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X.P. Guillonneau, J. Roger, O. Goureau; Characterization of Genes Regulated during Development of the Rat Retina . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1594.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: During development, progenitor cells give birth to the different subtypes of neurons and glial cells of the neural retina. Cell division and differentiation of these progenitors are under the control of endogenous factors produced within the retina, and exogenous factors such as CNTF. Identifying genes which expression is modulated during development is therefore a key step to understand the molecular events underlying retinal development, and also a way to characterized factors capable of modifying progenitor cell fate. Methods: In order to characterized such candidates, we used the Suppressive Subtractive Hybridization (SSH) technique to isolate genes which expression is modulated between Embryonic day 17 (E17) when neural retina is mostly composed of progenitors cells, and Post Natal day 0 (PN0), when the ganglionnary and amacrine cells and presumptive cone photoreceptors differentiate in neural retina. Therefore, the subtraction of E17 mRNA from PN0 mRNA by SSH should enriched the final cDNA pool with genes involved in the differentiation of these retinal cells. Results: A PN0-E17 library was construct from these subtracted cDNA and 196 clones were selected randomly and screened for differential expression by dot blot analysis using subtracted and control probes, and by reverse Northern blot analysis. After these successive selection steps, 11 candidates were sequenced and compared to the human genome, cDNA, and EST databases. Primers were designed to specifically amplified each of these 11 clones in RT-PCR experiments. On the basis of the level of expression of their corresponding mRNA in the retina at PNO and E17, 7 of them were dismissed, 2 clones presenting no identity with known proteins and/or sequences and 2 clones orthologue of mouse genes which functions are known were selected and their expression was further analyzed by RT-PCR and Northern blot. These clones presented either a retina specific pattern of expression or an expression restricted to PN0 and later stage of development. Conclusion: This library allowed the characterization of several genes known to be involved in neural differentiation as well as new genes expressed during retinal differentiation. In vivo and in vitro overexpression experiments will address the function of these genes. The characterization of genes involved in retinal progenitor differentiation is a challenging issue for cell therapy. Therefore E17-PN0 library analysis will be screened to isolate theses genes.
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