Creation of Engineered Human Embryonic Stem Cell Lines Using phiC31 Integrase

Abstract

It has previously been shown that the phage-derived phiC31 integrase can efficiently target native pseudo-attachment sites in the genome of various species in cultured cells, as well as in vivo. To demonstrate its utility in human embryonic stem cells (hESC), we have created hESC-derived clones containing expression constructs. Variant human embryonic stem cell lines BG01v and SA002 were used to derive lines expressing a green fluorescent protein (GFP) marker under control of either the human Oct4 promoter or the EF1␣ promoter. Stable clones were selected by antibiotic resistance and further characterized. The frequency of integration suggested candidate hot spots in the genome, which were mapped using a plasmid rescue strategy. The pseudo-attP profile in hESC differed from those reported earlier in differentiated cells. Clones derived using this method retained the ability to differentiate into all three germ layers, and fidelity of expression of GFP was verified in differentiation assays. GFP expression driven by the Oct4 promoter recapitulated endogenous Oct4 expression, whereas persistent stable expression of GFP expression driven by the EF1␣ promoter was seen. Our results demonstrate the utility of phiC31 integrase to target pseudo-attP sites in hESC and show that integrase-mediated site-specific integration can efficiently create stably expressing engineered human embryonic stem cell clones. STEM CELLS 2008;26:119 -126 Disclosure of potential conflicts of interest is found at the end of this article.

A regulatory toolbox of MiniPromoters to drive selective expression in the brain

“…Ultimately, the greatest impact of the Pleiades MiniPs is anticipated to be the added specificity for therapeutic gene delivery into the human brain. Although this may be accomplished using viruses, site-specific delivery to the human genome directly or in cell therapy is an area of active research (27,28) and the suitability of the Pleiades promoters for such therapeutic delivery will require further study. The availability of a large collection of new MiniPs will play an important role in research and treatment for incurable brain diseases.…”

Chromatin Insulator Elements Block Transgene Silencing in Engineered Human Embryonic Stem Cell Lines at a Defined Chromosome 13 Locus

“…The insertion of the plasmid to chromosome 13q32 at the second intron of the CLYBL gene was identified by plasmid rescue, which was described in [8]. The uninsulated retargeting plasmid pJTI-R4-EG that contained pEF1a-EmGFP was cloned as described [7].…”

“…Generation of the platform lines and the retargeted hESC lines was described previously [7,8]. Briefly, R4 cells were harvested using TrypLE and 1 · 10 6 cells were electroporated using a Neon microporator (Life Technologies) at 850 V, 30 ms, 1 pulse or ECM830 electroporator (BTX, 200 V at 10 ms, 2 pulses) with 10 mg of retargeting constructs.…”

“…We have previously established platform lines from a normal hESC line WA09 and an abnormal hESC line BG01V at the chromosome 13q32.3 locus by PhiC31 integrase-mediated recombination and created several reporter lines driven by an EF1a promoter [7,8,23]. Expression of reporters in these hESC lines was maintained in long-term culture at undifferentiated state.…”

“…We inserted transgenes [eg, an emerald green fluorescent protein (EmGFP) cassette] precisely and efficiently to this site via recombination mediated by integrases of the PhiC31 family [7,8]. We have shown that expression at the chromosome 13q32.3 locus remained constant and was not silenced through random differentiation by embryoid body (EB) formation and directed differentiation toward neural stem cells (NSCs).…”