High level transient gene expression in human lymphoid cells by SV40 large T antigen boost.
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.
Laboratoire INSERM U132, Hôpital Necker-Enfants-Malades, Paris, France.
R
R
de Chasseval
J P
JP
de Villartay
D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D018801:CD2 Antigens; D002460:Cell Line; D004261:DNA Replication; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008214:Lymphocytes; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011971:Receptors, Immunologic / Q000235:genetics; D014162:Transfection / Q000235:genetics*
D000945:Antigens, Differentiation, T-Lymphocyte; D000952:Antigens, Polyomavirus Transforming; D018801:CD2 Antigens; D011971:Receptors, Immunologic; D008156:Luciferases