000104999 001__ 104999
000104999 005__ 20180317094456.0
000104999 022__ $$a0006-3002
000104999 02470 $$2ISI$$a000188606100005
000104999 037__ $$aARTICLE
000104999 245__ $$aCalfection: a novel gene transfer method for suspension cells
000104999 260__ $$c2004
000104999 269__ $$a2004
000104999 336__ $$aJournal Articles
000104999 500__ $$aLaboratory of Cellular Biotechnology, Swiss Federal Institute of Technology, Lausanne, CH-1015, Switzerland.
000104999 520__ $$aWe 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.
000104999 6531_ $$aAnimals
000104999 6531_ $$aBioreactors
000104999 6531_ $$aCHO Cells
000104999 6531_ $$a*Calcium Chloride
000104999 6531_ $$aCattle
000104999 6531_ $$aCell Culture Techniques/*methods
000104999 6531_ $$aCell Line
000104999 6531_ $$aCricetinae
000104999 6531_ $$aDose-Response Relationship
000104999 6531_ $$aDrug
000104999 6531_ $$a*Gene Transfer Techniques
000104999 6531_ $$aGreen Fluorescent Proteins
000104999 6531_ $$aHumans
000104999 6531_ $$aHydrogen-Ion Concentration
000104999 6531_ $$aLuminescent Proteins/analysis/biosynthesis/genetics
000104999 6531_ $$aPlasmids/pharmacology
000104999 6531_ $$aSolutions
000104999 6531_ $$aTemperature
000104999 6531_ $$aTime Factors
000104999 700__ $$aLindell, J.
000104999 700__ $$0241450$$aGirard, P.$$g101639
000104999 700__ $$0241218$$aMuller, N.$$g144025
000104999 700__ $$0241449$$aJordan, M.$$g108539
000104999 700__ $$0240398$$aWurm, F.$$g107554
000104999 773__ $$j1676$$k2$$q155-61$$tBiochimica et biophysica acta
000104999 8564_ $$uhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14746910 $$zURL
000104999 909CO $$ooai:infoscience.epfl.ch:104999$$particle
000104999 909C0 $$0252009$$pLBTC$$xU10180
000104999 937__ $$aLBTC-ARTICLE-2004-012
000104999 970__ $$a22/LBTC
000104999 973__ $$aEPFL$$rREVIEWED$$sPUBLISHED
000104999 980__ $$aARTICLE