HIGH-LEVEL TRANSIENT GENE-EXPRESSION IN HUMAN LYMPHOID-CELLS BY SV40 LARGE T-ANTIGEN BOOST
HIGH-LEVEL TRANSIENT GENE-EXPRESSION IN HUMAN LYMPHOID-CELLS BY SV40 LARGE T-ANTIGEN BOOST
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DOI：
10.1093/nar/20.2.245
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发表时间：
1992-01-25
影响因子：
14.9
通讯作者：
DEVILLARTAY, JP
中科院分区：
文献类型：
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作者：
DECHASSEVAL, R;DEVILLARTAY, JP
淋巴样细胞的高水平瞬时基因表达一直是一个挑战，因为这类细胞很难有效地转染。这排除了任何试图克隆cDNA编码蛋白的手段，其特定的生物学功能在淋巴样细胞。我们开发了一种非常有效的瞬时真核表达系统，类似于众所周知的COS细胞表达系统。以萤火虫荧光素酶和人CD2基因为报告基因，克隆到含有巨细胞病毒强启动子和SV40复制起始点的真核穿梭载体pCDM8中，在表达SV40大T抗原的允许宿主细胞中进行自主质粒复制。将报告质粒与表达SV40 T Ag的质粒一起共转染，可使Luc活性或CD2标记的细胞表面表达在瞬时实验中扩增数倍。扩增水平取决于用于驱动SV40 T Ag表达的启动子的强度，并与转染细胞中报告质粒的自主复制程度相关。通过SV40 T Ag boost的高效瞬时基因表达适用于多种人类细胞系，使该系统成为表达克隆策略或其他需要高水平表达的基因转移应用的普遍兴趣。
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.