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Ruchi Sharma, Vaishakh Rajan, Qin Wan, Kapil Bharti; Engineering Isogenic Best Vitelliform Macular Dystrophy Patient iPS cell lines using TALEN Technology. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3987.
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Best Vitelliform Macular Dystrophy (BVMD) is an autosomal dominant form of retinal degeneration caused by the mutations in VMD2 gene. The disease is initiated by functional defects in Retinal Pigment Epithelium (RPE) leading to photoreceptor degeneration and vision loss. The purpose of this study was to generate patient specific induced pluripotent stem (iPS) cell lines, genetically engineer them using gene-specific TALEN to generate isogenic controls, and differentiate them into authenticated RPE to identify mechanisms involved in disease onset and progression.
Patient specific iPS cell lines were generated using non-integrating Sendai-Virus containing SOX2, OCT3/4, KLF4, and c-MYC factors. The lines were authenticated using pluripotency factor gene expression (qRT-PCR and immunofluorescence) and three germ layer differentiation assay. Passage 15 iPS cells were genetically engineered using VMD2 gene specific TALEN, which disrupts the open reading frame of both VMD2 isoforms. iPS cells were transfected with TALEN plasmids using Neon Transfection system using 24 different transfection parameters. Mutated clones were screened using a PCR based assay.
BVMD patient (Trp 24 to Cys) iPS cell line showed strong expression of several pluripotency markers and differentiated into all three germ layers. Out of twenty-four transfection parameters tested, ten parameters gave rise to healthy iPS cell colonies. Sequencing of PCR fragments from genomic DNA and cDNA showed that TALEN disrupted VMD2 open reading frame by non-homologous end joining thus resulting in the complete knockout of one VMD2 allele. This leads to two isogenic controls, one WT-heterozygous and the other mutant-heterozygous. BVMD (Trp 24 to Cys) iPS cells have been successfully differentiated into RPE at high efficiency. Currently, these isogenic iPS cell lines derived RPE are being used to determine BVMD disease mechanisms.
Isogenic iPS cells provide genetically matched controls for patient-derived cells thus allowing the possibility to identify disease cellular endophenotypes in the absence of genetic modifier effects. TALEN technology is a viable approach to generate gene knock-out using non-homologous end joining. BVMD isogenic iPS cell lines generated in this study will help better understand the dominant role played by VMD2 mutant allele.
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