Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection.
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.
Chenuet
Sebastien
S
Ecole Polytechnique Fédérale de Lausanne (EPFL), School of Life Sciences, Laboratory of Cellular Biotechnology, CHJ2-506, Station 6, CH-1015 Lausanne, Switzerland.
Martinet
Danielle
D
Besuchet-Schmutz
Nathalie
N
Wicht
Marianne
M
Jaccard
Nicolas
N
Bon
Anne-Charlotte
AC
Derouazi
Madiha
M
Hacker
David L
DL
Beckmann
Jacques S
JS
Wurm
Florian M
FM
D000818:Animals; D016466:CHO Cells; D002130:Calcium Phosphates / Q000737:chemistry / Q000494:pharmacology*; D011232:Chemical Precipitation; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D018628:Gene Dosage / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects*; D018390:Gene Targeting / Q000379:methods; D017930:Genes, Reporter / Q000187:drug effects; D005822:Genetic Vectors / Q000187:drug effects / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007202:Indicators and Reagents; D010957:Plasmids / Q000187:drug effects / Q000378:metabolism; D011094:Polyethyleneimine / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000187:drug effects
D002130:Calcium Phosphates; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D011094:Polyethyleneimine; D004247:DNA; C020243:calcium phosphate