Dilution enhancement of COS cell expression cloning

To search for an efficient expression cloning method, we mixed plasmid pmDA Tsv, which contains the mouse dopamine transporter (mDAT) cDNA, with a larg e amount of another plasmid prGlyTsv to mimic the situation of a cDNA libra ry and examined COS cell expression. Both plasmids have an SV40 replication origin and thus will be replicated to high copy numbers in COS cells. Afte r transfecting COS-7 cells with pmDATsv/prGlyTsv mixture at 1/1000 ratio, w e could not detect any cells expressing strong mDAT activity. In contrast, when prGlyTsv was replaced by prSERTsk (no SV40 origin) in the transfection mixture, we observed hundreds of cells expressing strong mDAT activity. Th e results suggested that in many cells low mDAT expression was not due to t he lack of pmDATsv plasmid but due to the presence of large numbers of repl icable prGlyTsv. Analysis with a mathematical model suggests that diluting cDNA libraries with other plasmids without the SV40 origin should improve t he detection of COS cells expressing target cDNAs. We tested this conclusio n with pmDATsv/ prGlyTsv mixture. When the mixture at 1/1000 ratio was dilu ted with prSERTsk and used for transfection, we could now easily detect cel ls expressing strong mDAT activity. (C) 2000 Academic press.