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) 

·        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.

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