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M. Saghizadeh, N. B. Akhmedov, C. K. Yamashita, D. B. Farber; A Novel Zinc-Finger Protein in Cone Photoreceptors of Human Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1115.
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© ARVO (1962-2015); The Authors (2016-present)
To isolate and characterize genes expressed in cone photoreceptors that may increase our understanding of these cells’ function and the molecular basis of cone-associated retinal diseases.
RDA combined with microarray screening was used to isolate a novel retinal gene, ZF-15. Northern blots verified the expression of the ZF-15mRNA in mouse and human retinas. Immunocytochemistry and Western blots determined the subcellular localization of ZF-15 using a polyclonal antibody generated against the ZF-15 N-terminus synthetic peptide. QPCR and Western blots were used to show the silencing of ZF-15 after transient transfection of SiRNA duplexes targeted to the to 5’ or 3’ regions of ZF-15 mRNA intoY79 retinoblastoma or HEK293 cells.
A novel gene, ZF-15, was isolated by microarray screening of an RDA library of enriched dog cone photoreceptor cDNAs obtained by subtraction of adult, cone photoreceptorless cd dog retinal mRNAs from normal dog retinal mRNAs. Northern blots revealed that orthologues of dog ZF-15 mRNA are expressed in mouse (single transcript, 5.3 Kb) and human (three transcripts, 6.5 Kb, 5.2 Kb and 3.7 Kb) retinas. QPCR of mRNA from mouse cone cells sorted by FACS activated flow cytometry, demonstrated ZF-15 mRNA expression in these cells. The complete coding region of ZF-15 was subcloned into the pcDNA4/HisMax vector and transiently transfected into HEK293 and Y79 retinoblastoma cells. Western blots and fluorescence microscopy showed that the ZF-15 protein was mostly localized to the nuclei of the transfected cells. However, immunocytochemistry showed nuclear as well as cytosolic localization of the protein in human and bovine cone cells. ZF-15 encodes a 135-kDa protein that has C2H2-type zinc finger motifs. QPCR and Western blot results showed the silencing of ZF-15 in HEK293 and retinoblastoma cells after 72 and 96 hours of transient transfection with three different SiRNAs. The morphology and the number of viable cells did not change after silencing ZF-15 in transfected cells.
We have isolated a novel human gene, which is expressed in the cone photoreceptors of human and mouse retinas. ZF-15 has two potential nuclear hormone receptor modules; thus, it may interact with nuclear hormone receptors directly or as a co-activator/co-repressor, binding to DNA sequences and stimulating or inhibiting gene expression.
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