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 β-galactosidase gene was chosen as a reporter gene, since it permits the determination of (i) the fraction of cell...

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Bibliographic Details
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
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1993.1060