TILLING the Zebrafish Genome: A Reverse Genetics Approach

描述（由申请人提供）：斑马鱼已经成为人类发育和疾病研究的首要模式生物。其最大的资产是，它是一个遗传系统，其中突变表型可以有效地解剖在有机体，细胞和亚细胞水平。许多正向遗传筛选已经确定了基因，并随后描述了它们在胚胎细胞命运规范、模式、形态发生和器官发生中的作用。然而，随着斑马鱼基因组的测序，我们现在知道许多基因从未被正向遗传学鉴定过，因此它们在发育中的作用仍然是一个悬而未决的问题。此外，最近对人类致病基因的鉴定激增，有可能将斑马鱼重新用作人类遗传疾病的主要模型，前提是这些基因的斑马鱼同源物可以在未来几年内发生突变。目前，人们正在对斑马鱼进行一些反向遗传学研究，这些研究有可能使斑马鱼基因组中的许多基因发生突变。然而，这些方法在很大程度上是没有针对性的，因此不能解决科学界当前和迫切的需求。我们已经建立了一个大型文库，包含15456条冷冻保存的ENU突变鱼，并估计该文库在任何平均大小的斑马鱼基因中至少包含一个无义等位基因，概率为97%。我们已经开发了一种方法来有效地筛选该文库中存在于单个杂合鱼中的突变，并且使用这些方法，我们已经确定了斑马鱼研究人员认为高度优先的80多个基因中的一个或多个无义突变。我们已经在社区内广泛分布了这些突变体，在过去的四年里发表了18篇论文。我们现在已经过渡到用于突变检测的创新和高通量Illumina测序方法，我们建议在三年内在300多个斑马鱼基因中识别一个或多个功能丧失突变，并通过ZIRC将它们交付给社区。

DESCRIPTION (provided by applicant): The zebrafish has emerged as a premier model organism for the study of human development and disease. Its greatest asset is that it is a genetic system in which mutant phenotypes can be effectively dissected at the organismal, cellular and subcellular levels. Many forward genetic screens have identified genes and subsequently delineated their roles in embryonic cell fate specification, patterning, morphogenesis and organogenesis. However with the sequencing of the zebrafish genome we now know that many genes have never been identified by forward genetics, so their role in development remains an open question. Furthermore, recent explosion in the identification of human disease-causing genes has the potential to re-purpose the zebrafish as a leading model for human genetic disease, provided the zebrafish homologs of these genes can be mutated within the next few years. A number of reverse genetics approaches are currently being pursued in the zebrafish, with the potential to generate knock-outs in many of the genes in the genome. However, these approaches are largely untargeted and therefore do not address the immediate and pressing needs of the scientific community. We have created a large library of 15,456 cryopreserved, ENU mutagenized fish and estimate that this library contains at least one nonsense allele in any average-sized zebrafish gene with 97% chance. We have developed methodologies to screen this library efficiently for mutations that are present in a single heterozygous fish, and using these methodologies we have identified one or more nonsense mutations in over 80 genes considered to be of high-priority by zebrafish researchers. We have distributed these mutants widely within the community, resulting in 18 publications in the past four years. We have now transitioned to innovative and high-throughput Illumina sequencing methodologies for mutation detection with which we propose to identify one or more loss-of-function mutation in over 300 zebrafish genes in three years, and to deliver them to the community via ZIRC. PUBLIC HEALTH RELEVANCE: The goal of this grant is to mutate zebrafish genes whose human homologs play important roles in development and disease, and to provide those mutants to the community. Using these mutants and the other attributes of this animal model (its fecundity, external development and optical qualities) researchers will be able to discover mechanisms underlying normal development and disease pathogenesis.