High-efficiency stable gene transfection using chloroquine-treated Chinese hamster ovary cells.
We describe a highly efficient stable gene transfection procedure for Chinese hamster ovary (CHO) cells using a modification of the calcium phosphate-DNA coprecipitation method. We have found that treatment of CHO cells with chloroquine increases the efficiency of gene transfer by up to 20-fold (from approx. 0.01% to approx. 0.2%) when transfection is done using the pSV2-neo plasmid. The optimized transfection procedure requires that CHO cells to be incubated with calcium phosphate-DNA coprecipitate and chloroquine (100 microM) for a total of 16 h. By using high-molecular-weight human genomic DNA as a DNA source for transfection, we show that this procedure is equally efficient for stably transferring a much larger gene, such as the 49-kb human hypoxanthine phosphoribosyltransferase gene.
Hasan
M T
MT
Department of Biochemistry, Dartmouth Medical School Hanover, New Hampshire 03756.
Subbaroyan
R
R
Chang
T Y
TY
D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells / Q000187:drug effects; D002130:Calcium Phosphates; D002738:Chloroquine / Q000494:pharmacology*; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D006801:Humans; D008247:Lysosomes / Q000187:drug effects; D014162:Transfection / Q000187:drug effects*
D002130:Calcium Phosphates; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; D002738:Chloroquine; D004247:DNA; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous