Previously, we created EF1α-EmGFP hESC lines integrated at chromosome 13q32.3, a predetermined genomic locus safe for transgene integration [7
]. Although GFP expression was maintained in various early lineage precursors, gene silencing was observed when these lines were terminally differentiated into somatic cell types, such as dopaminergic neurons. To circumvent this problem, we flanked the genes of interest by double-insulator sequences, providing a shelter for exogenous promoters from possible epigenetic modifications that might interfere with transgene expression. In these double-insulated reporter lines, GFP expression remained robust throughout the courses of directed differentiation into NSCs, dopaminergic neurons, pancreatic endoderm, and mesodermal lineages.
That GFP expression was maintained without any reduction through dopaminergic, pancreatic, and mesodermal differentiation not only shows that our double-insulated retargeting system is constitutive in various differentiated cell types, but also provides a powerful tool to further study in these lineages. A GFP-labeled, hESC-derived dopaminergic neuron population is highly desirable for investigating dopaminergic lineage development in human. Although TH+ cells derived from mouse ESCs or abnormal hESCs that are genetically tagged with GFP (driven by CMV or EF1α promoter) retain GFP expression [29
], such hESC lines with normal karyotype have not been created. Here, our double-insulated clone is able to be differentiated into TH+ GFP-expressing dopaminergic cells (). These cells have the potential to facilitate in vitro dopaminergic differentiation experiments and in vivo tracking after transplantation, making it feasible to directly monitor survival, integration, and striatal circuitry reconstruction of grafted cells, which may lead to identification of critical factors that modulate the degenerative brain environment, hence providing clues for hPSC-based therapy for Parkinson's patients.
Similar to dopaminergic derivation, the differentiation of hESCs toward pancreatic lineages is a lengthy in vitro process, which might alter transgenes genetically or epigenetically, causing gene silencing and preventing the application of genetic tags in such lineages. The data presented here offer a GFP-labeled population of pancreatic endoderm cells and provide a defined genomic locus and an effective engineering strategy for potential generation of pancreatic lineage-specific reporters in hPSCs in future investigations. Such reporters will be applied to optimization of differentiation protocols, obtaining purified insulin-producing cells from heterogeneous populations, as well as identification of key factors important for pancreatic endocrine differentiation in vitro.
Clues provided in previous reports suggest that insulators function through blocking de novo
DNA methylation to ensure stable and consistent transgene expression over extended culture [31
]. To pinpoint the mechanism that cHS4 insulator elements act in our hESC clones in particular, we compared the DNA methylation status of both endogenous and exogenous EF1α promoter in uninsulated and insulated EF1α-GFP clones. Our results indicate that prevention or reduction of DNA methylation of transgene promoters only contributes partially to the protection of insulator elements in this system (); other possible mechanisms such as changes of status in histone acetylation between the insulated and uninsulated clones after differentiation will be examined. Our system, with the ability to target only one copy of transgene at a defined genomic locus, has provided a platform allowing for further investigation along this line, since uncontrolled transgene copy number has also been reported to interfere with epigenetic modifications [32
]. Additional experiments to investigate the comprehensive mechanisms that different types of chromatin insulators establish to shield transgenes from silencing in hPSCs are of interest.
There are 2 major components of the integrating strategy that ensure appropriate regulation of transgenes in the retargeted line: (1) the identification of the safe integration locus at chromosome 13q32.3 (within the intron of the CLYBL gene), and (2) the use of chromatin insulator sequences to flank the gene of interest, supplying another layer of protection from undesired regulation that might cause gene silencing. These data indicate that chromatin insulators facilitate the appropriate transgene expression in hESCs and reduce the interference caused by adjacent strong promoters.
Of further interest is whether these chromatin insulator sequences function in a broader context, including hiPSC lines or other vector constructs for different genetic engineering purposes. The chromosome 13 site is the only genomic locus we have tested for insulating ability, although we have tested the same locus in at least 2 different hESC lines, H9 and BG01V (an abnormal hESC line with a karyotype of 48XY,
+17, but otherwise shares all of the hESC characteristics). Based on the data from episomal vectors () and previous reports from other groups, who used lentiviral [14
] or adenoviral transduction [12
], it would be reasonable to predict that the insulating strategy could be extended to other loci and various genetic engineering systems.
In conclusion, our current work and previous work [7
] show detailed and extensive characterization of a genetic manipulation system where a safe docking site is identified in the hESC genome and an optimized double-insulator retargeting strategy is provided. The system has the capacity to accept large, multigenic elements, and can be combined with Cre-Lox and Flp-FRT system to make versatile tools for additional genetic manipulations. The system ensures appropriate regulation of transgene expression and significantly reduces possible gene silencing as shown for a variety of hESC derivatives.