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P. Gonzalez, C. Luna, P. Challa, P. B. Liton, G. Li, D. L. Epstein; Screening for Promoters to Target Gene Expression to the Trabecular Meshwork (TM) in the Rat Eye. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2085.
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© ARVO (1962-2015); The Authors (2016-present)
A major limitation in the generation of transgenic models for glaucoma in rodents is the lack of known promoters capable of targeting expression to the cells of the outflow pathway without affecting other eye tissues including the ciliary body. Our objective was to identify genes preferentially expressed in the outflow pathway of the rat compared to other tissues in the anterior segment.
Twelve rats, 3 months old, were euthanased to obtain tissues from the anterior segment of the eye. The eyes were enucleated and stored in RNAlater (Ambion) until dissection. Five tissues were dissected: cornea, lens, trabecular meshwork (TM), iris and ciliary body. Three groups of 4 rats were pooled for RNA extraction. RNA was extracted with RNAeasy mini kit (Qiagen). RNA concentration and quality were measured with the total RNA pico assay. 15 to 40 nanograms of RNA per tissue were amplified using the Ovation Aminoallyl System (NuGen). The amplified cDNAs were hybridized with the version 3.0 of Rattus Novergicus microarrays that represents 22,012 genes and 27,044 transcripts. Each tissue was hybridized in triplicate. Data was analyzed using Genespring. Real-time Q-PCR reactions were performed using iQ SYBR Green Supermix (Biorad, Hercules, CA) in a BIO-RAD iCycler iQ system (BioRad, Hercules, CA).
Comparison between array data showed 45 genes with a significant 4 fold higher expression difference in the TM compared to the other four eye tissues. Five of these genes showed higher than 10 fold differential expression. Eight genes highly expressed in the TM and with values near to background levels in most of the other tissues were selected as possible promoter candidates and their expression was confirmed by real time Q-PCR. Some of the genes with higher fold expression differences between the TM and the other eye tissues included procollagen X alpha (fold difference between43.7 and 1024) and copine VII (fold differences between 9.8 and 157.6).
The promoters from some of the genes identified in this study have potential for targeting transgene expression to the TM of the rat without affecting the surrounding tissues of the eye. The identification of such promoters could provide the means for the generation of transgenic models relevant to glaucoma research.
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