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K. Kobayashi, R. Iwakiri, H. Kobayashi; Functional inhibition of Mitf in retinal pigment epithelium by small interfering RNA . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4781.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Mitf (microphthalmia–inducing transcription factor) encodes a basic helix–loop–helix–leucine–zipper protein that is known to function in the development of pigmented epithelial cells. The use of small interfering RNAs (siRNA) has become a powerful tool to knock down specific gene expression in a wide range of cells. To identify the functional mechanism of Mitf in differentiation, we used siRNA to specifically suppress Mitf expression in embryonic chick retinal pigment epithelium and investigate the expression of MMP115 (melanosomal matrix protein) in the transdifferentiation process. Methods: Nine–day–old embryonic chick retinal pigment epithelial cells were isolated and cultured. RNA duplexes of 21 nucleotides specific for chick Mitf were chemically synthesized by Dharmacon Research, Inc. Transfection of siRNA was carried out using RNAiFect (Qiagen). To monitor transfection efficiency, fluorescently labeled siRNA was viewed by fluorescence microscopy. RT–PCR and immunohistochemical study were employed to investigate suppression of Mitf and MMP–115. Results: Transfection efficiency of fluorescently labeled siRNA was 84.6%. The expression of Mitf was specifically reduced by siRNA Mitf at 75.0%. At day 7, the expression of MMP115 was extremely reduced and the number of differentiated cell was decreased, compared with the control. Conclusions: Mitf expression in embryonic chick retinal pigment epithelial cells was efficiently inhibited using RNA interference technology. Knockdown of Mitf in retinal pigment epithelial cells results in their promotion of transdifferentiation.
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