Efficient integration of transgenes into a defined locus in human embryonic stem cells

Abstract

Random integration is one of the more straightforward methods to introduce a transgene into human embryonic stem (ES) cells. However, random integration may result in transgene silencing and altered cell phenotype due to insertional mutagenesis in undefined gene regions. Moreover, reliability of data may be compromised by differences in transgene integration sites when comparing multiple transgenic cell lines. To address these issues, we developed a genetic manipulation strategy based on homologous recombination and Cre recombinase-mediated site-specific integration. First, we performed gene targeting of the hypoxanthine phosphoribosyltransferase 1 (HPRT) locus of the human ES cell line KhES-1. Next, a gene-replacement system was created so that a circular vector specifically integrates into the targeted HPRT locus via Cre recombinase activity. We demonstrate the application of this strategy through the creation of a tetracycline-inducible reporter system at the HPRT locus. We show that reporter gene expression was responsive to doxycycline and that the resulting transgenic human ES cells retain their self-renewal capacity and pluripotency.

Efficient Integration of Transgenes and Their Reliable Expression in Human Embryonic Stem Cells

“…Selection was performed for 10-14 days post-electroporation. Out of the 424 G418-resistant clones screened, six (1.42%) had undergone the desired homologous recombination event (Table 1) (Sakurai et al, 2010). HR was confirmed by PCR and a Southern blotting analysis (Sakurai et al, 2010).…”

“…Out of the 424 G418-resistant clones screened, six (1.42%) had undergone the desired homologous recombination event (Table 1) (Sakurai et al, 2010). HR was confirmed by PCR and a Southern blotting analysis (Sakurai et al, 2010). One targeted clone, named K1-HS, was randomly selected and used for subsequent gene replacement.…”

Embryonic stem cell application in drug discovery

“…Low-level residual expression of reprogramming factors may alter the differentiation potential of human induced pluripotent stem (iPS) cells. Indeed, both the viral vectors used for gene transfer and the encoded reprogramming factors are probably oncogenic and possess low transduction efficiency [43] . Some small molecules can replace transcription factors, and the combined activity of transcription factors can reprogram adult cells into iPS cells.…”

“…For example, one study using a high-throughput/content screen showed that a TGF-β inhibitor replaced Sox2 in reprogramming and produced unmodified iPS cell lines [44] . Therefore, small-molecule replacement of transcription factors may be one potential solution to lower the oncogenic potential and increase the reprogramming efficiency [39,43,44] .…”

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the <em>AAVS1</em> Locus

“…The mouse orthologous HPRT, despite its association with the loss-offunction Lesch-Nyhan Syndrome, and ROSA26 have been targeted in hPSCs. HPRT was reported to rapidly silence transgene expression in ESC and, like ROSA26, its ability to sustain transgene expression in terminally differentiated cells was not investigated [5][6][7] . Because a homozygous null mutation of the CCR5 gene appears to be well tolerated in humans, its value as safe harbor was assessed.…”