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Abstract
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.
Highlights
Human embryonic stem cells are powerful tools for the in vitro study of early human development, and provide a source of cells with therapeutic potential in regenerative medicine [1, 2]
We have developed a tetracycline-inducible short hairpin RNA interference for human embryonic stem cells and demonstrated doxycycline dose-dependent knockdown of the transcription factor OCT4 and the cell surface antigen b2-microglobulin
The induced knockdown of OCT4 resulted in rapid differentiation of human embryonic stem cells (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
Summary
Human embryonic stem cells (hESCs) are powerful tools for the in vitro study of early human development, and provide a source of cells with therapeutic potential in regenerative medicine [1, 2]. The molecular mechanisms controlling survival, self-renewal, and cell-fate decisions are not clearly defined, and strategies designed to control gene expression by either loss or gain-of-function are invaluable tools for their study. Problems associated with transfection efficiencies in hESCs [3, 4] can hinder the use of transient systems to control gene expression, making the generation of stablyexpressed transgenes a favorable option. Difficulties may arise if stably-expressed transgenes adversely regulate hESC survival, proliferation, or self-renewal. Inducible over expression of transgenes has been performed in hESCs using Tet [7, 8] and Cre recombinase-based systems [9, 10], but problems with the toxicity were encountered with constitutively expressed Tet activator and Cre in hESCs
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