Subtraction Analysis of Trabecular Meshwork Genes

Glaucoma is characterized by an elevated intraocular pressure (IOP) that is too high for the health of the eye. It is the second most important cause of nonremedial blindness in the United States. During normal physiological conditions, IOP is maintained by the fluids within the eye. The fluid in the anterior part of the eye (aqueous humor) is produced by cells in the so-called ciliary body and leaves the eye through a sponge-like tissue (called trabecular meshwork) situated in front of the eye. The reason for the increase in IOP in primary open-angle glaucoma (POAG) is an increase in aqueous humor outflow resistance in the trabecular meshwork. It is not clear which factors cause the glaucomatous impairment of trabecular meshwork function and the increase in trabecular meshwork outflow resistance in POAG. Our hypothesis is that trabecular meshwork cells in glaucoma do not express their normal set of genes anymore and that glaucomatous gene expression in this tissue leads to impairment of trabecular meshwork function. Genes that are changed in glaucoma could be more easily identified, if we would know what genes are normally expressed in this tissue. However, the complete set of characteristic trabecular meshwork genes is not known. A powerful way of identifying specific genes in a tissue is by a process called subtraction hybridization. This is a method which selects only those DNA sequences that are transcribed into RNA and are presumed to correspond to active genes. It is done by extracting the mRNA from cells and then making a complementary DNA (cDNA) copy of each mRNA molecule present. A second step is to enrich for genes that are characteristic for the specific tissue. This step takes advantage of the exquisite specificity of base-pairing interactions (“hybridization”) between two complementary nucleic acid molecules. cDNA copies are obtained from two closely related cell types. The cDNA from the cell type of interest is then hybridized with larger excess of cDNA from the other cell type. Those rare cDNA sequences that fail to find a partner are likely to represent mRNA sequences present only in the cell type of interest. Because these cDNAs remain unpaired after the hybridization, they can be purified with a relatively simply procedure and analyzed. We developed recently a cell line from mouse trabecular meshwork which has the unique feature that it can be kept under both differentiated and nondifferentiated conditions. Our hypothesis is that if we subtract cDNA from cells grown under these two conditions, we should be able to obtain for the first a complete array of characteristic trabecular meshwork genes We are confident that the identification of trabecular meshwork-characteristic and/or – specific genes will advance our understanding of trabecular meshwork function and its impairment in POAG.