Construction Of ShRNA Expression Vector Against Zebrafish VEGF And The Effect On Vascular Development In Zebrafish Embryos

VEGF (vascular endothelial growth factor) is a highly specific endothelial cell mitogen and it is critical for vascularization of tissues, including tumors. The central role of VEGF in angiogenesis makes it an attractive target for anti-tumor therapy. Recently, zebrafish has been generally considered as an excellent model in case of drug screening, disease model establishment, and vertebrate embryonic development study. However, almost all studies to date in zebrafish used chemically synthesized siRNA or in vitro transcribed siRNA, and the application of shRNA in zebrafish remains highly limited. To study the potential of shRNA for gene knockdown in zebrafish, we used a vector-based siRNA expression system, which overcomes the limitation of transience and instability of synthetic siRNA, to specially inhibit zebrafish VEGF gene expression.The potential shRNA target sites in the zebrafish were determined using the siRNA design program. Altogether four siRNAs targeting different locations of VEGF gene were designed. The forward and reverse synthetic oligonucleotides were annealed and inserted into the downstream of CMV promotor of the pSilencer 4.1-CMV neo vector to construct shRNA expression vectors, pS1-VEGF, pS2-VEGF, pS3-VEGF, and pS4-VEGF。The shRNA vectors were microinjected into zebrafish embryos at 1-2cell stages and total RNA was isolated from embryos at 48 hpf (hours post fertilization). RT-PCR analysis indicated that the vectors could down regulate the expression of VEGF to various degrees. VEGF mRNA levels were decreased by 80.5%, 42.8%, 12.5% and 40.7%, respectively. pS1-VEGF, which transcribing an shRNA against the common extron of VEGF165 and VEGF121 gene, was proved to be the most efficient one.Erythrocyte accumulation and pericardial edema, with reduced numbers of circulating red blood cells were observed following the injection of pS1-VEGF. Quantitative EAP assay showed that the shRNA could cause up to 31.8% reduction of zebrafish angiogenesis at the dose of 0.4 ng. Alkaline phosphatase staining using NBT and BCIP indicated that pSI-V1 could cause specific vascular defects in zebrafish in a dose-dependent manner. Absences of SIV and intersegmental vasculature were observed in 50% embryos following the injection of pS1-VEGF. A phenotypic class of embryos only with heart, yolk and head blood vessels was observed at the dose of 1 ng. Further study found that the expression of TS (thymidylate synthase) gene was not significantly affected after the injection of pS1-VEGF. Similarly, the expression of VEGF gene was not down regulated by control vector and siRNA expression vector against TS gene, as determined by RT-PCR. Dose-dependent study was then performed, and the result showed that pS1-VEGF could cause specific gene silencing in a dose-dependent manner at the concentration of 0-1.2 ng.In situ hybridization analysis indicated that silencing VEGF gene expression by shRNA resulted in down regulation of NRP1, a potent VEGF receptor. This provided evidence that the VEGF-mediated agiogenesis in zebrafish is dependent in part on NRP1 expression.The siRNA expressing vector in zebrafish have the potential to overcome the limitations of relying on transient regulation of gene targeting, and opening up the possibility of using zebrafish for functional gene analysis and anti-tumor therapy studies.