以双质粒逆病毒载体系统快速建立梯度过表达hMTA1稳转单克隆细胞系的方法  

A method to rapidly construct the sublines stably over-expressing hMTA1 and GFP in a non-fusion manner

作　　者：刘健[1] 王海娟[1] 张金龙[1] 常亚楠[2] 孟希亭[1] 张雪燕[1] 梁肖[1] 付明[1] 赵玫[1] 林晨[1] 黄常志[1] 钱海利[1] 

机构地区：[1]中国医学科学院、肿瘤医院肿瘤研究所、分子肿瘤学国家重点实验室,北京100021 [2]首都医科大学附属北京妇产医院,北京100006

出　　处：《癌症进展》2012年第3期205-210,共6页Oncology Progress

基　　金：国家自然科学基金项目(项目编号:81071773);北京协和医学院研究生创新基金(项目编号:2010-1001-014)

摘　　要：目的利用逆病毒表达载体快速构建梯度过表达人MTA1稳转单克隆细胞系。方法利用噬菌斑原位杂交筛选人肺噬菌体文库,以阳性克隆为模板,RT-PCR获得人MTA1完整CDS序列。以逆病毒表达载体pMX为基础,经过多次酶切连接,构建逆病毒表达载体pMX-MTA1-flag-IRES-EGFP。将构建好的逆病毒载体系统,转染293-gag/pol细胞,包装出含该表达载体的逆病毒颗粒。以含病毒上清多次感染HCT116细胞,获得稳定转染MTA1的HCT116多克隆细胞系。将该多克隆细胞系按合适比例稀释后接种至10cm大皿,获得大量单克隆细胞系,以GFP为筛选标志,筛选不同荧光强度的单细胞克隆进行扩增并分析MTA1与GFP表达水平的线性关系。结果测序结果显示成功克隆出人MTA1表达序列。免疫荧光、Western Blot结果证实成功构建出梯度过表达MTA1的稳转细胞系。相关性验证结果显示构建的非融合载体MTA1与GFP的表达呈明显的线性相关,相关系数r=0.932,P<0.01。结论利用逆病毒表达系统,快速成功构建并筛选出MTA1与GFP非融合梯度过表达稳转单克隆细胞系,为MTA1基因功能研究提供了良好模型。该逆转录病毒平台可用于快速建立其他基因的稳转单克隆细胞系。Objective To construct stable HCT116 sublines overexpressing MTA1 at gradient levels. Methods We cloned human MTA1 CDS by RT-PCR using the positive clones from a template screened by in situ plaque hybridization with the human lung phage library. The MTA1 CDS was cut out and ligated into the retroviral vector pMX （pMX-MTA1- flag-IRES-EGFP）. Retroviral particles were packaged by co-transfecting the constructed retroviral vector together with pM- DG plasmid into 293-gag/pol packaging cells. HCT116 cells were then infected with the supemant containing the generated retroviral particles to produce MTA1-expressing polyclonal cell pool. The polyclonal cell pool was diluted and seeded into a 10 cm-diameter dish at a very low density. About two weeks later, when the separated cells grew into clones, the MTA1- overexpressing subclones were selected with GFP as a screening marker. Results DNA sequencing verified the successful cloning of human MTA1 CDS. Immunofluorescence and Western Blot results screened a series of monoclonal sublines ex- pressing gradient MTA1. We also confirmed that levels of upstream MTA1 and downstream GFP, which were bridged with IRES sequence, showed a significant linear correlation （r = 0. 932, P 〈 0. 01 ）. Conclusion We have successfully es- tablished a method to rapidly develop a HCT116-based cancer cell line model expressing a gradient hMTA1 and this ap- proach can be extended to construction of cell lines over-expressing other genes.

关 键 词：MTA1 GFP 逆病毒 稳转 梯度过表达 单克隆 

分 类 号：Q78[生物学—分子生物学]