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PLoS One. 2012;7(6):e37680. doi: 10.1371/journal.pone.0037680. Epub 2012 Jun 5.

A novel RNAi lethality rescue screen to identify regulators of adipogenesis.

Author information

1
Department of Metabolic Diseases, Netherlands Metabolomics Centre, University Medical Centre Utrecht, Utrecht, The Netherlands.

Abstract

Adipogenesis, the differentiation of fibroblast-like mesenchymal stem cells into mature adipocytes, is tightly regulated by a complex cascade of transcription factors, including the nuclear receptor Peroxisome proliferator activator receptor γ (PPARγ). RNAi-mediated knock down libraries may present an attractive method for the identification of additional adipogenic factors. However, using in vitro adipogenesis model systems for high-throughput screening with siRNA libraries is limited since (i) differentiation is not homogeneous, but results in mixed cell populations, and (ii) the expression levels (and activity) of adipogenic regulators is highly dynamic during differentiation, indicating that the timing of RNAi-mediated knock down during differentiation may be extremely critical. Here we report a proof-of-principle for a novel RNAi screening method to identify regulators of adipogenesis that is based on lethality rescue rather than differentiation, using microRNA expression driven by a PPARγ responsive RNA polymerase II promoter. We validated this novel method through screening of a dedicated deubiquitinase knock down library, resulting in the identification of UCHL3 as an essential deubiquitinase in adipogenesis. This system therefore enables the identification of novel genes regulating PPARγ-mediated adipogenesis in a high-throughput setting.

PMID:
22679485
PMCID:
PMC3367974
DOI:
10.1371/journal.pone.0037680
[Indexed for MEDLINE]
Free PMC Article
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