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H. Huang, R. Adler; Cone– and Rod–Specific Transcriptional Regulators Identified by Single Cell cDNA Suppression Subtractive Hybridization . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3969.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Transcriptional regulators controlling the development of photoreceptor subtypes are largely unknown. The purpose of this study is to identify transcription factors expressed during the differentiation of cone and rod photoreceptor cells. Methods: Retinae from 18–day old chick embryos were dissociated after papain treatment. Single rods, green cones or red cones, identified by morphological criteria corroborated by immunocytochemistry, were individually captured with a capillary pipette, and microgram amounts of cDNA were synthesized and exponentially amplified from each cell (Wahlin et al, Mol Vis 2004;10:366–75). Cell identity was further verified by PCR amplification of cell type–specific genes. Suppression subtractive hybridization (SSH) was used to isolate cDNA species preferentially expressed in red cones versus rods. Subtracted libraries were constructed with the TOPO–T/A plasmid vector, and randomly–picked colonies were sequenced and subjected to BLAST search. Differential expression of candidate genes was verified by PCR and in situ hybridization. Results: Initial analysis was done with 250 clones from a subtracted red cone vs rod cDNA library. Thirty five clones (17%) were unknown genes, and 6 clones (3%) corresponded to transcriptional regulators, including a Y–box transcription enhancer, a transcription auxiliary factor containing YEATS domain, and four zinc finger transcription factors. The red cone specificity of 13 unknown genes and 3 zinc finger factors was verified by PCR. In addition, 10 of these 16 genes were shown to be developmentally regulated, and 2 genes were restricted to retina and brain. Ongoing studies include further characterization of these candidate genes by in situ hybridization, and analysis of a rod vs red cone subtracted library. Conclusions: The zinc finger transcription factors identified in this study are potential candidates for a role in the regulation of red cone differentiation, and illustrate the power of SSH and single cell techniques for the discovery of transcriptional regulators specifying retinal photoreceptor subtypes.
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