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Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection.
- Source :
-
Biotechnology and bioengineering [Biotechnol Bioeng] 2008 Dec 01; Vol. 101 (5), pp. 937-45. - Publication Year :
- 2008
-
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.
- Subjects :
- Animals
Calcium Phosphates chemistry
Chemical Precipitation
Cricetinae
Cricetulus
DNA analysis
DNA genetics
Female
Flow Cytometry
Gene Dosage drug effects
Gene Targeting methods
Genes, Reporter drug effects
Genetic Vectors drug effects
Genetic Vectors metabolism
Green Fluorescent Proteins biosynthesis
Green Fluorescent Proteins genetics
Indicators and Reagents
Plasmids drug effects
Plasmids metabolism
Polyethyleneimine chemistry
Recombinant Proteins biosynthesis
Transgenes drug effects
CHO Cells
Calcium Phosphates pharmacology
Gene Expression drug effects
Polyethyleneimine pharmacology
Recombinant Proteins genetics
Transfection methods
Subjects
Details
- Language :
- English
- ISSN :
- 1097-0290
- Volume :
- 101
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Biotechnology and bioengineering
- Publication Type :
- Academic Journal
- Accession number :
- 18781700
- Full Text :
- https://doi.org/10.1002/bit.21972