小鼠性激素结合球蛋白条件基因打靶载体的构建

摘要

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.%目的：构建小鼠性激素结合球蛋白（Shbg）条件基因打靶载体。方法运用ET克隆的方法构建Shbg基因第4⁃7号外显子两侧插入loxp位点的条件性剔除载体。首先以BAC DNA为模板克隆出Shbg及其上、下游基因并打靶质粒载体（pBR322⁃MK⁃AB），利用该质粒和BAC克隆，通过同源重组方式，获得套取质粒（pBR322⁃Shbg⁃Re）；再分别以PL452及PL451为模板扩增出含Neo片段，经过再一轮的Red/ET重组成功实现条件性基因敲除打靶载体（pBR322⁃MK⁃SHBG⁃cko）的构建。结果经多个限制性内切酶酶切鉴定和测序证实，构建的pBR322⁃MK⁃AB、pBR322⁃Shbg⁃Re、SHBG⁃Ln重组质粒和Shbg条件基因打靶载体（pBR322⁃MK⁃SHBG⁃cko）结构正确，与设计相符。结论成功构建了小鼠Shbg条件基因打靶载体，为后续Shbg基因条件敲除小鼠模型的构建奠定了基础。