Maximal Expression of Recombinant cDNAs in COS Cells for Use in Expression Cloning

• Published in: Analytical biochemistry 1993-02, Vol.208 (2), p.352-356
• Main Authors: Kluxen, F.W., Lubbert, H.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; beta-Galactosidase - genetics; Biological and medical sciences; Biotechnology; Cell Line; Cloning, Molecular - methods; DEAE-Dextran; DNA, Recombinant - genetics; Evaluation Studies as Topic; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic engineering; Genetic technics; Methods. Procedures. Technologies; Modification of gene expression level; Molecular Sequence Data; Plasmids; Transfection
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1993.1060

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 β-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 β-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 β-galactosidase positive cells but considerably reduced the total β-galactosidase activity. However, a 10% DMSO shock at the end of the transfection procedure simultaneously increased the number of transfected cells and the total β-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.