Abstract
The rabbit is a common animal model that has been employed in studies on various human disorders, and the generation of genetically modified rabbit lines is highly desirable. Female rabbits have been successfully cloned from cumulus cells, and the somatic cell nuclear transfer (SCNT) technology is well established. The present study generated hypoxanthine phosphoribosyltransferase (HPRT) gene knockout rabbits using recombinant adeno-associated virus-mediated homologous recombination and SCNT. Gene trap strategies were employed to enhance the gene targeting rates. The male and female gene knockout fibroblast cell lines were derived by different strategies. When male HPRT knockout cells were used for SCNT, no live rabbits were obtained. However, when female HPRT+/− cells were used for SCNT, live, healthy rabbits were generated. The cloned HPRT+/− rabbits were fertile at maturity. We demonstrate a new technique to produce gene-targeted rabbits. This approach may also be used in the genetic manipulation of different genes or in other species.
Highlights
Editing systems[14,15,16,17,18,19]
The recombinant adeno-associated virus (rAAV)-hrGFP vector was packaged into 293A cells, and GFP fluorescence was identified in most cells (Supplementary Fig. S1)
GFP fluorescence was identified in approximately 40% of fibroblasts (Supplementary Fig. S1), which indicated that rabbit fibroblasts are susceptible to rAAV infection and that our targeting protocol was effective
Summary
Editing systems[14,15,16,17,18,19]. each genetic manipulation technique has different benefits and disadvantages[20,21], establishing gene knockout animals via homologous recombination is still desirable because it could facilitate both the expression of an exogenous protein using an endogenous promoter and the development of a strategy for generating conditional gene knockouts. RAAV vectors were first successfully used in site-specific genetic modification[29] to target transgene insertion into the chromosomes of cultured human cells[28] This rAAV vector delivery system was well established for CFTR knockout in pigs[12] and ferrets[13]. HPRT gene knockout fibroblast cell lines of both genders were derived using different strategies and employed in SCNT, and healthy HPRT gene knockout rabbits were obtained from the female lines. This approach may be used to facilitate the efficient manipulation of different genes or in other species
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