Dilution enhancement of COS cell expression cloning.
To search for an efficient expression cloning method, we mixed plasmid pmDATsv, which contains the mouse dopamine transporter (mDAT) cDNA, with a large amount of another plasmid prGlyTsv to mimic the situation of a cDNA library and examined COS cell expression. Both plasmids have an SV40 replication origin and thus will be replicated to high copy numbers in COS cells. After transfecting COS-7 cells with pmDATsv/prGlyTsv mixture at 1/1000 ratio, we 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. The results suggested that in many cells low mDAT expression was not due to the lack of pmDATsv plasmid but due to the presence of large numbers of replicable prGlyTsv. Analysis with a mathematical model suggests that diluting cDNA libraries with other plasmids without the SV40 origin should improve the detection of COS cells expressing target cDNAs. We tested this conclusion with pmDATsv/prGlyTsv mixture. When the mixture at 1/1000 ratio was diluted with prSERTsk and used for transfection, we could now easily detect cells expressing strong mDAT activity.
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School of Medicine, Department of Pharmacology, Yale University, 333 Cedar Street, New Haven, Connecticut 06510, USA.
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D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D050483:Dopamine Plasma Membrane Transport Proteins; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008957:Models, Genetic; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis; D018741:Replication Origin; D013539:Simian virus 40 / Q000235:genetics
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins