title: CAPN5 gene silencing by short hairpin RNA interference.
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.
  
  
  RESULTS
  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.
authors.forename: Nnamdi G
authors.initials: NG
authors.lastname: Nelson
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Skeie
authors.forename: Hakim
authors.initials: H
authors.lastname: Muradov
authors.forename: Hannah A
authors.initials: HA
authors.lastname: Rowell
authors.forename: Seongjin
authors.initials: S
authors.lastname: Seo
authors.affiliation: Department of Ophthalmology and Visual Sciences, The University of Iowa Hospitals & Clinics, 200 Hawkins Drive, Iowa City, IA 52242, USA. mahajanlab@gmail.com.
authors.forename: Vinit B
authors.initials: VB
authors.lastname: Mahajan
mesh_terms: D002154:Calpain / Q000235:genetics*; D002460:Cell Line; D020868:Gene Silencing; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D034741:RNA, Small Interfering; D002154:Calpain; C494426:Capn5 protein, human