Efficient adenovirus transduction of 3T3-L1 adipocytes stably expressing coxsackie-adenovirus receptor.
3T3-L1 adipocytes have proven difficult to transfect with plasmid-encoded cDNAs or even infect with virally-derived cDNAs. We have developed and characterized a 3T3-L1 adipocyte cell line stably expressing the truncated receptor for coxsackievirus and adenovirus receptor (CAR) for its ability to be infected with adenoviruses at a low multiplicity of infection (m.o.i.). Using green fluorescent protein driven by the cytomegalovirus promoter in adenovirus fiber type 5 we compared infection efficiencies of CAR adipocytes versus the parental 3T3-L1 adipocytes. As assessed by immunofluorescence, CAR adipocytes were infected at approximately 100-fold greater efficiency than regular 3T3-L1 adipocytes. The efficiency of transduction for the CAR adipocytes was >90% at multiplicities of infection of 50 whereas standard adipocytes were poorly transduced even at an m.o.i. of 2000. Since many investigators studying insulin action use 3T3-L1 adipocytes, we compared CAR adipocytes versus regular adipocytes and showed that the two cell lines were similar with respect to insulin stimulation of insulin receptor, MAPK, and Akt phosphorylation and basal- and insulin-stimulated glucose transport. In addition, CAR adipocytes accumulated GLUT4 and SCD1 proteins during the adipogenesis program with the same time course as regular 3T3-L1 adipocytes. Lastly, CAR adipocytes produced and secreted the adipose-specific hormone Acrp30. These data suggest 3T3-L1CARDelta1 adipocytes are virtually indistinguishable from their parental cells, but demonstrate a significant advantage with improved efficiency of adenoviral transduction for gain or deletion of function studies.
Ross
Stuart A
SA
Pharmacia Corporation, Cardiovascular and Metabolic Diseases, St. Louis, MO 63167, USA. stuart.a.ross@pharmacia.com
Song
Xiaomin
X
Burney
Mary W
MW
Kasai
Yumi
Y
Orlicky
David J
DJ
D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics*; D017667:Adipocytes / Q000378:metabolism / Q000821:virology*; D000818:Animals; D002478:Cells, Cultured; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D051379:Mice; D011991:Receptors, Virus / Q000378:metabolism*; D014161:Transduction, Genetic
C568772:CLMP protein, mouse; D062727:Coxsackie and Adenovirus Receptor-Like Membrane Protein; D011991:Receptors, Virus