Nucleofection Mediates High-Efficiency Stable Gene Knockdown and Transgene Expression in Human Embryonic Stem Cells

Department of Biological Chemistry, Sue and Bill Gross Stem Cell Research Program, Center for Molecular and Mitochondrial Medicine and Genetics, University of California Irvine, Irvine, California 92697, USA.
Stem Cells (Impact Factor: 7.7). 07/2008; 26(6):1436-43. DOI: 10.1634/stemcells.2007-0857
Source: PubMed

ABSTRACT High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells. Disclosure of potential conflicts of interest is found at the end of this article.

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Available from: April D Pyle, Sep 23, 2014
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    • "H7 hESCs expressed significantly higher levels of the TSC1=TSC2 inhibitory complex compared to their differentiated progeny (Fig. 6B). To further demonstrate that restricting mTORC1 signaling is critical for the maintenance of pluripotency in hESCs, we introduced siRNA directed at TSC2 and Rictor in an attempt to upregulate mTORC1 activity using the Amaxa Nucleofector II system (Hohenstein et al., 2008). H7 hESCs nucleofected with either TSC2 or Rictor siRNA alone showed no change in colony formation, Oct4 expression, or increased p70 S6K activation, although hESC colonies were slightly smaller in size with Rictor siRNA (data not shown). "
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    • "The presence of these neurotrophins in culture medium increased clonal survival through antiapoptotic effects and stabilized chromosome ploidy after high-throughput passaging. Indeed, the presence of these factors significantly improved the stable transfection efficiency mediated by nucleofection (Hohenstein et al., 2008). A selective inhibitor of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, the Y-27632 compound, was also reported to increase survival of dissociated hESCs by reducing apoptosis, thus promoting clonogenicity (Watanabe et al., 2007). "
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