Grp78基因转染肝细胞癌SMMC-7721克隆筛选及鉴定.doc

文档介绍：Grp78基因转染肝细胞癌SMMC-7721克隆筛选及鉴定
(作者:___________单位: ___________邮编: ___________)
作者:谷燕娇, 高志安,苏荣健
【摘要】目的用Grp78基因转染人肝细胞癌SMMC-7721,筛选阳性克隆并鉴定。+Grp78转染SMMC-7721,+Grp78与转染试剂的比例(μg/μL)分别为2∶3、2∶4、2∶5、2∶6、2∶7、2∶8、2∶9, 400 μg/mL G418筛选阳性克隆后,传代扩增,Western Blot鉴定克隆的Grp78表达情况。。结果 Western Blot显示,转染后的SMMC-7721经G418筛选,除一个克隆不高表达外,其余克隆均高表达Grp78。结论 Grp78转染SMMC-7721的最佳转染效率是8∶2(转染试剂/DNA),由于假阳性克隆的存在,筛选后的克隆需要鉴定。
【关键词】 Grp78;基因转染;克隆筛选
Selecting and Verifying SMMC-7721 Clones Transfected by Grp78 GeneGU Yanjiao1, GAO Zhian1 ,SU Rongjian2
( Pathology Department of the First Affiliated Hospital of Liaoning Medical University;
Laboratory of Liaoning Medical University, Jinzhou 121001 China)Abstract:Objecti
ve This paper is to transfect Grp78 gene to SMMC-7721, select high Grp78 expression clone, and verify. Methods Using +Grp78 to transfect human hapatocellular carcinoma SMMC-7721, as control, DNA: Fugene HD (μg/μL) is 2∶3, 2∶4, 2∶5,2∶6,2∶7,2∶8,2∶9 and selecting positive clones by G418(400 μg/mL), amplifying, then verifying them by WB. Results Western Blot indicates that SMMC-7721 transfected by Grp78 gene could high express Grp78. Conclusions The optimized transfection efficiency of Grp78 transfecting SMMC-7721 is 8∶2(Fugene HD/DNA), as the existence of false positive clones, we need to verify all clones before using them.