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Conor Ramsden, Amanda-Jayne Francis Carr, Amelia Lane, Michael Powner, Britta Nommitse, Lyndon da Cruz, Pete Coffey; Characterisation of an induced pluripotent stem cell derived retinal pigment epithelial line derived from a subject with a MerTK defect hereditary retinopathy.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1836.
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© ARVO (1962-2015); The Authors (2016-present)
Hereditary retinopathy is now the most common cause for blind registration in the working population in the UK. In order to combat this heterogenous group of diseases, adequate disease models need to be developed in order to better understand physiology and to test potential therapies. Our subject is a previously genotyped with two mutations in the MerTK gene, an established premature stop mutation in exon 14 and a previously unreported mutation in intron 1 which has been predicted to interfere with splicing. The MerTK protein is expressed on the apical surface of the RPE cells and is regarded as an essential component of the phagocytic machinery.
Using plasmids, the Yamanaka factors were expressed in the fibroblasts of a patient with RP38 to generate pluripotent stem cells. Using established protocols, these cells were driven towards an RPE fate. The cells were interrogated with antibodies to RPE markers and gene expression checked with reverse transcription PCR.
The iPS derived RPE form pigmented monolayers and express the RPE genes PAX6, RPE65, OTX2, PMEL17, BEST, CRALBP and MERTK. Furthermore, all of these proteins were present except MerTK. The MerTK protein was noted to be present in a control line. The entire gene was amplified by PCR and the products of the PCR noted to be identical in size between the disease iPS line and the control line.
Using our tissue specific disease model, we dispute a previous prediction that the mutation in intron 1 interferes with splicing. Going forward, our intention is to use this disease model to further examine the role of MerTK in phagocytosis and test read through agents to gain expression of MerTK and ameliorate disease phenotype.
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