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。斑马鱼胚胎的透明性和外部发育允许详细和直接观察体内发生的血管生长。此外，斑马鱼可以进行各种不同的遗传操作，从而可以评估血管发育过程中的基因功能。尽管正向遗传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型的。此外，由于脊椎动物基因组的大小、世代时间和维持成本，筛选至饱和是困难的。越来越多的基因组和表达序列的可用性已经揭示了超过100个候选基因在内皮细胞中表达，并涉及血管发育，强调需要一个明确的反向遗传方法来确定这些基因的功能。最近，我们已经成功地应用锌指核酸酶在斑马鱼靶向基因失活。在本申请中，我们将建立在我们以前的工作，并在反向遗传筛选的背景下应用这项技术，以确定与血管发育有关的候选基因的功能。目的一：针对30多个内皮细胞表达基因的靶位点构建高特异性锌指蛋白。这些锌指蛋白将用于构建锌指核酸酶（ZFN），其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靶位点处携带无效突变的创始系。目标3将集中在详细的表型特征的突变胚胎携带突变的候选基因。特别是，我们将集中在血管形态发生，分化和功能的缺陷。比较突变体之间的缺陷，沿着与先前描述的突变体，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这些途径的更明确的遗传表征。ZFN在血管发育过程中重要基因的反向遗传筛选背景下的这种新应用将为类似方法提供框架，以解剖斑马鱼中的其他生物过程。此外，在该提案中应用的ZFN技术在斑马鱼社区的未来广泛使用将极大地促进新突变系集合的产生。 公共卫生相关性：在各种物种中的基因组测序工作已经导致鉴定出许多可能在发育和疾病中起重要作用的候选基因。然而，在许多模型系统中，这些基因的功能询问是有问题的。在这项计划中，我们将应用新技术在斑马鱼的30多个基因中产生敲除，以研究它们在血管发育过程中的功能。

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. PUBLIC HEALTH RELEVANCE: Genome sequencing efforts in a variety of species have led to the identification of numerous candidate genes that may play important roles in development and disease. However, in many model systems functional interrogation of these genes is problematic. In this proposal, we will apply new technology to generate zebrafish knockouts in more than 30 genes to investigate their function during vascular development.