Purchase this article with an account.
M. Saghizadeh, N.B. Akhmedov, E. Mendoza, S.F. Nelson, D.B. Farber; Characterization of a Gene Expressed in Cone Photoreceptor Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3072.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify genes expressed preferentially in cone photoreceptors that may be candidates for the cause of retinal degeneration in animals and humans. Previously, cd dog retinal mRNAs were subtracted from those of the normal dog retina using two rounds of a PCR–based method (RDA). The output of this subtraction was then subjected to microarray screening. Northern blots, relative quantitative RT–PCR (QPCR), and in situ hybridization (ISH) were used to verify the expression pattern in mouse retina of a subset of the identified genes. We are reporting here our preliminary studies on one of these genes (clone 15A15). Methods: Dissociated mouse retina cells were incubated with FITC–conjugated PNA. Stained cone cells were then sorted by FACS. Using the appropriate primers for α’– PDE (a cone marker), rod α–transducin, and α–PDE (both rod markers), we verified by RT–PCR and QPCR the expression of the 15A15 transcript in cone photoreceptors. To study the expression pattern of the mRNA product, its complete coding region was subcloned into the pcDNA4/HisMax vector and transiently expressed in HEK293 cells. Expression was analyzed by immunofluorescence (IF), using Anti Xpress FITC AB staining. Subcellular localization was determined after protein extract fractionation, followed by immunoblotting. Results: Northern blots and QPCR confirmed the expression of 15A15 cDNA in mouse retina. ISH using the 15A15 probe showed the same distribution pattern as the α’–PDE antisense probe confirming the localization of 15A15 mRNA in cone photoreceptors. Further, RT–PCR and QPCR on cone–sorted cells by FACS activated flow cytometry substantially supported the predominant expression of 15A15 in cone photoreceptors of mouse retina. Developmental studies using QPCR showed that 15A15 mRNA expression is already detected at postnatal day 4 (P4) and is maximal by P40. Transient expression of the gene product detected by IF showed the protein in the nuclei of HEK293 infected cells. This was corroborated by immunoblotting of the fractionated protein extract. The protein encoded by the gene corresponding to 15A15 has an approximate molecular weight of 135 kDa and DNA binding domains. Conclusions: These preliminary observations suggest that 15A15, encoded by a gene expressed in cone photoreceptors of the mouse retina, may be involved in regulation of cone mRNAs. Further characterization of this protein is in progress.
This PDF is available to Subscribers Only