微小RNA簇miR-302s条件性基因打靶小鼠的制备和鉴定  

Generation and Identification of miR-302s Conditional Gene Targeting Mice

作　　者：余飞[1] 钱丽萍[1] 丁利军[2] 

机构地区：[1]南京大学医学院附属鼓楼医院动物实验中心,南京210008 [2]南京大学医学院附属鼓楼医院生殖医学中心,南京210008

出　　处：《实验动物与比较医学》2016年第2期87-92,共6页Laboratory Animal and Comparative Medicine

基　　金：国家自然科学基金项目（30900847）,中央高校苗圃项目（20620140707）,南京市卫生杰出青年人才项目（JQ2014004）

摘　　要：目的制备微小RNA簇miR-302s条件性基因打靶小鼠。方法构建miR-302s打靶载体，通过电穿孔的方法转染胚胎干细胞（ES细胞），用正负筛选法筛选阳性Es细胞并进行PCR鉴定和Southern分析。利用显微注射技术将正确同源重组的ES细胞注射至B6（Cg）-Tyr〈sup〉c．2J〈／sup〉／J小鼠囊胚腔内，注射后的囊胚植入受体小鼠子宫。将得到的嵌合体小鼠与野生型雌鼠交配获得miR-302s-LoxP基因打靶小鼠。结果获得嵌合体小鼠11只，其中4只嵌合雄鼠嵌合率〉50％。利用嵌合体小鼠进行繁育交配，得到miR-302s基因打靶杂合子小鼠11只。结论成功建立miR-302s条件性基因打靶小鼠模型，为进一步研究miR-302s基因功能打下基础。Objective To generate microRNA miR-302s gene targeting mice. Methods The conditional gene targeting vector were generated by molecular cloning, and then the electroporation of embryonic stem （ES） cells were carried out. The targeted ES cells were screened by positive-negative selection and identified by PCR and Southern blot, and then the correct targeted ES cells were microinjected into blastula of B6（Cg）-Tyr〈sup〉c-2J〈/sup〉/J mice. Chimerical mice were generated after transplantation of the injected blastula into the host mice, and the miR-302s-LoxP gene recombinat mice were obtained after breeding. Results Eleven miR-302s chimeric mice were obtained including four male chimeras with a higher than 50% chimeric ratio. Eleven miR-302s heterozygous gene recombinat mice were generated after outbred and inbred. Conclusion The miR-302s conditional gene targeting mice were successfully obtained. This animal model provided the foundation for further study of miR-302s in vivo.

关 键 词：miR-302s 条件性基因打靶小鼠 胚胎干细胞 同源重组 

分 类 号：Q95-33[生物学—动物学]