A new expression system for mammalian cells based on putative replicator sequences of the mouse and a truncated thymidine kinase gene.
We have constructed a new expression vector for mammalian cells. The vector contains a truncated tk gene for amplification under selective conditions, a sequence putatively supporting the replication of plasmid DNA in eukaryotic cells (murine autonomously replicating sequence) and an expression cassette for the cDNA to be studied. As a model cDNA we have used that of human tissue-type plasminogen activator (t-PA). Analysis of Hirt supernatants and chromosomal DNA from L cells, prepared six weeks after isolation of the clones indicated a 50- to 500-fold amplification of the expression construct in the cells. Concomitantly, the expression of t-PA was dramatically increased. Our data are consistent with episomal persistence of the expression construct, with a head-to-tail mode of integration into the mouse genome and with coexistence of both episomal plasmids and head-to-tail integrates. In tk-deficient cell lines other then L-cells, such as mouse mastocytoma or rat hepatoma cells, a strong selection against the persistence of the expression construct was noted. After long-term propagation of the L-cells under selective conditions the expression of the indicator gene continually decreases, but finally a constant plateau level of expression is established. Expression could be restored to the original level by blocking more efficiently the de novo synthesis of nucleosides.
Weidle
U H
UH
Department of Genetics, Boehringer Mannheim GmbH, Penzberg, F.R.G.
Buckel
P
P
Grummt
F
F
D000818:Animals; D004274:DNA, Recombinant / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005784:Gene Amplification; D005796:Genes; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D007739:L Cells / Q000201:enzymology; D051379:Mice; D013937:Thymidine Kinase / Q000235:genetics*; D010959:Tissue Plasminogen Activator / Q000032:analysis / Q000235:genetics*; D014162:Transfection
D004274:DNA, Recombinant; D013937:Thymidine Kinase; D010959:Tissue Plasminogen Activator