Reverse Genetic Screening for Vascular Mutants Using Zinc Finger Nucleases

描述（申请人提供）：斑马鱼已成为研究胚胎发育过程中血管形成的理想模型。斑马鱼胚胎的透明和外部发育允许详细和直接地观察血管生长，因为它发生在体内。此外，斑马鱼可以适应各种不同的基因操作，这使得在血管发育过程中评估基因功能成为可能。尽管前向基因筛选在识别血管形成所需的新基因方面取得了成功，但这些方法是劳动和时间密集的。此外，由于脊椎动物基因组的大小，世代时间和维护成本，筛选到饱和是困难的。越来越多的基因组和表达序列已经揭示了100多个候选基因，这些基因在内皮细胞中表达，并与血管发育有关，强调需要明确的反向遗传方法来确定这些基因的功能。最近，我们成功地将锌指核酸酶应用于斑马鱼的靶向基因失活。在这项应用中，我们将建立在我们之前的工作基础上，并将这项技术应用于反向遗传筛选的背景下，以确定与血管发育有关的候选基因的功能。在Aim 1中，我们将构建针对30多个内皮细胞表达基因靶位的高特异性锌指蛋白。这些锌指蛋白将用于构建锌指核酸酶（ZFNs），该酶将在斑马鱼胚胎中进行功能验证。在目标2中，我们将利用ZFNs在候选基因的靶位点产生具有零突变的创始系。目标3将集中于携带候选基因突变的突变胚胎的详细表型特征。我们将特别关注血管形态发生、分化和功能方面的缺陷。比较突变体之间的缺陷，以及先前描述的突变体，将允许这些基因进入遗传途径的初步组装。随后的上位性实验将允许对这些途径进行更明确的遗传表征。这种ZFNs在血管发育过程中重要基因的反向遗传筛选中的新应用将为类似方法解剖斑马鱼的其他生物过程提供框架。此外，该提案中应用的ZFN技术未来在斑马鱼群落的广泛获取将极大地促进新突变系的产生。

DESCRIPTION (provided by applicant): The zebrafish has become an ideal model to study blood vessel formation during embryonic development. The transparency and external development of the zebrafish embryo allows detailed and direct observation of blood vessel growth as it occurs in vivo. Furthermore, the zebrafish is amenable to a variety of different genetic manipulations making it possible to assess gene function during vascular development. Despite the success of forward genetic screens in identifying novel genes required for blood vessel formation, these approaches are labor- and time-intensive. Furthermore, due to the size of the vertebrate genome, generation time, and maintenance costs, screening to saturation is difficult. The increasing availability of genomic and expressed sequences has revealed more than 100 candidate genes that are expressed in endothelial cells and are implicated in vascular development, underscoring the need for a definitive reverse genetic approach to determine the function of these genes. Recently, we have successfully applied zinc finger nucleases for targeted gene inactivation in the zebrafish. In this application, we will build on our previous work and apply this technology in the context of a reverse genetic screen to determine the function of candidate genes implicated in blood vessel development. In Aim 1, we will construct high- specificity zinc finger proteins against target sites in more than 30 endothelial cell-expressed genes. These zinc finger proteins will be used to construct zinc finger nucleases (ZFNs) that will be functionally validated in zebrafish embryos. In Aim 2, we will utilize ZFNs to generate founder lines that bear null mutations at targets sites within candidate genes. Aim 3 will focus on the detailed phenotypic characterization of mutant embryos bearing mutations in candidate genes. In particular, we will focus on defects in vascular morphogenesis, differentiation and function. Comparison of defects between mutants, along with previously described mutants, will allow preliminary assembly of these genes into genetic pathways. Subsequent epistasis experiments will allow more definitive genetic characterization of these pathways. This novel application of ZFNs in the context of a reverse genetic screen for genes important during vascular development will provide a framework for similar approaches to dissect other biological processes in the zebrafish. Furthermore, the future widespread access of the ZFN technology applied in this proposal to the zebrafish community will greatly facilitate the generation of collections of new mutant lines.