小鼠LRP16基因打靶载体的构建和胚胎干细胞同源重组克隆鉴定  

Construction of LRP16 Gene Targeting Vector and Identification of Homologously Recombinant Clone of Embryonic Stem Cells

作　　者：伍志强[1] 韩为东[1] 赵亚力[1] 司艺玲[1] 母义明[1] 孟元光[1] Masatoshi Nomura 

机构地区：[1]解放军总医院,北京100853 [2]日本九州大学医学院第三内科

出　　处：《中国实验动物学报》2006年第3期167-171,共5页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金(编号:30471813);国家自然科学基金(编号:30471990);北京市自然科学基金(编号:7052061)

摘　　要：目的既往研究表明LRP16基因是一个雌激素反应基因,其表达水平与乳腺癌细胞增殖及侵袭密切相关。为对该基因进行生理功能研究,本文构建了针对小鼠LRP16基因的打靶载体,转染胚胎干细胞(embryonicstem cell,ES cells)并筛选同源重组克隆。方法PCR方法筛选129品系小鼠基因组文库中LRP16克隆,在第5外显子内插入SA-RIES-βgeo序列,构建插入失活型打靶载体并转染ES细胞,经G418筛选,挑取抗性克隆,Southern blot方法鉴定同源重组ES细胞克隆。结果将包含第5至11外显子的LRP16基因组片段亚克隆到pBluescript SKⅡ+载体,SA-IRES-βgeo序列的正确插入第五外显子中,打靶载体成功转染ES细胞,Southern blot结果显示具有一个打靶序列同源重组型插入的ES细胞克隆。结论成功构建了第五外显子插入失活型LRP16基因打靶载体并筛选到同源重组型ES细胞,为下一步建立LRP16缺失型小鼠并为从整体水平研究LRP16基因的生理功能奠定基础。Objective Previous studies have demonstrated that LRP16 is an estrogen-responsive gene, its expression level is closely related with proliferation and invasive growth of human breast cancer cells. To further explore the physiological function of LRP16, here, we constructed a LRP16 targeting vector and got an embryonic stem （ES） cell clone with inactive LRP16 gene. Method Targeting sequence of LRP16 gene was obtained from 129 mouse genomic Bacterial Artificial Chromosomes Library based on polymerase chain reaction screening. The SA-RIES-βgeo fragment was designed to insert within the LRP16 fifth exon to inactivate LRP16. ES ceils were screened with G418 and the homologously recombinant clone was identified by Southern blot analysis. Results The LRP16 fragment including exons 5 to 11 was then subcloned into the pBluescript SK Ⅱ vector. Restriction map demonstrated that the SA-IRES-βgeo fragment was correctly inseted into the LRP16 fifth exon. Southern blot results showed that there was an ES clone with targeting sequence homolougously inserted. Conclusion A LRP16 gene-targeting vector was constructed and a homologous recombinant clone of ES cells is obtained. The study laid a foundation for preparing LRPI6 knockout mouse models.

关 键 词：LRP16 基因打靶 ES细胞 

分 类 号：R349.6[医药卫生—基础医学]