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Debora B Farber, Edouard Baulier; Generation of stem cell lines that can model human Ocular Albinism Type 1 by carrying a mutated or deleted OA1 gene.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2876.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the OA1 gene cause Ocular albinism type 1, a disease characterized by giant melanosomes in the RPE and a reduction in the ipsilateral projections of the optic nerve axons to the brain. The goal of our study was to establish an iPSC line resulting from reprogramming skin fibroblasts of an Ocular albinism patient, and H1 ESC lines with a knocked out OA1 gene.
A skin biopsy was obtained from a patient with an intronic mutation in the OA1 gene leading to an aberrant OA1 transcript (Vetrini, 2006). Sanger sequencing confirmed the mutation in the biopsy fibroblasts, which were reprogrammed using Sendai virus vectors. The resulting iPSCs (SEIi001-A) were karyotyped and analyzed using immunocytochemistry, flow cytometry and RT-PCR. After cell passage 10, neither Sendai virus-specific transcripts nor mycoplasma contamination were detected.To obtain H1 ESCs carrying a knocked out OA1 gene, gRNAs were combined in vitro with the Cas9 nuclease prior to transfection with the NEON electroporation system into H1 cells. Transfected cells were subjected to clonal selection. After an expansion phase, 62 clones each containing a unique colony originating from a single H1 cell were screened for OA1 and GAPDH (control) gene expression on agarose gels.
SEIi001-A iPSCs had typical embryonic stem cell-like phenotype and, after expansion, showed expression of SOX2, NANOG, TRA1-81, OCT4, SSEA4 and TRA1-60. No chromosomal abnormalities were observed in the cells’ karyotype, and they could differentiate in vitro to endoderm, mesoderm and ectoderm lineages. The aberrant OA1 transcript was detected in the iPSCs. STR analyses performed on the patient’s fibroblasts and the iPSCs at 16 different loci showed a perfect match between the two cell lines.Absence of the OA1 band while the GAPDH band was still present in 3 out of the 62 H1 ESC clones screened indicated that OA1 had been successfully knocked out in those cells.
The SEIi001-A iPSC line that we have generated containing a mutation in the OA1 gene as well as the 3 H1-ESC lines with the knocked-out OA1 gene are new tools that will allow us to carry out screenings in a dish for potential therapeutic candidates for Ocular Albinism and also studies of pathways/mechanisms that will increase our understanding of this disease.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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