Growth of HeLa S3 cells cotransfected with plasmids containing a c-fos gene under the control of the SV40 promoter complex, pRSVcat, and G418 resistance.
HeLa S3 cells, which have been fractionated into sequential and synchronous cell cycle phase-specific fractions, express c-fos at twice the basal levels in the earliest part of G1 phase. To determine whether this peak in c-fos synthesis has regulatory significance, a DNA construct was prepared which contained the human c-fos gene under the transcriptional control of the SV40 promoter complex. The pc-fos(human)-1 gene (9 kilobases) was inserted into the eukaryotic expression vector pSG5 (4.076 kilobases) at the EcoRI site. Electroporation with an exponentially decaying pulse was employed to cotransfect this construct into HeLa S3 cells along with the plasmids pRSVcat and the neomycin-resistance plasmid pF beta fos3' neo. The level of transient expression of each plasmid was determined. Transfection efficiency was determined as percentage fluorescent cells by measurement of immunofluorescence with a chloramphenicol acetyltransferase (CAT) antibody. Efficiency of transfection ranged up to approximately 5% of the cells. Transfected cells were selected on the basis of resistance to Geneticin (G418) at 400 micrograms/mL. CAT fluorescence and Geneticin resistance were employed to select permanently transformed cell lines. Compared with exponentially growing cells, successfully transfected cell lines expressed more than twice the level of c-fos mRNA as determined by dot-blot analysis and 16 times more of the 62-kilodalton c-fos protein as determined by Western blot analysis. As all cells in the population were not stable c-fos transfectants, this value is likely to be an underestimate of the overexpression level. In addition, expression was under the control of a strong serum induction insensitive promoter, unlike the native c-fos promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
Pai
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Department of Pathobiology, Auburn University, AL 36849.
Bird
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D000123:Acetyltransferases / Q000235:genetics; D016895:Culture Media, Serum-Free; D015870:Gene Expression; D005813:Genes, Synthetic; D016762:Genes, fos; D005822:Genetic Vectors; D005839:Gentamicins / Q000494:pharmacology; D006367:HeLa Cells / Q000378:metabolism*; D006801:Humans; D007399:Interphase; D007613:Kanamycin Resistance; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection
D016895:Culture Media, Serum-Free; D005839:Gentamicins; D016760:Proto-Oncogene Proteins c-fos; D011993:Recombinant Fusion Proteins; C010680:antibiotic G 418; D000123:Acetyltransferases; C019681:aminoglycoside acetyltransferase