Abstract
Human embryonic stem cells (hESCs) have great promise as a source of unlimited transplantable cells for regenerative medicine. However, current progress on producing the desired cell type for disease treatment has been limited due to an insufficient understanding of the developmental processes that govern their differentiation, as well as a paucity of tools to systematically study differentiation in the lab. In order to overcome these limitations, cell-type reporter hESC lines will be required. Here we outline two strategies using Transcription Activator Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated protein (Cas) to create OCT4-eGFP knock-in add-on hESC lines. Thirty-one and forty-seven percent of clones were correctly modified using the TALEN and CRISPR-Cas9 systems, respectively. Further analysis of three correctly targeted clones demonstrated that the insertion of eGFP in-frame with OCT4 neither significantly impacted expression from the wild type allele nor did the fusion protein have a dramatically different biological stability. Importantly, the OCT4-eGFP fusion was easily detected using microscopy, flow cytometry and western blotting. The OCT4 reporter lines remained equally competent at producing CXCR4+ definitive endoderm that expressed a panel of endodermal genes. Moreover, the genomic modification did not impact the formation of NKX6.1+/SOX9+ pancreatic progenitor cells following directed differentiation. In conclusion, these findings demonstrate for the first time that CRISPR-Cas9 can be used to modify OCT4 and highlight the feasibility of creating cell-type specific reporter hESC lines utilizing genome-editing tools that facilitate homologous recombination.
Highlights
Embryonic stem cells (ESCs) are pluripotent cells located in the inner cell mass of early embryos that have the capacity for long-term self-renewal and the ability to form all cell types of the embryo proper
One potential method to address both of these issues is the generation of reporter Human embryonic stem cells (hESCs) lines that facilitate the study of human development in culture and to allow for high throughput, high content screens to uncover factors that drive differentiation
While several landmark papers have described the generation of reporter lines using these technologies [20,21,22], no studies have thoroughly investigated the effects of the genomic modification on stem cell characteristics or directed differentiation potential
Summary
Embryonic stem cells (ESCs) are pluripotent cells located in the inner cell mass of early embryos that have the capacity for long-term self-renewal and the ability to form all cell types of the embryo proper. A better approach is to knock-in a reporter gene downstream, but in-frame with the protein of interest, allowing for marker expression driven by the endogenous promoter without altering expression of the targeted gene This strategy was previously difficult in hESCs due to the low rate of homologous recombination and the requirement for very large homology arms [16]. With the recent advent of three high efficiency genome editing technologies, Zinc Finger Nucleases (ZFNs), Transcription Activator Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPRAssociated protein (Cas), genome editing is fast becoming a reality in human ESCs [17,18,19] These technologies utilize sequence-specific (10–30 bp in length) nucleases to create a double stranded break in the DNA, which dramatically increases the frequency of homologous recombination through homology directed repair. While several landmark papers have described the generation of reporter lines using these technologies [20,21,22], no studies have thoroughly investigated the effects of the genomic modification on stem cell characteristics or directed differentiation potential
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