小鼠LRP16基因打靶载体的构建和同源重组型胚胎干细胞筛选(英文)

Construction of a LRP16 gene targeting vector and screening of homologously recombinant clone of embryonic stem cells

背景: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。目的:构建针对小鼠LRP16基因的打靶载体,转染胚胎干细胞(EmbryonicStemCell)并筛选同源重组克隆。设计:通过SA-RIES-βgeo插入小鼠LRP16基因组DNA构建插入失活型打靶载体。单位:日本九州大学第三内科生物调控研究室与解放军总医院分子生物室。材料:本实验所用主要材料由日本九州大学生物调控研究室提供。实验所用鼠基因组文库(mousegenomiclibraryinpBeloBAC11Vector)购自invitrogen公司,TopF10感化态细菌购自北京天为时代公司,pCDNA3.1(+)由本室保存。鼠胚胎干细胞株由日本九州大学提供。方法:实验主要工作于2004-11/2005-05在日本九州大学生物调控研究室与解放军总医院分子生物室完成。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胚胎干细胞,经G418筛选,挑取抗性克隆,Southernblot方法鉴定同源重组胚胎干细胞克隆。主要观察指标:具有同源重组的胚胎干细胞克隆。结果:将包含第5至11外显子的LRP16基因组片段亚克隆到pBluescriptSKII+载体,SA-IRES-βgeo序列的正确插入第五外显子中,打靶载体成功转染胚胎干细胞,Southernblot结果显示具有一个打靶序列同源重组型插入的胚胎干细胞克隆。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胚胎干细胞。

BACKGROUND： Previous studies have demonstrated that LRP16 is an estrogen-responsive gene. Its expression level is strongly associated with the proliferation and invasive growth of human breast cancer cells. OBJECTIVE： To construct a LRP16 targeting vector and screen mouse embryonic stem cell clones with homolougous recombination of an inactive LRP 16 gene. DESIGN： Constructing an inserting inactivation target by inserting SA-RIES- β geo expression cassette. SETTING： Bioregulatory Laboratory of the Third Medical Department of Kyushu University in Japan and Department of Molecular Biology, General Hospital of Chinese PLA. MATERIALS： The materials used here were mainly provided by the Bioregulatory Laboratory, the Third Medical Department of Kyushu University in Japan. The mouse genomic library in pBeloBAC11 Vector was purchased from Invitrogen Corp. The competent TopF10 was purchased from Beijing Tiangen B iotech Corp. pcDNA3.1（＋） vector was kept in our laboratory. Mouse ES cells were provided by Kyushu University. METHODS： The experiment was performed in Kyushu University and Department of Molecular Biology of PLA General Hospital from November 2004 to May 2005. Targeting sequence of LRP16 gene was obtained from 129 mouse genomic Bacterial Artificial Chromosomes library based on polymerase chain reaction （PCR） screening. The SA-RIES- β geo fragment was inserted within LRP16 fifth exon to inactivate LRP16. ES cells were screened with G418 and the homologously recombinant clone was identified by Southern blot analysis. MAIN OUTCOME MEASURES： Clones with homologous recombination. RESULTS： The LRP16 fragment including exon 5 to 11 was subcloned into the pBluescript SK Ⅱvector. Restriction map demonstrated that the SA-IRES- β geo fragment was correctly inserted into the LRP16 fifth exon. Southern blot results showed that there was an ES clone with targeting sequence homologously inserted. CONCLUSION： A LRP 16 gene targeting vector is constructed and a homologous recombinant is obtained.