TY - JOUR
T1 - High efficiency DNA transfection in murine embryonal carcinoma cells
T2 - Expression of pSV3neo in wild type and retinoid-resistant cell lines
AU - Purpus, E. J.
AU - McCue, P. A.
PY - 1993
Y1 - 1993
N2 - 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 μg per plate resulted in over 600 colonies per 106 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.
AB - 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 μg per plate resulted in over 600 colonies per 106 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.
KW - Animals
KW - Cell Differentiation
KW - Cell Line
KW - Dosage Compensation, Genetic
KW - Drug Resistance/genetics
KW - Embryonal Carcinoma Stem Cells
KW - Gene Expression Regulation, Viral/genetics
KW - Liposomes
KW - Mice
KW - Neoplastic Stem Cells
KW - Promoter Regions, Genetic
KW - Simian virus 40/genetics
KW - Teratoma/genetics
KW - Transfection/methods
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M3 - Article
C2 - 8389573
SN - 0214-6282
VL - 37
SP - 117
EP - 124
JO - International Journal of Developmental Biology
JF - International Journal of Developmental Biology
IS - 1
ER -