Calfection: a novel gene transfer method for suspension cells
We have developed a novel method called Calfection for gene delivery to and protein expression from suspension-cultivated mammalian cells. Plasmid DNA was simply diluted into a calcium chloride solution and then added to the cell culture for transfection. We evaluated and optimized this approach using suspension-adapted HEK293 cells grown in 12-well plates that were shaken on an orbital shaker. Highest expression levels were obtained when cells were transfected at a density of 5x10(5) cells/ml in the presence of 9 mM calcium and 5 microg/ml of plasmid DNA while maintaining a culture pH of 7.6 at the time of transfection. Suspension-adapted BHK 21 and CHO DG 44 cells could also be transfected using this method. Calfection differs from the widely known calcium phosphate coprecipitation technique. The physico-chemical composition of the DNA interacting complexes is not yet known. The transfection cocktail, DNA in a calcium chloride solution, remained highly efficient during long-term storage at temperatures ranging from room temperature to -80 degrees C. In contrast, calcium phosphate-DNA cocktails are only efficient for gene transfer when prepared fresh. Furthermore, passing the calcium-plasmid DNA mixture through a 0.2-microm filter did not compromise protein expression, whereas calcium phosphate-DNA coprecipitates were retained by the filter. High protein expression levels, a limited number of manipulations and the possibility to filter the cocktail make the Calfection approach suitable for both large-scale transfection in bioreactors and for high-throughput transfection experiments in microtiter plates.
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- URL: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14746910
Keywords: Animals ; Bioreactors ; CHO Cells ; *Calcium Chloride ; Cattle ; Cell Culture Techniques/*methods ; Cell Line ; Cricetinae ; Dose-Response Relationship ; Drug ; *Gene Transfer Techniques ; Green Fluorescent Proteins ; Humans ; Hydrogen-Ion Concentration ; Luminescent Proteins/analysis/biosynthesis/genetics ; Plasmids/pharmacology ; Solutions ; Temperature ; Time Factors
Laboratory of Cellular Biotechnology, Swiss Federal Institute of Technology, Lausanne, CH-1015, Switzerland.
Record created on 2007-06-05, modified on 2016-08-08