 gallons of blood through the body.

Share

13. Your highest blood flow is in your kidneys

The highest blood flow isn't in your heart, liver, or brain - it's in your kidneys. That's because kidneys are the body's natural filtration system.

Share

12. You can produce enough saliva to fill two bathtubs a year

Yikes!

Share

11. Healthy lungs are pink lungs

Healthy lungs look pink and rubbery on the outside. The more you smoke though, the more tarry and black they become. This can have a serious effect on quality of life, and can even cause bubbles (called blebs) rupturing in your lungs (a condition called spontaneous pneumothorax)

Share

10. Dreaming in black and white

12% of people dream in black and white. What's weirder is that human beings used to sleep and dream in black and white even more before color tv (~15%). The average person still dreams in color, but it's interesting to see how the human brain is susceptible to media.

Share

Share on facebook
Share on twitter
Share on linkedin
Share on pinterest

9. Your blood vessels could circle the globe

Though people have small blood vessels, the networking is amazingly long. If they were laid out they would measure more than 60,000 miles. Please don't do that though, it's good to keep your vessels in your body.

Share

8. The average human adult has 2000-4000 taste buds

They aren't only located on the tongue. These tiny sense organs that give us the ability to taste are also located in the back of your throat, your nose, and your esophagus. That's right, your nose doesn't just smell - it can taste too! The sense of smell is a very important component of tasting.

Share

7. Your body has more than 600 muscles

Your strongest muscle is located in your jaw known as the masseter muscle. The stapedius muscle, located in the middle of your ear, is your weakest muscle.

Share

6. In time your brain has a harder time with long-term memories

In time, your brain will have a harder time with long-term memory because the brain is unable to filter and remove old memories - which prevent it from absorbing new ideas. That means the brain has to work harder to produce memories over the average lifetime.

Share

5. Your skin has 1000 different species of bacteria on it

Your skin is vital in protection of the body against bacteria (the skin itself has 1000 different species of bacteria on it at any given time), which is why the outer layer of your skin continually renews itself. The entire process of skin cell renewal takes about 28 days.

Share

4. your strongest and longest bone is your femur

The smallest bone is one of the ossicles which are located in the ear. One quarter of your body's bones are located in your feet.

Share

3. your fingernails grow faster than your toenails

That's probably why you trim them more often.

Share

2. You're probably more like your dad

Researchers showed that in the scales between the female egg and male sperm that your genes tend to favor your dad, especially over multiple generations. This phenomena is known as "gene expression."

Share

Share on facebook
Share on twitter
Share on linkedin
Share on pinterest

1. Don't sweat these amazing facts

Don't sweat these amazing facts! You have between 2 and 5 MILLION sweat glands, and the more active you are, the more you sweat. That's because the body recognizes that you need to cool off so you can continue to work out. Stress sweat and regular sweat are composed of different things. Stress fat has fatty acids in it, where regular sweat doesn't. Weird to think how your brain controls that, huh?

There you have it. The body is a totally amazing thing. If you love this weird and wacky thing you call your body, then make sure you treat it right. Eat well, exercise, and make sure you get enough sleep! Take care of that body and it'll take care of you too.

# data[].attributes.attachment_link.title
Do You Know These Fun Facts About the Human Body?

# data[].attributes.attachment_link.description
There are things that go on in our bodies, things we have no idea about as we go on about our day. Be it your new found height in the morning or how many times your heart beats per day, here are 15 fascinating facts about your body.

# data[].attributes.body
https://foodprint.org/real-food/papayas/

# data[].attributes.attachment_link.readable_text
The origins of papaya ( Carica papaya) are lost to time. Even the definitive "The Oxford Companion to Food" says very little of its pre-Columbian history.

Believed to be native to Central America, Spanish invaders quickly took to the salmon-orange colored fruit and planted it throughout the Caribbean and South America. By the 17th Century, Dutch and Portuguese colonists had followed suit and brought papaya to Africa and then onto the Far East, where it became an integral part of the cuisines of Southeast Asia.
<<View All Real Food Encyclopedia Entries
What to Look for When Buying Papayas

Choose a papaya whose skin is slightly soft when you press into it, like an avocado. Avoid fruit with dark spots, overly soft areas or shriveled skin.

Papayas come in two shapes - the smaller, pear shaped Hawaiian variety and the bigger, oblong variety grown in Mexico and Central America and commonly found in supermarkets. On average they can weigh in around three to five pounds. Slice open the yellow-green papaya and at the center is a mass of distinctive black seeds with a gelatinous coating. The color of the fruit ranges from salmon hued to a deep, ruddy orange. In Asian markets and grocery stores you may find green papaya, which is distinctive for is green skin and hard, pale interior and white seeds.

