A Novel RNAi Lethality Rescue Screen to Identify Regulators of Adipogenesis

PLoS One

Olivier van Beekum¹  Ruud Berger¹  Eric Kalkhoven¹  Yuan Gao¹  Arjen Koppen¹ 

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

关键词: MicroRNAs;    Small interfering RNAs;    Adipocytes;    Library screening;    Cell differentiation;    Adipocyte differentiation;    RNA interference;    Regulator genes;   

DOI  :  10.1371/journal.pone.0037680

学科分类：医学（综合）

来源: Public Library of Science

【 摘 要 】

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 atractive 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.

【 授权许可】

CC BY