Nucleic Acids Research, Vol 24, Issue 4 596-601, Copyright © 1996 by Oxford University Press
M Jordan, A Schallhorn and FM Wurm
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.
ARTICLES
Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation
Genentech Inc., South San Francisco, CA 94080, USA.
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