부산대학교 도서관

Background The purpose of this project was to identify short hairpin RNA (shRNA) sequences that can suppress expression of human CAPN5 in which gain-of-function mutants cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). We created HEK293T cells that stably express an ADNIV disease allele, CAPN5-p.R243L. Transfection protocols were optimized for neuroblastoma SHSY5Y cells. The gene silencing effect of four different shRNA plasmids that target CAPN5 was tested. RNA and protein expression was determined using quantitative RT-PCR and immunoblot analysis. Findings Two of four shRNA plasmids reduced mutant CAPN5 RNA in a stable cell line. Similar knockdown was observed in SH-SY5Y cells that natively express CAPN5. Lactose dehydrogenase assays showed that down-regulation of CAPN5 was not cytotoxic. Conclusions CAPN5 expression can be suppressed by shRNA-based RNA interference. Further testing in ADNIV models will determine the potential of gene silencing as a strategy to treat, delay, or prevent blindness in ADNIV patients. Keywords: Autosomal dominant neovascular inflammatory vitreoretinopathy, ADNIV, CAPN5, shRNA, Gene therapy
Author(s): Nnamdi G Nelson[sup.1,2] , Jessica M Skeie[sup.1,2] , Hakim Muradov[sup.1,2] , Hannah A Rowell[sup.1,2] , Seongjin Seo[sup.1] and Vinit B Mahajan[sup.1,2] Background CAPN5 mutations are the cause of Autosomal [...]