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C.-Y. Hu, C.-H. Yang, H.-Y. Huang, C.-T. Tu, H.-J. Tsai; Vascular Endothelial Growth Factor Over-Expression in Transgenic Zebrafish Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3421.
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We intended to establish a zebrafish model of vascular endothelial growth factor (VEGF) over-expression in retina, so that it may become a useful animal model of retinal or choroidal neovascularization.
We micro-injected a construct of zebrafish VEGF driven by retina-specific rhodopsin promoter and an additional construct of red fluorescent protein (RFP) driven by heart-specific cmlc2 promoter into the embryos derived from zebrafish transgenic line Tg(fli1:EGFP), which highly expresses enhanced green fluorescent protein (EGFP) in vascular endothelium. We cultured the larvae with heart-specific RFP expression and mated them with each other after sexual maturation. We used polymerase chain reaction (PCR) to identify the existence of rhodopsin promoter and VEGF coding sequence in the genome of F1 progeny to prove successful inheritance of transgene. Semi-quantitative reverse transcription PCR (RT-PCR) was applied to compare the expression levels of VEGF between the Tg(fli1:EGFP) larvae and the Tg(rhodopsin:VEGF) larvae. The alterations in ocular tissue were confirmed by histological examination.
Hundreds of embryos from line Tg(fli1:EGFP) were micro-injected with the linearized plasmid DNA. The putative founders expressing RFP in heart were cultured for 3 to 4 months. PCR analysis confirmed the stable existence of transgene by identifying the injected sequence in the genomic DNA of F1 larvae. Semi-quantitative RT-PCR also demonstrated that the VEGF expression level of Tg(rhodopsin:VEGF) larvae was higher than that of Tg(fli1:EGFP) larvae. Histological examination of the PCR-confirmed F1 adult fish showed increased number and size of blood vessels in choroid and vitreous cavity, but the sensory retina did not change apparently.
We successfully established a zebrafish model of VEGF over-expression in retina. Its long-term influence on retinal and choroidal vasculogenesis deserves further more detailed investigation.
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