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Majlinda Lako, Joseph Collin, Carla Bernadette Mellough, Birthe Dorgau; Using zinc finger nuclease technology to generate reporter-labelled human pluripotent stem cells as a tool to optimize photoreceptor transplantation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1822.
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© ARVO (1962-2015); The Authors (2016-present)
To construct human pluripotent cell lines in which the Green Fluorescent protein (GFP) reporter gene is introduced into the endogenous loci of a key transcription factor namely Cone-Rod Homeobox (CRX) shown to be expressed in post-mitotic photoreceptor precursors.<br />
Sixteen Zinc Finger Nuclease (ZFN) pairs were designed to target the 3’UTR of human CRX gene without disrupting its coding sequence. The most efficient ZFN pair was selected using a mismatch sensitive enzyme assay and subsequently transfected into human embryonic stem cell line (H9) along with a donor construct containing homology to the target region and a puromycin selection cassette. Following puromycin selection, analysis of six resistant clones indicated integration of reporter cassette into the 3’UTR of CRX gene, five containing a heterozygous insertion and one homozygous. Sequencing of the insertion site showed targeted integration and copy variant analysis suggested that the homozygous clone contained no other copies inserted elsewhere within the genome or as tandem repeats at the 3’UTR. This clone was selected for further analysis and differentiation towards the retinal lineage using a 3D protocol developed by our group.<br />
Analysis of the homozygous CRX-GFP labelled clone showed that insertion of the reporter cassette does not interfere with maintenance of pluripotency and genomic stability. Flow activated cell sorting was used to purify GFP+ and GFP- cell fractions, which were further analysed by quantitative RT-PCR. This analysis and immunocytochemistry of sections obtained from embryoid bodies indicated a significant correlation between GFP expression and endogenous CRX expression. Immunocytochemistry analysis with various markers labelling photoreceptors precursors, RPE, retinal ganglion cells and inner nuclear layer retinal neurones indicated clear expression only in the photoreceptor precursors throughout the 90 day differentiation time course.<br />
This approach could enable the isolation of stage-specific photoreceptor precursors from human pluripotent stem cell differentiations, allowing characterisation of their transcription and proteomic profile and subsequent testing of their transplantation potential.<br />
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