Sustainability of Papayas

Papaya was one of the first fruits on the front lines in the debate over genetically engineered (GE) food. In 1998, it became the first transgenic fruit sold in the United States, and to this day papaya is a source of contention in Hawaii where a large percent of what's grown is GE and pollen drift has even cross contaminated presumably organic papayas. While the future of GE papaya is uncertain, especially in Hawaii, the genie is out of the bottle.

Papaya is also a thirsty plant, needing moist soil to thrive. The Water Footprint Network, which divides water into three types, calls this "green" water or the "volume of rainwater consumed during the production process." In fact, papaya has a water footprint of 55 gallons per pound. But when you compare papaya against other thirsty plants like coffee, chocolate, mango or even wheat, it has a relatively low water footprint.

Pesticides and Papayas

According to the Environmental Working Group, papayas are part of the " Clean Fifteen," meaning that of the fruits and vegetables it tested for pesticide residue, papaya was one of fifteen with the lowest chemical load. That's good news! Well, sort of. As a precaution against foodborne pathogens and invasive pests, papaya is now irradiated before export in some countries.

Eating Papayas

Storing Papayas

Papayas are highly perishable. Once they ripen, they won't last long unless refrigerated, and even then, no longer than a week or two.

Cooking with Papayas

Most people eat papaya raw. All you have to do is slice up the fruit into cubes, discarding the skin and seeds. You can even dress it with lime juice and a sprinkling of cayenne pepper. Fancy! In Thailand, they shred green papaya raw and serve in a salad called Som Tam with fish sauce, lime, sprouts, vegetables and fresh herbs. If you're feeling adventurous, you can dry and grind papaya seeds and use as a black pepper substitute. Other uses for papaya include folk remedies to treat wounds, bites and Dengue fever.

Preserving Papayas

The easiest way to save your papaya for later is to freeze in cubes, which can then be added to smoothies. But there is a long tradition in warmer climes of making papaya into jam and papaya pickles. Try dried papaya for a healthy snack when traveling.

Papaya Nutrition

Papaya is rich with vitamins, minerals and reduces inflammation. In fact, a cup of raw papaya contains 150 percent of the recommended daily intake of Vitamin C as well as 31 percent of Vitamin A and 10 percent of potassium. Papaya is high in beta carotene, which has been known to cause carotenemia, a harmless and temporary yellowing of the skin if you eat a ton of it. Additionally, papaya contains latex and may cause a reaction in those who are allergic.

# data[].attributes.attachment_link.description
The origins of papaya ( Carica papaya) are lost to time. Even the definitive "The Oxford Companion to Food" says very little of its pre-Columbian history. Believed to be native to Central America, Spanish invaders quickly took to the salmon-orange colored fruit and planted it throughout the Caribbean and South America.

# data[].attributes.body
https://www.everydayhealth.com/diet-nutrition/diet/papayas-nutrition-benefits-risks-how-eat-more/

# data[].attributes.attachment_link.title
Papaya 101: Nutrition, Benefits, Risks, How to Eat, More | Everyday Health

# data[].attributes.attachment_link.description
Papaya is a yellow-orange fruit with edible seeds. Papaya's benefits include lowering blood sugar, reducing inflammation, and helping with weight loss. Learn about papaya fruit here: what's in it, how to prepare and eat it, and who should avoid it!

# data[].attributes.body
1. Human interactions give rise to the formation of different kinds of opinions in a society. The study of formations and dynamics of opinions has been one of the most important areas in social physics. The opinion dynamics and associated social structure leads to decision making or so called opinion consensus

2.   Opinion formation is a process of collective intelligence evolving from the integrative tendencies of social influence with the disintegrative effects of individualisation and therefore could be exploited for developing search strategies. Here, we demonstrate that human opinion dynamics can be utilised to solve complex mathematical optimization problems

# data[].attributes.body
The need of humans that this would meet is that it would be genetically modified for different taste buds, like different genes for the taste. However, the gene for vanilla is genes encoding a phenylalanine ammonia-lyase (VpPAL1) and a cinnamate 4-hydroxylase (VpC4H1) were identified in vanilla pods and studied during maturation. Which the gene would have to change in order for the cake to taste like cabbage. This can be done by finding the genes for cabbage and the genes for cake and combining them together like breeding!!
Then, the cake would then have a different taste which would be cabbage for our situation.

# data[].attributes.body
1 Pro - You will get to try something different and it would probably make a impact on new food. Which would help other people like new things for those who argues about picky people, they may like this!!!

1 Con - It could make someone sick or hurt them. It could not turn out as a good food item and get banned from the state, and be warned to not try this out because it could kill/sicken people in our world.