Efficient adenovirus transduction of 3T3-L1 adipocytes stably expressing coxsackie-adenovirus receptor

• Published in: Biochemical and biophysical research communications 2003-03, Vol.302 (2), p.354-358
• Main Authors: Ross, Stuart A, Song, Xiaomin, Burney, Mary W, Kasai, Yumi, Orlicky, David J
• Format: Article
• Language: English
• Subjects: 3T3 Cells; 3T3-L1 adipocytes; Adenoviridae - genetics; Adenovirus; Adipocytes - metabolism; Adipocytes - virology; Animals; Cells, Cultured; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Mice; Receptors, Virus - metabolism; Transduction; Transduction, Genetic
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/S0006-291X(03)00180-3

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 ∼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-L1CARΔ1 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.