TILLING the Zebrafish Genome: A Reverse Genetics Approach

描述（由申请人提供）：斑马鱼已成为研究人类发育和疾病的首要模式生物。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，并随后描绘了它们在胚胎细胞命运特化、模式化、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识别，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，最近在鉴定人类致病基因方面的爆炸性发展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。目前正在斑马鱼中进行一些反向遗传学方法，有可能在基因组中的许多基因中产生敲除。然而，这些方法在很大程度上没有针对性，因此不能解决科学界的直接和迫切需要。我们已经创建了一个包含15，456条冷冻保存的ENU诱变鱼的大型文库，并估计该文库在任何平均大小的斑马鱼基因中含有至少一个无义等位基因，概率为97%。我们已经开发了有效筛选该文库中存在于单个杂合鱼中的突变的方法，并且使用这些方法，我们已经在超过80个基因中鉴定出一个或多个无义突变，这些基因被斑马鱼研究人员认为是高优先级的。我们在社区内广泛分发了这些突变体，在过去四年中发表了18篇论文。我们现在已经过渡到创新和高通量的Illumina测序方法来进行突变检测，我们计划在三年内在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将它们提供给社区。 公共卫生相关性：这项资助的目标是突变斑马鱼基因，这些基因的人类同源物在发育和疾病中起重要作用，并将这些突变体提供给社区。利用这些突变体和该动物模型的其他属性（其繁殖力，外部发育和光学品质），研究人员将能够发现正常发育和疾病发病机制的潜在机制。

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.