小分子干扰RNA对斑马鱼VEGF基因的沉默作用  

Knock-down of VEGF expression by siRNA in zebrafish embryos

作　　者：杨少丽[1] 郑兰红[1] 阎松[2] 王征[1] 林秀坤[1] 

机构地区：[1]中国科学院海洋研究所实验海洋生物学重点实验室 [2]大连交通大学环境与化学工程学院,大连116028

出　　处：《高技术通讯》2007年第12期1278-1282,共5页Chinese High Technology Letters

基　　金：国家自然科学基金(40506031);山东省自然基金(2004C07)资助项目

摘　　要：运用RNA干扰技术,针对斑马鱼血管内皮生长因子(VEGF)基因设计并合成了特异性小分子干扰RNA(siRNA),并从血管形成水平研究了siRNA对VEGF基因表达的影响。为了得到能在体内稳定表达的siRNA,进一步以pSilencer4.1-CMVVector为载体,合成了短发夹RNA(shRNA)表达模板并定向克隆到CMV启动子下游,构建了shRNA表达载体。定量碱性磷酸酶染色显示注射合成的siRNA后胚胎新血管生成(angiogenesis)为71.8%,NBT/BCIP血管染色显示表达载体可以引起斑马鱼胚胎肠下静脉、节间血管发育缺陷,原位杂交结果显示胚胎头部及前原肾管部位VEGF表达异常。An siRNA （small interfering RNA） targeting zebrafish VEGF （vascular endothelial growth factor） gene was designed and synthesized by chemical synthesis. Its effect on zebrafish angiogenesis was evaluated. To obtain stable regulation of VEGF expression, shRNA （ short hairpin RNA） transcription vector was constructed by inserting the annealed forward and reverse synthetic oligonucleotides into the downstream of CMV promoter. Quantitative endogenous alkaline phosphatase （EAP） assay showed that 71.8 % of angiogenesis was detected following the injection of synthetic siRNA. Alkaline phosphatase staining using NBT and BCIP indicated that the vector constructed could cause specific subintestinal veins （SIV） and intersegmental vessels （Se） vascular development defects. In situ hybridization showed that the expression of VEGF in the brain and anterior pronephric ducts region were anomaly.

关 键 词：RNA干扰 斑马鱼 血管形成 VEGF基因 

分 类 号：Q78[生物学—分子生物学]