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CAPN5 gene silencing by short hairpin RNA interference
Journal article   Open access   Peer reviewed

CAPN5 gene silencing by short hairpin RNA interference

Nnamdi G Nelson, Jessica M Skeie, Hakim Muradov, Hannah A Rowell, Seongjin Seo and Vinit B Mahajan
BMC research notes, Vol.7(1), pp.642-642
09/12/2014
DOI: 10.1186/1756-0500-7-642
PMCID: PMC4169796
PMID: 25216694
url
https://doi.org/10.1186/1756-0500-7-642View
Published (Version of record) Open Access

Abstract

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.
 Gene Therapy ADNIV Autosomal dominant neovascular inflammatory vitreoretinopathy CAPN5 shRNA Technical Note

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