High efficiency DNA transfection in murine embryonal carcinoma cells: expression of pSV3neo in wild type and retinoid-resistant cell lines.
Embryonal carcinoma cells provide a convenient and manipulable model for early embryogenesis. Like their counterparts in the inner cell mass, they are refractory to infection by several viruses. In their undifferentiated state, EC cells are resistant to calcium-phosphate DNA transfection. This resistance is compounded by the inefficient and/or actively inhibited expression of transfected genes driven by certain viral promoters. Conversely, the differentiated derivatives do not share this resistance and readily express virally promoted genes. We have developed a protocol for liposome-mediated gene transfer in EC cells and compared its efficiency in wild-type and retinoid-resistant variants. Dose response experiments with the EC cell line PCC4.aza1R showed a linear progression of colony formation when transfected with the vector pSV3neo and selected in medium containing the antibiotic G418. DNA concentrations of 10 micrograms per plate resulted in over 600 colonies per 10(6) cells. This represents a 20-30 fold greater efficiency over reported values for calcium-phosphate methods even though the neomycin resistance gene in this plasmid is driven by the SV40 viral promoter. The retinoid-resistant line PCC4(RA)-2 also showed enhanced transformation by lipofection, but despite the relatively high efficiency, colony formation rate for the differentiation-defective cells was less than 25% of the parental line. Our data indicates that there is no absolute block of genes driven by the SV40 early region promoter in murine EC cells if enough DNA is introduced to titrate out negative regulatory factors.
Purpus
E J
EJ
Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107.
McCue
P A
PA
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D004303:Dosage Compensation, Genetic; D004351:Drug Resistance / Q000235:genetics; D054278:Embryonal Carcinoma Stem Cells; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D008081:Liposomes; D051379:Mice; D014411:Neoplastic Stem Cells; D011401:Promoter Regions, Genetic; D013539:Simian virus 40 / Q000235:genetics; D013724:Teratoma / Q000235:genetics* / Q000473:pathology; D014162:Transfection / Q000379:methods*
D008081:Liposomes