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MW Sharp, JS Friedman, V Raymond, MA Walter; Creation of Human Eye cDNA Libraries for Differential Screening and Yeast Two-Hybrid Analysis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2437.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Glaucoma can be caused by anterior chamber malformations and/or misfunctions in the developing and adult eye. To discover genes involved in glaucoma, we are creating cDNA libraries from human eye-tissues. Applications of these cDNA libraries include identifying differentially-expressed ocular genes and discovering proteins that interact with known glaucoma genes. Methods: 50 human eyes were dissected from fetuses (9-13 weeks gestation) and total RNA was extracted. The mRNA was then isolated from the fetal eye total RNA with a poly-A RNA extraction kit and was used as the starting material for cDNA synthesis. With protocol alterations to the Cytotrap library construction system (Stratagene), the cDNA was size-fractionated and cloned into a modified two-hybrid prey vector. The pMyr vector was mutagenisized to include recombinational cloning sites facilitating the shuttling of the cDNA library or specific cDNA clones into other vectors while maintaining the frame of the cDNA inserts. PCR was initially performed on the cDNA library to determine the presence or absence of glaucoma-associated genes. Results: The human fetal eye cDNA library includes approximately 3.8 x 104 primary colony forming units (cfu). The PCR analysis indicated the presence of FOXC1, Oculoglycan, and PITX2 transcripts as expected. Human adult trabecular meshwork tissue collection, dissection and the total RNA extraction have already been completed. We intend to create and test a human adult trabecular meshwork cDNA library in the same fashion. Conclusion: These eye-specific cDNA libraries will be excellent and unique tools for use in identifying genes involved in ocular devlopment and function. Two-hybrid analyses of the fetal eye cDNA library using known glaucoma genes as bait are in progress.
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