Construction of Targeting Vector for Conditional Knockout of Murine Shbg / 中国医科大学学报

ABSTRACT

Objective To construct the targeting vector for conditional gene knockout of sex hormone?binding globulin(Shbg)in mice. Methods Based on Red/ET,two LoxP were inserted into both sides of extron 4 and extron 7 for conditional gene knockout of murine Shbg. Firstly,the Shbg gene and its upstream,downstream genes obtained from BAC DNA by PCR were cloned into plasmid pBR322?MK,which was named pBR322?MK?AB. The retrieve plasmid(pBR322?Shbg?Re)was obtained by homologous recombination between the plasmid and BAC.Then a great quantity of Neo fragments obtained from PL452 and PL451 were inserted into the targeting vector after another round of Red/ET and then the final targeting vec?tor(pBR322?MK?SHBG?cko)was achieved. Results The correct structures of the targeting vectors such as pBR322?MK?AB,pBR322?Shbg?Re, SHBG?Ln and pBR322?MK?Shbg?cko were confirmed by restriction enzyme digestion and sequencing analysis. Conclusion The targeting vector for conditional knockout of murine SHBG was successfully constructed. The construction of targeting vector paved the way for conditional knockout mouse strain generated by targeted mutation of Shbg.