Abstract: Objective To construct a knockout HPRT targeting vector and a promoterless knockin targeting vector,which can be used for making rabbit HPRT gene knockout models and transgenic rabbit in the future. Methods The rabbit full length HPRT gene BAC clone LBNL1-304M19 is used as the template. A 12.5kb rabbit HPRT gene fragment，which does not include promoter, is cloned into pKS-plasmid to form pKS-rHPRT recombinant plasmid via Gap-Repair by Red recombination system. Then, the PCR was used to obtain the IRES-eGFPCre-Frt/Neo/Frt fragments flanking with 50bp homologous arms for homologous recombination of rabbit HPRT gene on the basis of pKS-rHPRT and pIGCN21 plasmids. A knockout HPRT taigeting vector by replacing the last three exons with a IRES-eGFPCre-Frt/ Neo/Frt cassette and a promoterless knockin targeting vector by inserting a IRES-eGFPCre-Frt/Neo/Frt cassette in the 3’UTR of the HPRT gene were rapidly constructed. Results The identification of the vectors with PCR,enzyme restriction and sequence showed two vectors were constructed successfully. Conclusions We rapidly constructed a knockout HPRT taigeting vector and a promoterless knockin targeting vector, the efficiency of deleting or inserting DNA fragment by homologous recombination technology has also been studied.

Key words: Red recombination, PCR technology, HPRT gene, Targeting-vector