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Multiple factors should be considered when designing a strategy focused on generating a broad antibody response. These include (1) preparation and presentation of the immunogen, (2) choice of adjuvant, (3) immunization method, and (4) the impact of T-cell tolerance.

When preparing immunogens, we focus on strategies that are the least likely to disrupt the native structure of the antigen and destroy important epitopes. Our goal is to produce at least three orthogonal types of immunogens for each target: soluble protein, cells expressing the target on the membrane, and DNA expression constructs

adjuvants are absolutely required for generating a robust immune response and diverse antibody repertoires in XenoMouse

Genetic immunization offers many advantages compared with traditional methods. Minimally, it requires only plasmid DNA encoding the antigen of interest which, upon delivery, is taken up by cells and expressed in vivo. The simplicity and native-like expression of this method increases the probability that the natural conformation of the antigen is maintained. This strategy is particularly attractive for complex membrane proteins

we have exploited observations that tolerance can often be overcome by adding T-cell epitopes (TCEs) to antigens.

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1. Overexpression of PD dominant traits (LRRK2 and α-Syn) or knockout of dominant-negative genes (GBA and VPS35) in fly has been consistently demonstrated to mimic the essential PD signs such as DA neurodegeneration and beha- vioral deficits.

2. missense mutation A53T in the SNCA gene and the insoluble aggregated α-Syn forms as the major component of LBs, a pathological hallmark of PD, heralded a new era in PD research

3. the combination roles of the genetic and environmental factors such as oxidative stress plays a role in progression of parknsons disease

4. It has shown that sorafenib, curcumin, or GW5074 significantly suppressed LRRK2 PD-like phenotypes in Drosophila

5. Geldamamycin, an Hsp90 inhibitor and chaperone inductor, was able to protect α-Syn-expressing neurons in Drosophila (104). Nicotinamide, the principal form of niacin (vitamin B3), has been demonstrated to improve the motor dysfunction in α-Syn transgenic flies through improvement of oxidative mitochondrial dysfunction

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1. The use of sitespecific recombinases and integrases for targeted transgene insertion into an animal genome has a regulatory significance in the case of transgenic animals intended for practical use, due to the significant simplification of the characterization of the transformation event

2. CRISPR/Cas9 system was a revolutionary breakthrough in this field, due to the simplicity of its practical implementation in combination with high efficiency.

3. Using mutant Cas9 nuclease (nCas9, nickase) introducing distant single-strand DNA breaks into the plus and minus chains of a target genomic locus

4. NHEJ-based repair of breaks by ligating the complementary overlapping single-strand DNA ends of the genomic target site and the repair template comprising a transgene, which are generated by a ZFN nuclease pair upon cleavage of the targeted sequences of the genome and repair template.

5. CRISPR/Cas9 technology enables the generation of transgenic animals with transgene integration into a specified genomic site; together with the use of homologous recombination.

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1. Regularly immunize large cohorts of mice to maximize exploration and sampling of the theoretical antibody sequence space.

2. Toll-like receptor (TLR) agonists have received significant attention because of the important role of TLRs in the activation of innate signaling pathways

3. Genetic immunization offers many advantages compared with traditional methods. Minimally, it requires only plasmid DNA encoding the antigen of interest which, upon delivery, is taken up by cells and expressed in vivo. The simplicity and native-like expression of this method increases the probabil- ity that the natural conformation of the antigen is maintained.

4. Exploited observations that tolerance can often be overcome by adding T-cell epitopes (TCEs) to antigen.

5. Using human-mouse chimeras and grafting the ECLs of multipass membrane proteins onto related proteins that share a common structure with the target of interest

6. Design an immunization campaign that alternates between the human and orthologous antigen to steer the antibody repertoire toward functional antibodies that cross-react to both.

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1. Targeted genome editing technologies significantly increase the efficiency in transgene integration into a pre-determined site of the genome, which, in some cases, eliminates the use of selective markers and, most importantly, enables highly efficient targeted transgene integration directly into the genome of a zygote, followed by the generation of transgenic animals

2. Transgenic animals can serve as bioreactors for the synthesis of the recombinant proteins secreted into milk, which enables the production of recombinant proteins in substantially larger amounts and at much lower costs than the production of proteins in eukaryotic cell cultures

3. Promoters that enable production of the target protein at a sufficiently high level in milk (up to tens of grams per liter of milk) include promoters of goat and cow β-casein, cow α-s1-casein, rabbit whey acidic protein (WAP), human α-lactalbumin, and sheep β-lactoglobulin genes

4. The use of so-called “safe harbors” as transgene integration sites which are genomic loci that are, on the one hand, insignificant for the development and functioning of the organism, which enables harmless transgene insertion into this locus and, on the other hand, provide a high level of transgene expression in the presence of appropriate regulatory elements in transgene

5. Promoters of one animal species can provide effective transcription of the transgene in the mammary gland cells of another animal species due to conservatism of the transcription factors regulating the production of milk proteins in mammary cells

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Drosophila models have provided significant contributions to our understanding of the mechanisms of PD pathogenesis in a comparatively short time frame and cost effective mode.

