[Recombinant type 1 adeno-associated virus-mediated transfection of ECV304 cells with enhanced green fluorescent protein gene in vitro].
OBJECTIVE
To assess the feasibility of recombinant type 1 adeno-associated virus (rAAV1) as a vector for gene therapy of corneal neovascularization.


METHODS
The rAAV1 vector carrying enhanced green fluorescence protein (EGFP) gene (rAAV1-EGFP) was transfected into ECV304 cells at different multiplicities of infection (MOI=5 x 10(3), 5 x 10(4), 5 x 10(5)). EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP-positive cell percentage determined by flow cytometry. MTT assay was used to assess the proliferation of the transfected cells.


RESULTS
The cells with rAAV1-EGFP transfection at MOI of 5 x 10(5) began to exhibit GFP expression 2 days after transfection, and the fluorescence intensity increased with the MOI used for transfection. GFP expression reached the maximum on day 7, at the point of which the transduction efficiency of rAAV1-EGFP in ECV304 cells was 45.90%, 58.56% and 68.31% corresponding to MOIs of 5 x 10(3), 5 x 10(4), and 5 x 10(5), respectively. MTT assay did not reveal significant difference in the absorbance between the transfected cells and the control cells at 72 and 96 h after transfection.


CONCLUSIONS
arAAV1-EGFP gene can be stably and efficiently expressed in ECV304 cells without causing cell growth inhibition, suggesting the potential of rAAV1 as a safe and efficient vector for gene therapy of corneal neovascularization.
Tang
Ming-fang
MF
Department of Ophthalmology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. tmf1901@163.com
Lu
Xiao-he
XH
Gao
Ji-min
JM
Zhong
Yan-yan
YY
Luo
Wei
W
Zhou
Ming-qian
MQ
D002460:Cell Line; D016510:Corneal Neovascularization / Q000628:therapy; D000229:Dependovirus / Q000235:genetics*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins