多用途基因捕获C3H/10T1/2细胞阳性克隆库的构建及鉴定  

Establishment and identification of gene trap clones from C3H/10T1/2 cells transfected with rosafary vector

作　　者：王明科[1] 邹仲敏[1] 罗成基[1] 粟永萍[1] 

机构地区：[1]第三军医大学军事预防医学院放射医学教研室,全军复合伤研究所,创伤,烧伤与复合伤国家重点实验室,重庆400038

出　　处：《第四军医大学学报》2008年第6期508-512,共5页Journal of the Fourth Military Medical University

基　　金：国家重点基础研究发展规划资助项目(973项目)(2005CB522605);军队"十一五"归国人员基金(06H029)

摘　　要：目的：以10T1／2细胞为间充质干细胞模型，构建用于间充质干细胞分化及恶性转化分子机制等研究的基因捕获阳性克隆库．方法：Dra I线性化逆转录病毒基因捕获载体ROSAFARY后，与pIRES2-EGFP用脂质体共转染phoenix细胞，LacZ染色证明转染成功后收集病毒上清感染10T1／2细胞．经潮霉素筛选获得阳性克隆，为排除假阳性克隆用LacZ部分片段PCR进行鉴定．结果：ROSAFARY脂质体转染phoenix细胞后48h，GFP指示的转染效率约为20％～30％，LacZ染色有大约10％的阳性率．150mg／L潮霉素筛选病毒感染的10T1／2细胞，挑取药物抗性克隆，PCR鉴定后获得43个阳性克隆，LacZ染色5个为捕获基因高表达细胞克隆．结论：基因捕获技术建立非胚胎干细胞的克隆是可行的，成功建立的基因捕获阳性克隆库为应用基因捕获技术揭示成体干细胞分化及恶性转化分子机制奠定了基础．AIM： To construct a library of gene trapped clones based on rosafary-transfected C3H/10T1/2 cell line in order to provide a new platform for study of the mechanism of differentiation or malignant transformation of mesenchymal stem cells. METHODS： Retroviral gene-trap vector, ROSAFARY, was line- arized with Dra I and co-transfected with pIRES2-EGFP into packaging cell line phoenix by Lipofectamine. The retrovirus particles containing gene-trap construct were collected to infect 10TI/2 cells and LacZ staining was used to prove the result of transfec- tion. Genomic PCR targeting LacZ sequences in ROSAFARY vector were performed to further identify the drug-resistant 10T1/2 clones. RESULTS： At 48 h after the transfection, GFP co-trans- fection indicated that the transfection efficiency reached 20% 30% and about 10% of positive LacZ staining was achieved in packaging cells. By hygromycin selection with the concentration of 150 mg/L, total 43 clones were confirmed by PCR, including 5 clones with positive LacZ staining, which indicated the active promotet activity of trapped gene. CONCLUSION： It is feasible to construct a library of gene trapped clones by gene trap technique in cells other than embryonic stem cells and the library provids a new platform for screening genes related with adult stem cell differentiation and malignant transformation.

关 键 词：基因捕获 C3H/10T1/2细胞 聚合酶链反应R β-半乳糖苷酶类 

分 类 号：R33-34[医药卫生—人体生理学]