Adenovirus-mediated gene transfer in primary murine adipocytes.
The transfer of genes into primary murine adipocytes using an adenovirus system has been developed. A recombinant adenovirus was constructed (expressing green fluorescent protein [GFP] under the control of the strong cytomegalovirus [CMV] promoter and a luciferase reporter gene under the control of the weak adipocyte promoter keratinocyte lipid-binding protein [KLBP/FABP5]) and incubated with primary adipocytes from C57BL/6J mice. Analysis of infected cells by confocal microscopy detected GFP expression in both the cytoplasm and nucleus of adipocytes with a 64% efficiency of infection. To demonstrate the applicability of this method in the study of gene regulation, adenovirus-infected adipocytes exhibited significant levels of luciferase activity even from a weak promoter. TPA treatment of infected adipocytes increased luciferase activity, consistent with previous studies indicating that the KLBP/FABP5 gene is up-regulated by phorbol esters. These results provide an efficient, convenient, and sensitive method to transiently infect primary murine adipocytes, facilitating protein expression or permitting analysis of reporter gene activity from both viral and endogenous promoters.
Hertzel
A V
AV
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul 55108, USA.
Sanders
M A
MA
Bernlohr
D A
DA
D000256:Adenoviridae; D017667:Adipocytes / Q000166:cytology; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002478:Cells, Cultured; D050556:Fatty Acid-Binding Proteins; D005786:Gene Expression Regulation / Q000187:drug effects; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008156:Luciferases / Q000235:genetics; D018996:Myelin P2 Protein / Q000096:biosynthesis* / Q000235:genetics; D009363:Neoplasm Proteins; D010703:Phorbol Esters / Q000494:pharmacology
D002352:Carrier Proteins; D050556:Fatty Acid-Binding Proteins; D018996:Myelin P2 Protein; D009363:Neoplasm Proteins; D010703:Phorbol Esters; D008156:Luciferases