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RL Bradford, AS Hackam, DJ Zack, R Adler; Gene Discovery in the Embryonic Chick Retina: A Microarray-based Approach . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2677.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The chick embryo offers many advantages for the experimental analysis of retinal development, but those studies are frequently hindered by limited availability of information about chicken genes in general, and in the chick embryo retina in particular. The purpose of this study, therefore, is to identify and characterize genes expressed in the chick embryo retina that may be relevant for the development of photoreceptors and other retinal cell types. Methods: RNA from embryonic day (ED) 18 White Leghorn chick embryo retinae was used to generate a lambda phage cDNA library, which was subsequently converted into a plasmid library. Five thousand clones were individually grown in microwells, and arrayed onto nylon membranes using the Microgrid II robot (BioRobotics). Replicate membranes were hybridized with 33P-labeled cDNA probes synthesized from poly(A)+ mRNA from ED18 retina, brain and liver. Inserts from clones preferentially expressed in retina were purified, sequenced, and identified by homology searches using public databases. Results: We have so far identified 208 clones that are preferentially expressed in retina, and have obtained useful sequence information on 108. Of these, 28 are previously cloned chick genes, 41 are chick homologs of known genes from other organisms, 28 are novel genes with no known homologs in other species, and 11 correspond to chicken ESTs. Several interesting clones for which sequence homologies suggested possible roles in transcriptional regulation, apoptosis and intracellular signaling were tested by in situ hybridization. The corresponding mRNAs were found to be expressed in the embryonic retina in topographically specific, developmentally regulated patterns, suggesting that they may be involved in key events during embryogenesis. Conclusion: We have demonstrated that a combination of microarray analysis and in situ hybridization provides a high-throughput strategy for the identification and characterization of novel genes expressed in the developing chick retina.
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