剔除护骨素基因的小鼠胚胎干细胞杂合子模型的建立
基因敲除；胚胎干细胞；护骨素；骨质疏松,,],基因敲除；胚胎干细胞；护骨素；骨质疏松,1材料与方法,2结果,3讨论,[参考文献]
[摘要] 目的 建立护骨素(OPG)基因剔除小鼠胚胎干细胞杂合子模型，为建立用于研发抗骨质疏松药物的基因剔除动物模型奠定基础。方法 PCR方法扩增两条同源臂，将两条用于同源重组的片段插入XpPNT载体，构建OPG基因敲除打靶载体。线性化及纯化以后通过电穿孔转导小鼠胚胎干细胞，并用G418和Gancyclovir进行双药筛选培养，得到双药抗性克隆后抽提基因组DNA，采用PCR和southern杂交方法确定同源重组的胚胎干细胞克隆。 结果 经双药筛选得到124个双药抗性克隆，PCR和southern杂交鉴定获得一个发生同源重组的胚胎干细胞克隆。结论 总之，本研究成功获得了OPG(-/+)杂合子小鼠胚胎干细胞克隆，为进一步通过显微注射及交配育种获得OPG基因剔除小鼠，在活体水平研究OPG基因的功能打下基础，同时也使建立用于研发抗骨质疏松药物的基因剔除动物模型成为可能。[关键词] 基因敲除；胚胎干细胞；护骨素；骨质疏松
Establishing the heterozygous model of OPG gene knockout ES cell
QIAO Jianou,ZHOU Longnv,NING Guang,et al.The 9th Peoples Hospital Affiliated to Shanghai Jiaotong University,Shanghai 200232,China
[Abstract] Objective To obtain the homologous recombinant embryonic stem cell (ES cell )with their OPG gene knocked out. Methods A targeting vector pointing to OPG exon 2 and a little part of intron 2 was built with 3.3kb Xbal/BamHI fragment as short arm and 3.9kb NotI/SalI fragment as long arm after two homology arms was got by PCR.The linearized and purified targeting vector DNA was transfected into ES cells through electroporation and then some of these ES cell lines survived G418 and Gancyclovir selection.Twodrugresistant clones were expanded and identified by PCR and Southern blot. Results Totally 124 twodrugresistant clones were harvested and one of them was confirmed as positive. Conclusion The result of this experiment made basis for the further establishment of the OPG knockout mouse model and the deeper study of OPG in vivo. ......
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