利用Tol2转座子介导的增强子诱捕技术获得血管相关转基因斑马鱼系

心血管系统形成于胚胎发育极早期并为其他器官的发育、维持、修复所必需,血管生长异常可造成多种疾病.然而,由于研究对象所限,胚胎血管的发育机制尚未完全阐明,调控血管发育的基因也所知有限.通过Tol2转座子介导的大规模增强子诱捕筛选到26个血管特异表达绿色荧光蛋白(EGFP)报告基因的转基因斑马鱼系,其中有一些品系在胚胎的某些特异血管结构中表达绿色荧光.通过linker-mediated PCR克隆到22个鱼系中Tol2插入位点附近的斑马鱼基因组序列,其中有17个鱼系的Tol2插入可定位到现有的斑马鱼基因组中的单一位点.通过整体胚胎原位杂交对插入位点附近的基因进行表达谱分析,得到8个表达谱与转基因鱼系一致的基因,涵盖了9个鱼系,其中dusp5基因对应于2个不同的鱼系.这8个基因中包括hhex、ets1a和dusp5等3个功能已知的基因,但是大部分(5个)基因在斑马鱼中尚无功能研究,分别为zvsg1、micall2a、arl8b(1of2)、zgc:73355以及hecw2(1of2).hhex和ets1a基因对血管与血细胞前体的发育具有重要作用,所获得的EGFP报告基因受hhex或ets1a基因增强子控制的转基因斑马鱼(mp378b和mp430c-2)为国际首例,为深入研究这两个基因在血管与血液发育中的作用机制提供了新的机遇.筛选到的功能未知基因可以用来进一步研究其在血管发育中的功能;同时,利用所获得的转基因鱼系,可以实现实时、动态观察成血管细胞的起源、分化与基因表达调控,并可用于高通量小分子药物筛选等重要研究.

Generation and Characterization of Blood Vessel Specific EGFP Transgenic Zebrafish via Tol2 Transposon Mediated Enhancer Trap Screen

Cardiovasculature forms during early stages of embryonic development and enables other organs to develop, maintain and regenerate. Imbalanced growth of blood vessels can give rise to numerous pathological disorders. However, the genes involved in blood vessel development remain largely elusive. Zebrafish (Danio rerio) is an ideal vertebrate model organism for the study of developmental processes, especially for that of cardiovascular formation. 26 transgenic fish lines with blood vessel-specific EGFP expression were identified via a large scale enhancer trap screen mediated by Tol2 transposon in zebrafish. The EGFP expression in some of these lines shows different and unique patterns in different part of blood vessels. The genomic sequences flanking the Tol2 insertion sites have been successfully cloned from 22 lines via linker-mediated PCR, among which 17 sequences could be mapped to a unique location within current zebrafish genome assembly. Expression of 8 flanking genes from 9 transgenic lines was confirmed to recapitulate the expression of EGFP reporter gene in their corresponding lines via RNA whole mount in situ hybridization (ISH). Three of these genes, hhex, ets1a and dusp5 are known to be important for vasculargenesis. Since hhex and ets1a are also expressed in hematopoietic precursors, these transgenic zebrafish should be very useful for the study of both hematopoiesis and vasculargenesis. The rest of these genes, namely zvsg1, micall2a, arl8b (1 of 2), zgc:73355 and hecw2 (1 of 2), are either novel or functionally unknown in zebrafish. Further investigation of these fish lines and corresponding genes will give important insights of blood vessel developmental mechanisms including hemangioblast formation and differentiation, as well as genes and enhancer elements important for cardiovascular system. In addition, these transgenic fish lines could also make invaluable contributions to small molecule screen for drug discovery.