Efficient transformation and frequent single-site, single-copy insertion of DNA can be obtained in mouse erythroleukemia cells transformed by electroporation.
Electroporation has recently been shown to have advantages over the commonly used method of calcium phosphate precipitation for obtaining DNA-mediated transformation of certain types of cells. Although mouse erythroleukemia cells and other cells of hematopoietic origin are not transformed at useful frequencies by calcium phosphate-DNA precipitation methods, we obtained high frequencies of transformation (approximately 10(-5)) of these cells with electroporation. Even higher transformation frequencies (approximately 10(-3)) were obtained with human fibroblasts. Another advantage of electroporation was found when analysis of Southern blots of DNA from 243 transformed erythroleukemia cell colonies indicated that, under appropriate conditions, about 79% of the transformed cells had the exogenous DNA integrated in single copies at single sites. Under conditions of higher DNA and lower cell concentrations using fibroblasts, cotransformation was obtained with two plasmids that confer HAT or G418 resistance when integrated into cellular DNA. About 23% of the transformed cells developed both types of resistance. We describe a simple, inexpensive apparatus for carrying out electroporation.
Boggs
S S
SS
Gregg
R G
RG
Borenstein
N
N
Smithies
O
O
D000818:Animals; D002463:Cell Membrane Permeability; D004273:DNA, Neoplasm / Q000235:genetics*; D004274:DNA, Recombinant; D005347:Fibroblasts / Q000032:analysis; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000235:genetics*; D051379:Mice; D010957:Plasmids; D014170:Transformation, Genetic
D004273:DNA, Neoplasm; D004274:DNA, Recombinant