The genetic LRRK2 Drosophila models provide promising in vivo platforms for inhibitor identification and validation, and drug development

Protein quality control systems, oxidative stress, mitochondrial function, and DA biosynthesis pathways are potential targets for developing therapeutic agents for α-Syn toxicity

Overexpression of PD dominant traits (LRRK2 and α-Syn) or knockout of dominant-negative genes (GBA and VPS35) in fly has been consistently demonstrated to mimic the essential PD signs such as DA neurodegeneration and behavioral deficits.

One of the limitations is drosophila is a relatively simple model organism, far less complex brain circuit than humans

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·        the number of B cells in a mouse at any given moment is 10₈, and the theoretical diversity of the human antibody repertoire exceeds 10₁₁, it is reasonable to conclude that an individual mouse can only harbor a fraction of the potential human repertoire

·        adjuvants are absolutely required for generating a robust immune response and diverse antibody repertoires in XenoMouse

·        Genetic immunization offers many advantages compared with traditional methods. Minimally, it requires only plasmid DNA encoding the antigen of interest which, upon delivery, is taken up by cells and expressed in vivo. The simplicity and native-like expression of this method increases the probability that the natural conformation of the antigen is maintained. This strategy is particularly attractive for complex membrane proteins

·        The ability to overcome immune tolerance remains a central problem for antibody discovery using transgenic platforms. It is shown from the results that tolerance can often be overcome by adding T-cell epitopes (TCEs) to antigens.

·        using human-mouse chimeras and grafting the ECLs of multipass membrane proteins onto related proteins that share a common structure with the target of interest is shown to be successful. Grafting the target loops onto a scaffold that retains key intramolecular interactions raises the likelihood that the correct conformational epitopes are retained.

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CLASSICAL TRANSGENESIS OF ANIMALS AND THE GENETIC ELEMENTS REQUIRED FOR THE PRODUCTION OF RECOMBINANT PROTEINS

the transgene is randomly integrated into the recipient’s genome; in this case, the number of integrated transgene copies, including incomplete transgene fragments, is uncontrolled. The key determinant that provides tissue specificity in transgene expression is the promoter. Inclusion of introns into the transgene.

ALTERNATIVES TO CLASSICAL TRANSGENESIS OF ANIMALS

SCNT technology is used to produce transgenic animals by replacing the oocyte nucleus with somatic cell nucleus carrying the genetic modification and inducing embryo development. Disadvantages are Animals are often unviable and die prematurely, which is associated with the side effects of somatic cell nuclear transfer, in particular with defects in the development of extraembryonic tissues and epigenetic reprogramming.

Embryonic Stem cells used to generate a genetically modified animal, stem cells carrying a genetic modification are injected into blastocysts, with their subsequent implantation and creation of transgenic mosaic animals. Disadvantages are potential loss of pluripotent properties by stem cells upon generation of genetically modified clones during cultivation.

SITE-SPECIFIC RECOMBINASES FOR TARGETED TRANSGENE INSERTION INTO THE GENOME

the concept of usage of site-specific recombinases is based on the generation of a line of transgenic animals carrying recognition site(s) for recombinase in the genome. lines of transgenic animals with different variants of transgene localization in the genome are used for selection, where lines with the transgene integrated into the genomic site that provides the required properties of transgene expression are selected. This animal line is then used as a universal recipient for the insertion of different transgenes into a specific genomic site via site-specific recombination

APPLICATION OF TARGETED GENOME EDITING TECHNOLOGIES IN ANIMAL TRANSGENESIS

Targeted transgene integration using site-specific nucleases is based on a significant increase in the site-specific efficiency of transgene integration into the recipient genome during the repair of double- or single-strand DNA breaks. There are several classes of artificial nucleases that are used for targeted genome editing and the production of transgenic animals such as Zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), artificial meganucleases and hybrid artificial nucleases. Lastly, the promising one which is CRISPR/Cas9 system. In the CRISPR/Cas9 system, the target is recognized as a consequence of the complementary interaction between CRISPR RNA (crRNA)

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- The Drosophila Gal4/UAS system is a powerful tool for targeted transgene expression and has been used to direct selective expression of mutant PD genes.

- Overexpression of PD dominant traits (LRRK2 and α-Syn) or knockout of dominant-negative genes (GBA and VPS35) in fly has been consistently demonstrated to mimic the essential PD signs such as DA neurodegeneration and behavioral deficits.

- Drosophila models as platforms to identify potential therapeutic compounds for PD.

- Drosophila models so far are one of the major model systems to study GBA and LRRK2 functions in PD.

- The combination roles of the genetic and environmental factors such as oxidative stress have been explored in PD through Drosophila models.