Abstract
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.
Highlights
The transfer of genes into primary murine adipocytes using an adenovirus system has been developed
In this article we report the methods necessary to achieve a high-efficiency transfer of genes into murine adipocytes by adenovirusmediated gene transfer and, using this technique, we demonstrate that phorbol ester transcriptionally regulates the keratinocyte lipid-binding protein (KLBP/FABP5) gene
All animal protocols were approved by the Institutional Animal Care and Use Committee. Adipocytes isolated in this manner were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 1.5-mL Eppendorf tubes covered with aluminum foil
Summary
Primary adipocytes were prepared from C57BL/6J mice as follows: gonadal fat pads were dissected from a minimum of three mice, minced, and digested with collagenase (type II collagenase, 1 mg/mL; Sigma, St. Louis, MO) for 1 h at 37ЊC in Krebs– Ringer–HEPES (KRH) buffer [4,5] supplemented with bovine serum albumin (BSA, 10 mg/mL) and 5 mm glucose. Adipocytes were isolated by repeated (three or four) washes with supplemented KRH buffer followed by centrifugation at 4,000 rpm for 10 min. The floating adipocytes were recovered and subjected to a final wash in Dulbecco’s modified Eagle’s medium (DMEM). The viability of the adipocytes was determined by tr ypan blue exclusion analysis. All animal protocols were approved by the Institutional Animal Care and Use Committee
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
