Abstract: Objective To construct a rabbit HPRT gene targeting vector. Methods The 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 recombi-nant plasmid via Gap-Repair by Red recombination system. Then we construct a GFP and neo selective targeting vector pKS-HGPRT-GFP-neo on the basis of pKS-HGPRT and pEGFP-Cl-SD1211 plasmids.Results Linearized targeting construct DNA was introduced into the rabbit fibroblasts by Lipofectamine^TM 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. Constructions The knockout HPRT targeting vector was rapidly constructed and the results set a good basis for the establishment of HGPRT-knockout rabbit by gene target and nuclear transfer methods.

Key words: Knockout, Rabbit fibroblast, HGPRT, Cell isolation