With the advent of endonuclease methods of genome editing, particularly CRISPR/Cas9, it has become possible to obtain genetically modified rabbits by microinjection of zygotes. These highly effective human disease models can be used for various purposes. The present review aims to consider modern achievements in the creation of rabbit biomodels of human diseases using the technologies of genetic editing. It is concluded that Russian laboratories should intensify research in the development of genetically modified rabbits that can be used for various biomedical studies and biomodelling.
The survival rate of rabbit embryos microinjected by the plasmid form of CRISPR/Cas9 components specific to the sour whey protein gene was evaluated. At high concentrations of plasmid components, embryo survival decreased slightly, possibly because the WAP gene does not belong to the housekeeping genes. After microinjection of a genetic construct with a sequence of green fluorescent protein under a cytomegalovirus promoter, the embryo survival significantly decreased. This is most likely due to the superexpression of GFP at the 2-16 cell stage of development.
The high content of whey acidic protein in rabbit milk makes the gene of this protein a promising candidate for its replacement by the gene of pharmacologically active protein using the CRISPR/Cas9 system. The plasmid that contains 5’ and 3’ arms of homology to the rabbit WAP gene was created. A fragment containing a green fluorescent protein gene under the CMV promoter has been integrated into this site. A strategy of making double-stranded cuts in the gene WAP and receiving four pX330 plasmids encoding the endonuclease Cas9 and guide RNAs was developed. The plasmid containing a fragment cmvEGFP was designed for site-specific integration by homologous recombination into the gene WAP to assess the effectiveness of site-specificity of components of the CRISPR/Саѕ9 in vitro.
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