A cassette system to study ES cell differentiation by inducible RNA interference

¹ Institute for Medical Microbiology, Department of Biological and Clinical Sciences, University of Basel, Switzerland
² Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

^* To whom correspondence should be addressed. E-mail: Christoph.Moroni{at}unibas.ch.

	Abstract

While differentiation of pluripotent embryonic stem cells is restricted by a hierarchy of transcription factors, little is known whether posttranscriptional mechanisms similarly regulate early embryoid differentiation. We developed a system where shRNAs can be induced in ES cells from a defined locus following integration by Flp recombinase-mediated DNA recombination. To verify the system, the key transcription factor Stat3 which maintains pluripotency was downregulated by shRNA and the expected morphological and biochemical markers of differentiation were observed. Induction of shRNA specific for the posttranscriptional regulator Brf1 (Zfp36L1) amplified the cardiac markers with strong stimulation of cardiomyocyte formation within embryoid bodies. These findings identify Brf1 as a novel potential regulator of cardiomyocyte formation and suggest that posttranscriptional mechanisms are of importance to early development and possibly to regenerative medicine. The inducible RNA interference system presented here should also allow assignment of function for candidate genes with suspected roles in ES cell development.

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