The Study Of Construction Rabbit Hgprt Gene-targeting Vector And Expression Of Gfp Gene In Transgenic Fibroblast

Compared to mouse, rabbits possess characters of physiology, anatomy and evolution closer to human race. Rabbits have a relatively less breeding cost, making them suitable for studies of the cardiovascular, pulmonary and metabolism diseases. Thus, gene-modified rabbit model should find wide applications and market value in medical research. Gene targeting is an approach to introduce targeted changes into the genome which is based on the principle of homologous recombination. Combined with the somatic cell nuclear transfer technology, it has increased an original strategy to research gene functions in vivo and establish experimental animal models associated with human disease.This study is dedicated to smooth the way of establishing the HGPRT-knockout rabbit model. The work includes two parts. In the first part, rabbit full length HGPRT gene BAC clone LBNL1-304M19 is used as the template. A 12.5kb rabbit HGPRT gene fragment, which does not include promoter, is cloned into pKS-plasmid to form pKS-HGPRT recombinant plasmid via Gap-Repair by Red recombination system. Then construct a GFP and neo selective targeting vector pKS-HGPRT-GFP-neo on the basis of pKS-HGPRT and pEGFP-C1-SD1211 plasmids. Linearized targeting construct DNA was introduced into the rabbit fibroblasts by LipofectamineTM 2000. The positive-negative selection was performed and survival clones were screened by PCR and sequencing, and eight colonies with homologous recombination were obtained. The targeted colonies would further confirmed by Southern blotting. Then,culture the transgenic fibroblast using four different levels of G418,such as 0μg/mL,200μg/mL,400μg/mL,600μg/mL.Afterwards,we study the expression of exogenous genes in fibroblast through analyzing cell cycle, counting chromatosome,flow cytometry.The studies are succeeded in constructing HGPRT expression plasmid with the gene of GFP,the studies even discover that the expression level of GFP is becoming lower in the transgenic fibroblast cells during the process of serial subcultivation.These experiments form the basis for studying HGPRT-associated diseases, exploring the gene targeting in rabbit fibroblast cells and embryonic stem cells, generating the HGPRT-knockout rabbit model. The results set a good basis for the establishment of HGPRT-knockout rabbit by gene target and nuclear transfer methods.