Regulation of G418 selection efficiency by cell-cell interaction in transfection.
We attempted to establish the optimum conditions for the calcium phosphate (CaPO4) precipitation protocol by counting G418 resistant (G418r) colonies after transfection of pSV2-neo DNA into BALB 3T3 cells. The amount and molecular size of carrier DNA, number of plating cells, treatment period of DNA-CaPO4 precipitates and expression time of G418 selection were found to be important factors in the induction of G418r colonies. Six G418r clones were derived from BALB 3T3, NIH 3T3 and FRSK cells, and cocultured with G418 sensitive (G418s) parent cells in G418 medium. The colony formation capacity of all G418r cell clones decreased with the increasing number of plated G418s cells. Cell-cell contact appeared to be necessary to reduce the colony formation of G418r cells, and contact-dependent G418r cell killing was probably not related to gap junction formation. Contact-mediated cell killing is a likely explanation for the observation that induction of G418r colonies is often reduced under conditions of high-density plating, long treatment of DNA-CaPO4 precipitates, and long expression time of G418 selection. These results suggest that in some instances transfection efficiency using pSV2-neo DNA should be carefully evaluated because culture conditions can mask the induction of G418r colonies.
Sasaki
K
K
Department of Cell Biology, Hatano Research Institute, Food and Drug Safety Center (FDSC), Kanagawa, Japan.
Mizusawa
H
H
Ishidate
M
M
Tanaka
N
N
D000818:Animals; D002130:Calcium Phosphates; D002450:Cell Communication; D002452:Cell Count / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004274:DNA, Recombinant / Q000235:genetics; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D005839:Gentamicins / Q000494:pharmacology*; D007365:Intercellular Junctions / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D002130:Calcium Phosphates; D004274:DNA, Recombinant; D005839:Gentamicins; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; C010680:antibiotic G 418; C485829:calcium phosphate, dibasic, anhydrous; D013755:Tetradecanoylphorbol Acetate; D004121:Dimethyl Sulfoxide