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

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.%Objiective To conslrucl slable HCT116 sublines overexpressing MTA1 al gradienl levels.Methods Werncloned human MTA1 CDS by RT-PCR using the posilive clones from a lemplale screened by in situ plaque hybridization wilh ihe human lung phage library. The MTA1 CDS was cul oul and ligaled inlo the relroviral veclor pMX ( pMX-MTAl-flag-IRES-EGFP). Relroviral panicles were packaged by co-lransfecting the constructed relroviral veclor logelher wilh pM-DG plasmid inlo 293-gag/pol packaging cells. HCT116 cells were then infecled wilh the supernanl conlaining the generaledrnrelroviral particles Lo produce MTA1-expressing polyclonal cell pool. The polyclonal cell pool was diluled and seeded inlo a 10 cm-diameler dish al a very low densily. AbouL Lwo weeks later, when ihe separated cells grew inlo clones, ihe MTA1-overexpressing subclones were selecled wilh GFP as a screening marker.Results DNA sequencing verified ihe successfulrncloning of human MTA1 CDS. Immunofluorescence and Weslern Blol resulls screened a series of monoclonal sublines expressing gradienl MTA1. We also confirmed lhal levels of upslream MTA1 and downslream GFP, which were bridged wilh IRES sequence, showed a significant linear correlation ( r = 0. 932, P < 0. 01) .Conclusion We have successfully es-rnlablished a melhod lo rapidly develop a HCT116-based cancer cell line model expressing a gradienl hMTAl and ihis approach can be exlended lo construction of cell lines over-expressing olher genes.