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Serial Analysis of Gene Expression (SAGE) in the Rat Limbal and Central Corneal Epithelium

¹From the Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland; and the ²International Graduate Center of Evolution, Institute of Evolution, University of Haifa, Haifa, Israel.

PURPOSE. To identify genes preferentially expressed in the stem-cell–rich limbal epithelium of the rat cornea.

METHODS. The limbal and central corneal epithelial cells of 6-week-old rats were isolated by microdissection. Serial analysis of gene expression (SAGE) libraries were constructed and analyzed, and in situ hybridization, reverse transcription–polymerase chain reaction (RT-PCR) and cDNA cloning were conducted by conventional procedures.

RESULTS. The rat limbal and central corneal epithelial SAGE libraries consisted of 41,894 and 40,691 tags, respectively. After annotation, this was reduced to 759 transcripts specific for the limbal library and 844 transcripts specific for the central corneal library; 2292 transcripts overlapped. Transcripts encoding proteins with metabolic functions comprised the major functional category in both libraries. In situ hybridization and/or RT-PCR results of 12 of the most abundant, highly enriched transcripts in the limbal epithelium were in general agreement with the SAGE data and showed that these proteins are also expressed in the conjunctival epithelium. Interesting limbal-enriched transcripts encode WDNM1-like protein (similar to WDNM1/Expi, a putative secreted proteinase and inhibitor of metastasis), mesothelin (a cancer marker), marapsin (a trypsin-like serine protease that may control cell growth and migration), K4 and K15 (both cytokeratins), and membrane-spanning four-domain subfamily A member 8B. WDNM1-like protein was cloned and confirmed as a member of the four-disulfide core family.

CONCLUSIONS. The SAGE results extend the database of genes expressed in the rodent cornea and suggest an association between several genes preferentially expressed in the limbal epithelium with cellular proliferation and migration.

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