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

Détails

ID Serval
serval:BIB_C3C9C19B9A5F
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection.
Périodique
Biotechnology and Bioengineering
Auteur(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
Statut éditorial
Publié
Date de publication
2008
Peer-reviewed
Oui
Volume
101
Numéro
5
Pages
937-45
Langue
anglais
Notes
Publication types: Comparative Study ; Journal Article ; Research Support, Non-U.S. Gov't - Publication Status: ppublish
Résumé
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.
Mots-clé
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
Création de la notice
20/02/2009 14:23
Dernière modification de la notice
03/03/2018 21:12
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