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。斑马鱼胚胎的透明度和外部发育允许详细和直接地观察体内发生的血管生长。此外，斑马鱼可以接受各种不同的基因操作，这使得评估血管发育过程中的基因功能成为可能。尽管正向基因筛查在识别血管形成所需的新基因方面取得了成功，但这些方法是劳力和时间密集型的。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。基因组和表达序列的日益可获得性已经揭示了100多个在内皮细胞中表达并与血管发育有关的候选基因，这突显了确定这些基因功能的明确的反向遗传方法的必要性。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，我们将在以前工作的基础上，将这项技术应用于反向基因筛查，以确定与血管发育有关的候选基因的功能。在目标1中，我们将构建针对30多个内皮细胞表达基因的靶点的高特异性锌指蛋白。这些锌指蛋白将被用来构建锌指核酸酶(ZFN)，并将在斑马鱼胚胎中进行功能验证。在目标2中，我们将利用ZFN来产生在候选基因内的目标位置具有零突变的方正品系。目标3将侧重于携带候选基因突变的突变胚胎的详细表型特征。特别是，我们将关注血管形态发生、分化和功能方面的缺陷。对突变体之间的缺陷进行比较，以及之前描述的突变体，将允许这些基因初步组装成遗传途径。随后的上位性实验将允许对这些途径进行更明确的基因特征描述。ZFN在反向遗传筛选血管发育重要基因方面的新应用将为研究斑马鱼其他生物学过程的类似方法提供一个框架。此外，这项建议中应用的ZFN技术未来将广泛应用于斑马鱼群落，这将极大地促进新突变品系的收集。

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.