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Journal of Lipid Research, Vol. 48, 465-471, February 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

RNAi-based gene silencing in primary mouse and human adipose tissues

Vishwajeet Puri^*, Abhijit Chakladar^*, Joseph V. Virbasius^*, Silvana Konda^*, Aimee M. Powelka^*, My Chouinard^*, G. Nana Hagan^*, Richard Perugini and Michael P. Czech^1,*

^* Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605
Department of Surgery, University of Massachusetts Medical School, Worcester, MA 01605

Published, JLR Papers in Press, November 8, 2006.

¹ To whom correspondence should be addressed. e-mail: Michael.Czech{at}umassmed.edu

Cultured adipocyte cell lines are a model system widely used to study adipose function, but they exhibit significant physiological differences compared with primary cells from adipose tissue. Here we report short interfering RNA-based methodology to selectively attenuate gene expression in mouse and human primary adipose tissues as a means of rapidly validating findings made in cultured adipocyte cell lines. The method is exemplified by depletion of the PTEN phosphatase in white adipose tissue (WAT) from mouse and humans, which increases Akt phosphorylation as expected. This technology is also shown to silence genes in mouse brown adipose tissue. Previous work revealed upregulation of the mitochondrial protein UCP1 in adipose cells from mice lacking the gene for the transcriptional corepressor RIP140, whereas in cultured adipocytes, loss of RIP140 has a little effect on UCP1 expression. Application of our method to deplete RIP140 in primary mouse WAT elicited markedly increased oxygen consumption and expression of UCP1 that exactly mimics the phenotype observed in RIP140-null mice. This ex-vivo method of gene silencing should be useful in rapid validation studies as well as in addressing the depot- and species-specific functions of genes in adipose biology.

Supplementary key words RNA interference • mitochondrial proteins • receptor-interacting protein 140 • Akt

Abbreviations: BAT, brown adipose tissue; siRNA, short interfering RNA; WAT, white adipose tissue

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