Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection.

Details

Serval ID
serval:BIB_C3C9C19B9A5F
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection.
Journal
Biotechnology and Bioengineering
Author(s)
Chenuet S., Martinet D., Besuchet-Schmutz N., Wicht M., Jaccard N., Bon A.C., Derouazi M., Hacker D.L., Beckmann J.S., Wurm F.M.
ISSN
0006-3592
Publication state
Published
Issued date
2008
Peer-reviewed
Oui
Volume
101
Number
5
Pages
937-45
Language
english
Notes
Publication types: Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't - Publication Status: ppublish
Abstract
Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.
Keywords
Animals, CHO Cells, Calcium Phosphates, Cricetinae, Cricetulus, DNA, Female, Flow Cytometry, Gene Dosage, Gene Expression, Gene Targeting, Genes, Reporter, Genetic Vectors, Green Fluorescent Proteins, Indicators and Reagents, Plasmids, Polyethyleneimine, Precipitation, Recombinant Proteins, Transfection, Transgenes
Pubmed
Web of science
Create date
20/02/2009 13:23
Last modification date
20/08/2019 15:39
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