利用新型DNDF7和携带LoxP位点的LPL4质粒快速构建S-Sox5基因打靶载体

Construction of a knockout vector targeting S-Sox5 gene using DNDF7 vector and a novel LPL4 plasmid carrying two LoxP sites

目的体外实验已证实S-SOX5蛋白转录调节运动纤毛基因Spag6，为进一步探索其在体内的作用，拟构建S-Sox5基因的敲除载体。方法利用新型DNDF-7和携带LoxP位点的LPL4载体，选择S-Sox5基因第1外显子作为条件性敲除目的片段，SV129系Es细胞DNA为模板分步扩增包括S-Sox5第1外显子的中间段（middle piece），上游同源左臂4．8kb（upper arm）及下游同源右臂2．5kb（down arm）的基因片段；构建upper-arm-DNDF7、down-arm-DNDF7和middle-piece-LPL4质粒，将middle-piece-LPL4的酶切产物LoxP—middle—piece．LoxP和down-arm-DNDF7酶切产物相连，形成携带LoxP位点的middle-down-arms-DNDF7重组质粒，该质粒与upper-arm-DNDF7酶切产物相连，获得S-Sox5基因打靶载体。结果经酶切鉴定及测序证实，构建的upper-arm-DNDF7、middle-piece-LPL4、down-arm-DNDF7重组质粒和S-Sox5基因打靶载体结构正确，与设计相符。结论成功构建小鼠S-Sox5条件基因打靶载体，为进一步建立S-Sox5条件敲除小鼠，在体内研究其功能打下基础。

Objective It has been shown that S-SOX5 transcriptionally regulates sperm associated antigen 6 （Spag6）, a gene essential for ciliary function. The aim of this study is to construct a knockout vector targeting S-Sox5 gene to investigate its function in vivo. Methods The DNDF7 vector and the LPIA vector carrying two LoxP sites were used to generate the S-Sox5 targeting vector. Briefly, exon 1 of S-Sox5 was selected as the targeting region （middle piece）, and DNA from ES cells （SV129 background） was used as a template to amplify upper arm and down arm flanking the targeting region. The middle piece was cloned into LPL4 vector. Then, the middle piece, together with the two LoxP sites were released from the LPIA vector and ligated to DNDF7 plasmid. The upper arm and down arm were also cloned into DNDF7 vector subsequently to create the final construct targeting exon 1 of S-SoxS. Results The S-Sox5 KO vector was confirmed by series digestion with restriction enzymes and DNA sequencing. Conclusion Successfully constructing the S-Sox5 KO vector provides a tool to make a floxed S-Sox5 mice model and study the role of S-Sox5 in specific cells/tissues in vivo.