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.
Lindell
Jeanette
J
Laboratory of Cellular Biotechnology, Swiss Federal Institute of Technology, Lausanne, CH-1015, Switzerland.
Girard
Philippe
P
Müller
Natalie
N
Jordan
Martin
M
Wurm
Florian
F
D000818:Animals; D019149:Bioreactors; D016466:CHO Cells; D002122:Calcium Chloride; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000494:pharmacology; D012996:Solutions; D013696:Temperature; D013997:Time Factors
D008164:Luminescent Proteins; D012996:Solutions; D049452:Green Fluorescent Proteins; D002122:Calcium Chloride