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  4. Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation
 
research article

Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation

Jordan, M.  
•
Schallhorn, A.
•
Wurm, F. M.  
1996
Nucleic acids research

DNA-calcium phosphate co-precipitates arise spontaneously in supersaturated solutions. Highly effective precipitates for transfection purposes, however, can be generated only in a very narrow range of physico-chemical conditions that control the initiation and growth of precipitate complexes. The concentrations of calcium and phosphate are the main factors influencing characteristics of the precipitate complex, but other parameters, such as temperature, DNA concentration and reaction time are important as well. An example for this is the finding that almost all of the soluble DNA in the reaction mix can be bound into an insoluble complex with calcium phosphate in <1 min. Extending the reaction time to 20 min results in aggregation and/or growth of particles and reduces the level of expression. With improved protocols we gained better reproducibility and higher efficiencies both for transient and for stable transfections. Up to 60% of cells stained positive for beta-gal and transient production of secreted proteins was improved 5- to 10-fold over results seen with transfections using standard procedures. Similar improvements in efficiency (number of recombinant cell colonies) were observed with stable transfections, using co-transfected marker plasmids for selection. Transient expression levels 2 days after DNA transfer and titers obtained from stable cell lines, emerging weeks later, showed strong correlation.

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Type
research article
DOI
10.1093/nar/24.4.596
PubMed ID

8604299

Author(s)
Jordan, M.  
Schallhorn, A.
Wurm, F. M.  
Date Issued

1996

Publisher

Oxford University Press

Published in
Nucleic acids research
Volume

24

Issue

4

Start page

596

End page

601

Subjects

Animals

•

Calcium Phosphates

•

Cells

•

Cultured

•

Cricetinae

•

DNA/*genetics

•

Transfection/*methods

Note

Genentech Inc., South San Francisco, CA 94080, USA.

National Licences

Editorial or Peer reviewed

REVIEWED

Written at

EPFL

EPFL units
LBTC  
Available on Infoscience
June 5, 2007
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/7661
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