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Qubo Zhu, Wenyu Sun, Krzysztof Palczewski; Sponge Transgenic Mouse Model Reveals the Important Roles of Mir-183 Cluster in Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):507.
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© ARVO (1962-2015); The Authors (2016-present)
MiR-96, miR-182 and miR-183 constitute a polycistronic miRNA cluster called miR-183 cluster, which are highly expressed in photoreceptors and interneurons in retina. Although the in vitro data of other investigators have demonstrated the important role of miR-183 cluster in retina, its biological activities in vivo were still obscure. To better understand the roles of miR-183 cluster in retinal development, maintenance and light-adaptation function, we generated a sponge transgenic mouse model to knockdown the expression of these three microRNAs at the same time in retina.
To generate miR-183 cluster sponge elements, we introduced 10 copies of complementary sequences to miR-96, miR-182 and miR-183 into the 3' UTR of EGFP in a pNOVA expression vector, which is regulated by a rhodopsin promoter and ended with a SV40 poly A tail. The 6.8 kb target sequence was cut out, and then injected into the fertilized eggs of BL6/SJL hybrid mice using pronuclear injection method by Case Transgenic and Targeting Facility in CWRU.
Although the morphology showed no differences between transgenic and wild type mice in normal condition, the sponge transgenic mice got serious retinal degeneration after 30 min 10,000 Lux light induction. Histological studies showed that the ONL thickness was dramatically decreased in the superior site of transgenic mouse retina. Real time PCR results in both the sponge transgenic mice model and different microRNA stable cell lines demonstrated that Arrdc3, Neurod4, Casp2 maybe the downstream genes of miR-183 cluster; and this result was also confirmed by luciferase assay. Further studies showed that both miR-183 clusters and Casp2 were increased during light induction. Expression level of Casp2 was enhanced in transgenic mice after light induction because of the inhibition of miR-183 cluster induction by sponge elements.
Our research reveals the important roles of miR-183 cluster in retinal degeneration in vivo; and the mechanism is because miR-183 cluster down-regulates the Casp2 gene, which is up-regulated under light induction, and thus protects the photoreceptors from apoptosis. This work will benefit the future studies of the relationship between microRNA and retinal diseases.
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