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: 6.52). 07/2008; 26(6):1436-43. DOI: 10.1634/stemcells.2007-0857
Source: PubMed


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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    • "Transgenic stem cells using the homologous recombination technique were first reported in mouse embryonic stem cells by Thomas & Capecchi in 1987 [19]. Subsequently, researchers have successfully delivered transgenes into pluripotent stem cells using several methods, including electroporation [20], liposomal [21] and viral vectors [22], [23], and nucleofection [24]. However, stably introducing transgenes in these cells has proven difficult because of the low efficiency and cytotoxic side effects. "
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    ABSTRACT: Because of the physiological and immunological similarities that exist between pigs and humans, porcine pluripotent cell lines have been identified as important candidates for preliminary studies on human disease as well as a source for generating transgenic animals. Therefore, the establishment and characterization of porcine embryonic stem cells (pESCs), along with the generation of stable transgenic cell lines, is essential. In this study, we attempted to efficiently introduce transgenes into Epiblast stem cell (EpiSC)-like pESCs. Consequently, a pluripotent cell line could be derived from a porcine-hatched blastocyst. Enhanced green fluorescent protein (EGFP) was successfully introduced into the cells via lentiviral vectors under various multiplicities of infection, with pluripotency and differentiation potential unaffected after transfection. However, EGFP expression gradually declined during extended culture. This silencing effect was recovered by in vitro differentiation and treatment with 5-azadeoxycytidine. This phenomenon was related to DNA methylation as determined by bisulfite sequencing. In conclusion, we were able to successfully derive EpiSC-like pESCs and introduce transgenes into these cells using lentiviral vectors. This cell line could potentially be used as a donor cell source for transgenic pigs and may be a useful tool for studies involving EpiSC-like pESCs as well as aid in the understanding of the epigenetic regulation of transgenes.
    PLoS ONE 08/2013; 8(8):e72184. DOI:10.1371/journal.pone.0072184 · 3.23 Impact Factor
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    • "For RNAi in hES cells, nucleofection was performed as previously described (Hohenstein et al., 2008). hES cells were transfected with 100 μM of short interfering RNAs (siRNAs) corresponding to SOX2 and β2M (Qiagen, Germantown, MD). "
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    ABSTRACT: Human embryonic stem (hES) cells have the dual ability to self-renew and differentiate into specialized cell types. However, in order to realize the full potential of these cells it is important to understand how the genes responsible for their unique characteristics are regulated. In this study we examine the regulation of the tropomyosin-related kinase (TRK) genes which encode for receptors important in hES cell survival and self-renewal. Although the TRK genes have been studied in many neuronal cell types, the regulation of these genes in hES cells is unclear. Our study demonstrates a novel regulatory relationship between the TRKC gene and the transcription factor SOX2. Our results found that hES cells highly express full-length and truncated forms of the TRKC gene. However, examination of the related TRKB gene showed a lower overall expression of both full-length and truncated forms. Through RNA interference, we knocked down expression levels of SOX2 in hES cells and examined the expression of TRKC, as well as TRKB. Upon loss of SOX2 we found that TRKC mRNA levels were significantly downregulated but TRKB levels remained unchanged, demonstrating an important regulatory dependence on SOX2 by TRKC. We also found that TRKC protein levels were also decreased after SOX2 knock down. Further analysis found the regulatory region of TRKC to be highly conserved among many mammals with potential SOX binding motifs. We confirmed a specific binding motif as a site that SOX2 utilizes to directly interact with the TRKC regulatory region. In addition, we found that SOX2 drives expression of the TRKC gene by activating a luciferase reporter construct containing the TRKC regulatory region and the SOX binding motif.
    Stem Cell Research 03/2012; 8(2):206-14. DOI:10.1016/j.scr.2011.10.003 · 3.69 Impact Factor
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    • "Studies of the molecular mechanisms regulating hESC pluripotency could be helpful in this regard. Several transcription factors are known to be important regulators of pluripotency and self-renewal in hESC, including Oct4, Sox2 and Nanog [3], [4], [5], [6]. These three transcription factors are able to regulate the expression of each other, effectively forming a core transcriptional network governing pluripotency of hESC [7]. "
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    ABSTRACT: Human embryonic stem cells (hESC) are stem cells capable of differentiating into cells representative of the three primary embryonic germ layers. There has been considerable interest in understanding the mechanisms regulating stem cell pluripotency, which will ultimately lead to development of more efficient methods to derive and culture hESC. In particular, Oct4, Sox2 and Nanog are transcription factors known to be important in maintenance of hESC. However, many of the downstream targets of these transcription factors are not well characterized. Furthermore, it remains unknown whether additional novel stem cell factors are involved in the establishment and maintenance of the stem cell state. Here we show that a novel gene, L1TD1 (also known as FLJ10884 or ECAT11), is abundantly expressed in undifferentiated hESC. Differentiation of hESC via embryoid body (EB) formation or BMP4 treatment results in the rapid down-regulation of L1TD1 expression. Furthermore, populations of undifferentiated and differentiated hESC were sorted using the stem cell markers SSEA4 and TRA160. Our results show that L1TD1 is enriched in the SSEA4-positive or TRA160-positive population of hESC. Using chromatin immunoprecipitation we found enriched association of Nanog to the predicted promoter region of L1TD1. Furthermore, siRNA-mediated knockdown of Nanog in hESC also resulted in downregulation of L1TD1 expression. Finally, using luciferase reporter assay we demonstrated that Nanog can activate the L1TD1 upstream promoter region. Altogether, these results provide evidence that L1TD1 is a downstream target of Nanog. Taken together, our results suggest that L1TD1 is a downstream target of Nanog and represents a useful marker for identifying undifferentiated hESC.
    PLoS ONE 04/2011; 6(4):e19355. DOI:10.1371/journal.pone.0019355 · 3.23 Impact Factor
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