Transfection of DNA into isolated rat adipose cells by electroporation: evaluation of promoter activity in transfected adipose cells which are highly responsive to insulin after one day in culture.
Isolated adipose cells are among the most insulin responsive cells with respect to glucose transport and metabolism. However, molecular biological techniques such as transfection of DNA have heretofore not been applied successfully in these cells in primary culture. We report a method for transfection of DNA into rat adipose cells by electroporation. Six shocks at 800 V and 25 microF in a 0.4 cm gap cuvette results in efficient transfection. We compared the ability of five promoters to drive expression of a luciferase reporter gene in transfected adipose cells. After one day in culture, promoter activity ranged from no expression to a very high level of expression. These transfected, cultured cells also displayed a 10-fold increase in 3-O-methylglucose transport with maximal insulin stimulation. The ability to transfect DNA into adipose cells which remain insulin responsive after one day in primary culture may be helpful for understanding adipose cell-specific gene regulation and elucidating the molecular mechanisms of insulin action.
Quon
M J
MJ
Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Zarnowski
M J
MJ
Guerre-Millo
M
M
de la Luz Sierra
M
M
Taylor
S I
SI
Cushman
S W
SW
D019325:3-O-Methylglucose; D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D004558:Electric Stimulation / Q000379:methods; D015870:Gene Expression / Q000187:drug effects; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008757:Methylglucosides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection / Q000379:methods*
D007328:Insulin; D008757:Methylglucosides; D011994:Recombinant Proteins; D019325:3-O-Methylglucose; D008156:Luciferases; D011972:Receptor, Insulin