de Chasseval R, de Villartay J P
Laboratoire INSERM U132, Hôpital Necker-Enfants-Malades, Paris, France.
Nucleic Acids Res. 1992 Jan 25;20(2):245-50. doi: 10.1093/nar/20.2.245.
High level transient gene expression in lymphoid cells has always been challenging because of the difficulty to efficiently transfect such cells. This has precluded any attempt to clone cDNA encoding proteins by means of their specific biological function in lymphoid cells. We have developed a very efficient transient eukaryotic expression system analogous to the well-known expression system in COS cells. Firefly luciferase and human CD2 genes were used as reporter genes and cloned into the eukaryotic shuttle vector pCDM8 which contains the strong cytomegalovirus promoter and the SV40 origin of replication for autonomous plasmid replication in permissive host cells that express the large SV40 T Antigen. Co-transfection of the reporter plasmids together with an SV40 T Ag expressing plasmid resulted in the several fold amplification of either the Luc activity or the cell surface expression of the CD2 marker in a transient assay. The level of amplification was dependent on the strength of the promoter used to drive the SV40 T Ag expression and was correlated with the extent of autonomous replication of the reporter plasmid in transfected cells. This highly efficient transient gene expression by SV40 T Ag boost was suitable to several human cell lines, making this system of general interest for expression cloning strategies or other gene transfer application that need high level expression.
由于难以高效转染淋巴细胞，在淋巴细胞中进行高水平瞬时基因表达一直具有挑战性。这使得通过淋巴细胞中蛋白质的特定生物学功能来克隆编码这些蛋白质的cDNA的任何尝试都无法进行。我们开发了一种非常高效的瞬时真核表达系统，类似于COS细胞中著名的表达系统。萤火虫荧光素酶和人CD2基因用作报告基因，并克隆到真核穿梭载体pCDM8中，该载体含有强巨细胞病毒启动子和SV40复制起点，用于在表达大SV40 T抗原的允许宿主细胞中进行自主质粒复制。在瞬时分析中，将报告质粒与表达SV40 T抗原的质粒共转染，导致荧光素酶活性或CD2标志物的细胞表面表达增加了几倍。扩增水平取决于用于驱动SV40 T抗原表达的启动子的强度，并且与报告质粒在转染细胞中的自主复制程度相关。通过SV40 T抗原增强实现的这种高效瞬时基因表达适用于几种人类细胞系，使得该系统对于表达克隆策略或其他需要高水平表达的基因转移应用具有普遍意义。
