CAPN5 gene silencing by short hairpin RNA interference

被引：8
作者：
Nelson N.G. [1 ,2 ]
Skeie J.M. [1 ,2 ]
Muradov H. [1 ,2 ]
Rowell H.A. [1 ,2 ]
Seo S. [1 ]
Mahajan V.B. [1 ,2 ]
机构：
[1] Department of Ophthalmology and Visual Sciences, University of Iowa Hospitals and Clinics, 200 Hawkins Drive, Iowa City, 52242, IA
[2] Omics Laboratory, University of Iowa Hospitals and Clinics, Iowa City, IA
来源：
BMC Research Notes | / 7卷 / 1期
基金：
美国国家卫生研究院;
关键词：
ADNIV; Autosomal dominant neovascular inflammatory vitreoretinopathy; CAPN5; Gene therapy; shRNA;
D O I：
10.1186/1756-0500-7-642
中图分类号：
学科分类号：
摘要：
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. © 2014 Nelson et al.; licensee BioMed Central Ltd.
引用
收藏
相关论文
共 23 条
  • [1] Mahajan V.B., Skeie J.M., Bassuk A.G., Fingert J.H., Braun T.A., Daggett H.T., Folk J.C., Sheffield V.C., Stone E.M., Calpain-5 mutations cause autoimmune uveitis, retinal neovascularization, and photoreceptor degeneration, PLoS Genet, 8, (2012)
  • [2] Mahajan V.B., Lin J.H., Lymphocyte infiltration in CAPN5 autosomal dominant neovascular inflammatory vitreoretinopathy, Clin Ophthalmol, 7, pp. 1339-1345, (2013)
  • [3] Goll D.E., Thompson V.F., Li H., Wei W., Cong J., The calpain system, Physiol Rev, 83, pp. 731-801, (2003)
  • [4] Rowell H.A., Bassuk A.G., Mahajan V.B., Monozygotic twins with CAPN5 autosomal dominant neovascular inflammatory vitreoretinopathy, Clin Ophthalmol, 6, pp. 2037-2044, (2012)
  • [5] Tlucek P.S., Folk J.C., Sobol W.M., Mahajan V.B., Surgical management of fibrotic encapsulation of the fluocinolone acetonide implant in CAPN5-associated proliferative vitreoretinopathy, Clin Ophthalmol, 7, pp. 1093-1098, (2013)
  • [6] Wert K.J., Skeie J.M., Bassuk A.G., Olivier A.K., Tsang S.H., Mahajan V.B., Functional validation of a human CAPN5 exome variant by lentiviral transduction into mouse retina, Hum Mol Genet, (2013)
  • [7] Maguire A.M., Simonelli F., Pierce E.A., Pugh E.N., Mingozzi F., Bennicelli J., Banfi S., Marshall K.A., Testa F., Surace E.M., Rossi S., Lyubarsky A., Arruda V.R., Konkle B., Stone E., Sun J., Jacobs J., Dell'Osso L., Hertle R., Ma J.X., Redmond T.M., Zhu X., Hauck B., Zelenaia O., Shindler K.S., Maguire M.G., Wright J.F., Volpe N.J., McDonnell J.W., Auricchio A., High K.A., Bennett J., Safety and efficacy of gene transfer for Leber's congenital amaurosis, N Engl J Med, 358, pp. 2240-2248, (20
  • [8] Bennett J., Ashtari M., Wellman J., Marshall K.A., Cyckowski L.L., Chung D.C., McCague S., Pierce E.A., Chen Y., Bennicelli J.L., Zhu X., Ying G.S., Sun J., Wright J.F., Auricchio A., Simonelli F., Shindler K.S., Mingozzi F., High K.A., Maguire A.M., AAV2 gene therapy readministration in three adults with congenital blindness, Sci Transl Med, 4, (2012)
  • [9] Jiang L., Li T.Z., Boye S.E., Hauswirth W.W., Frederick J.M., Baehr W., RNAi-mediated gene suppression in a GCAP1(L151F) cone-rod dystrophy mouse model, PLoS ONE, 8, (2013)
  • [10] Wert K.J., Sancho-Pelluz J., Tsang S.H., Mid-stage intervention achieves similar efficacy as conventional early-stage treatment using gene therapy in a pre-clinical model of retinitis pigmentosa, Hum Mol Genet, 23, pp. 514-523, (2014)
123