G.Z. and S.R.A. contributed equally to this work.
Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 15 JAN 2009
Copyright © 2009 AlphaMed Press
Volume 27, Issue 4, pages 776–782, April 2009
How to Cite
Zafarana, G., Avery, S. R., Avery, K., Moore, H. D. and Andrews, P. W. (2009), Specific Knockdown of OCT4 in Human Embryonic Stem Cells by Inducible Short Hairpin RNA Interference. STEM CELLS, 27: 776–782. doi: 10.1002/stem.5
Author contributions: G.Z.: conception and design, collection and assembly of data, data analysis and interpretation; S.R.A.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; K.A.: collection and assembly of data; H.D.M.: provision of study material, final approval of manuscript; P.W.A.: financial support, data analysis and interpretation, manuscript writing, final approval of manuscript.
First published online in STEM CELLSExpress January 15, 2009; available online without subscription through the open access option.
- Issue online: 6 APR 2009
- Version of Record online: 15 JAN 2009
- Accepted manuscript online: 15 JAN 2009 12:00AM EST
- Manuscript Accepted: 20 DEC 2008
- Manuscript Received: 21 JUL 2008
- Biotechnology and Biological Sciences Research Council
- ESTOOLS Consortium under the Sixth Research Framework Program of the European Union. Grant Number: LSHG-CT-2006-018739
- Human embryonic stem cells;
- RNA interference;
- Inducible short hairpin RNAi;
Manipulation of gene function in embryonic stem cells by either over expression or downregulation is critical for understanding their subsequent cell fate. We have developed a tetracycline-inducible short hairpin RNA interference (shRNAi) for human embryonic stem cells (hESCs) and demonstrated doxycycline dose-dependent knockdown of the transcription factor OCT4 and the cell surface antigen β2-microglobulin. The induced knockdown of OCT4 resulted in rapid differentiation of hESCs with a significant increase in transcription of genes associated with trophoblast and endoderm lineages, the extent of which was controlled by the degree of induction. Transgene toxicity, which may occur in conditional over-expression strategies with hESCs, was not observed with wild-type Tet repressor protein. The system allows efficient, reversible, and long-term downregulation of target genes in hESCs and enables the generation of stable transfectants for the knockdown of genes essential for cell survival and self-renewal, not necessarily possible by nonconditional shRNAi methods. STEM CELLS 2009;27:776–782