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Wei Liu, Albert Lowe, Ales Cvekl; Generation of a VSX2::EGFP reporter cell line for modeling human retinal differentiation. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4572.
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© ARVO (1962-2015); The Authors (2016-present)
In murine retinal development, homeodomain transcription factor Vsx2 (also known as Chx10) is a specific marker for newly specified retinal progenitor cells (RPCs) in the inner layer of optic cups. In our in vitro retinal differentiation system, newly specified VSX2+ RPCs self-organize into apically convex epithelium, and substantially pure VSX2+ epithelium has an intrinsic propensity to differentiate into stratified neuroretina in floating cultures (Lowe et al., 2016). Accordingly, the generation and isolation of VSX2+ epithelium is the critical step in retinal differentiation in vitro. This study aims to generate VSX2::EGFP H1 hESCs to investigate human retinal differentiation.
Genetic engineering of undifferentiated H1 hESCs using CRISPR/Cas9 technology was performed. The engineered hESCs were used for retinal differentiation following a novel procedure that was recently established in our laboratory.
VSX2::EGFP-Puro H1 hESCs were generated by nucleofection with a homology directed repair (HDR) vector containing a 2A-EGFP-Puro cassette targeting the VSX2 locus immediately before the stop codon and a PX458 vector containing sgRNA. Puromycin was used for selecting electroporated H1 hESCs. Targeted hESCs were identified using PCR with primer pairs detecting homologous recombination at the left and right homology arm, respectively. Homozygous VSX2::EGFP-Puro H1 hESCs were identified when the native allele was absent. Then, VSX2::EGFP hESCs were generated after CRE-mediated removal of Puro cassette. VSX2::EGFP H1 hESCs were used for retinal differentiation following our procedure. In the retinal cultures, VSX2::EGFP recapitulates VSX2 expression, which is revealed by dual immunostaining. Live imaging shows that VSX2::EGFP is specifically present in retinal organoids following Dispase-mediated cell detachment, confirming self-organization of VSX2+ epithelium we described recently (Lowe et al., 2016).
We have generated a VSX2::EGFP reporter cell line that has multiple applications in modeling human retinal differentiation and disease.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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