Study on transfection of adeno associated virus 2-ND4 gene into mitochondria
10.3760/cma.j.issn.2096-0160.2014.08.005
- VernacularTitle:腺相关病毒2-ND4基因转染细胞线粒体的研究
- Author:
Shuo, YANG
;
Lei, LIU
;
Han, PEI
;
Xing, WAN
;
Duoduo, LU
;
Weikun, HU
;
Bin, LI
- Publication Type:Journal Article
- Keywords:
Optic atrophy,hereditary,Leber/therapy;
Genetic therapeutic;
DNA,mitochondrial/genetics;
Transfection;
Adeno-associated virus;
Retinal ganglion cell;
Cell line;
Y03 double fluorescent quantum dot
- From:
Chinese Journal of Experimental Ophthalmology
2014;32(8):693-695
- CountryChina
- Language:Chinese
-
Abstract:
Background Leber hereditary optic neuropathy (LHON) is mitochondrial DNA (mtDNA) disease and mainly leads to optical nerve degeneration.Its primary mechanism is synthesis disorder of DN4 protein due to variation of mtDNA 11778 locus.So to construct a vector with exogenous normal ND4 and transfect into mitochondria is a key of gene therapy for LHON.Objective This study was to investigate the in vitro transfection of adeno-associated virus (AAV)-ND4 gene into mitochondria.Methods Human renal epithelial cell lines transfected adenovirus E1A (293 cells) were regularly cultured and divided into two groups.Framework plasmids of recombinant AAV-ND4 or simple AAV2 were added to the cell medium respectively.The expression of ND4 in cells were located 12,24,36 and 48 hours after transfected by Y03 dual fluorescent quantum dots staining.The positive response for ND4 showed the green fluorescence.Results Cultured 293 cells grew well with 80％ confluence.Abundant green fluorescence particles were seen in cytoplasm in the AAV-ND4 transfected group,but only red fluorescence from mitochondrial protein was seen in the simple AAV transfected group under the fluorescence microscope.Conclusions Exogenous ND4 protein can been successfully transfected into mitochondria using the ND4 gene constructed AAV.This result provides experimental evidence for the further study on gene therapy of LHON.
