Maximal expression of recombinant cDNAs in COS cells for use in expression cloning.
In the process of establishing an expression cloning system for cell surface receptors we examined parameters which influence the expression of foreign genes in COS cells. The bacterial beta-galactosidase gene was chosen as a reporter gene, since it permits the determination of (i) the fraction of cells transfected as well as (ii) the total activity of the synthesized enzyme in parallel experiments. This renders it possible to calculate the enzyme activity per individual cell. In transfected COS cells, the plasmid pXMgal directed a 20- and 10-fold higher beta-galactosidase activity than pCH110 and pCDLgal, respectively. DEAE-dextran-mediated DNA uptake and protoplast fusion were found to result in higher expression rates than lipofection and electroporation. A coincubation of the cells with chloroquine during the DEAE-dextran transfection protocol caused, as reported, an increase of beta-galactosidase positive cells but considerably reduced the total beta-galactosidase activity. However, a 10% DMSO shock at the end of the transfection procedure simultaneously increased the number of transfected cells and the total beta-galactosidase activity, thus maintaining the high expression per single cell. Using these optimized conditions, COS-1 cells expressed higher amounts of recombinant protein than COS-7 cells.
Kluxen
F W
FW
Sandoz Pharma Ltd., Basel, Switzerland.
Lübbert
H
H
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D003637:DEAE-Dextran; D004274:DNA, Recombinant / Q000235:genetics*; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D008969:Molecular Sequence Data; D010957:Plasmids; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D004274:DNA, Recombinant; D003637:DEAE-Dextran; D001616:beta-Galactosidase