High efficiency DNA-mediated transformation of human diploid fibroblasts.
Normal human diploid fibroblasts (HDF) have only rarely been used as recipients in DNA transfection experiments even though they have the potential to add to our knowledge about the genes that control normal cell proliferation. A major impediment to the use of HDF has been their poor transfection frequency. In this paper, we show that IMR-90 human diploid fibroblasts can be stably transfected at high efficiency with pRSVneo at an average frequency of 2 x 10(-3) (0.2%) using a modified calcium phosphate-DNA coprecipitation transfection protocol. Prior to this report, the best transfection frequencies of HDF were on the order of 10(-5)-10(-4). In the present protocol it is very important to maintain relatively low cell densities, particularly during the selection process post-transfection. Using this protocol other neo-containing plasmids also yield high transfection frequencies in IMR-90. IMR-90, which are readily available from the NIH Aging Cell Repository, may be one of the best HDF to use as recipients because they are five times as efficient as are WI-38, which is another well characterized HDF.
Donahue
L M
LM
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder.
Stein
G H
GH
D002130:Calcium Phosphates / Q000494:pharmacology; D002452:Cell Count; D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000473:pathology; D006801:Humans; D014162:Transfection
D002130:Calcium Phosphates; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous