Tilling the Zebrafish Genome: A Reverse Genetic Approach

斑马鱼基因组的耕耘：反向遗传方法

• 批准号: 6934500
• 负责人: Cecilia B Moens
• 金额: $ 50.41万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-22 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6934500
• 关键词: alleles; biotechnology; cryopreservation; developmental genetics; gene mutation; genetic library; genetic polymorphism; genetic screening; high throughput technology; method development; neurogenetics; nitrosourea; nonmammalian vertebrate embryology; phenotype; polymerase chain reaction; rhombencephalon; sperm; tumor suppressor genes; zebrafish

DESCRIPTION (provided by applicant): The zebrafish has become the model system of choice for a growing number of investigators interested in understanding mechanisms of vertebrate development, disease and evolution. While forward genetic screens for mutations induced by chemical mutagenesis have uncovered the functions of many zebrafish genes, so far no reverse genetic technology has been developed that is both inexpensive and high-throughput. This application proposes to develop a method for identifying N-ethyI-N-nitrosourea (ENU)-induced mutations in genes of interest in zebrafish. The approach, termed TILLING, uses the CELl enzyme, which cuts DNA at the site of single basepair mismatches, to detect ENU-induced mutations in PCR-amplified genomic DNA fragments. Preliminary data shows that this approach can effectively identify ENU-induced mutations in zebrafish genomic DNA at a rate of 2.03 x 10" mutations per base pair, or one mutation per 493 kb. To determine the feasibility of this approach in zebrafish on a larger scale, a library will be constructed consisting of 10,000 ENU mutagenized fish, preserved as frozen sperm and genomic DNA, which can in principle be screened for mutations in any gene of interest. This library will be used to generate an allelic series including loss-of-function and reduction-of function mutations in 50 zebrafish genes. Targets include genes involved in embryonic patterning, with particular emphasis on genes that are hypothesized to control the patterning and segmentation of the developing hindbrain. In order to demonstrate the general applicability of this approach to the broader interests of the zebrafish community, mutations will also be identified in zebrafish genes that are inaccessible to three-generation forward genetic screens or antisense approaches currently available in the zebrafish. These include maternal effect genes, genes that are function in the adult nervous system, and putative tumor suppressor genes. The long-term objectives of the project are to optimize the efficiency of mutation detection by TILLING in zebrafish, to use mutations generated by TILLING to understand the genetic basis of zebrafish neural patterning, and above all to determine the feasibility of this approach as a general method for reverse genetics in zebrafish.

描述（由申请人提供）：斑马鱼已成为越来越多的研究人员选择的模型系统，这些研究人员对了解脊椎动物发育、疾病和进化机制感兴趣。虽然化学诱变诱导突变的正向遗传筛选已经揭示了许多斑马鱼基因的功能，但迄今为止还没有开发出既便宜又高通量的反向遗传技术。本申请提出开发用于鉴定斑马鱼中感兴趣的基因中的N-乙基-N-亚硝基脲（ENU）诱导的突变的方法。该方法称为TILLING，使用CEL 1酶，其在单个碱基对错配位点切割DNA，以检测PCR扩增的基因组DNA片段中的ENU诱导的突变。初步数据表明，这种方法可以有效地识别斑马鱼基因组DNA中的ENU诱导的突变，每碱基对2.03 × 10 - 16个突变，或每493 kb一个突变。为了确定这种方法在更大规模的斑马鱼中的可行性，将构建一个由10，000条ENU诱变鱼组成的文库，保存为冷冻精子和基因组DNA，原则上可以筛选任何感兴趣基因的突变。该文库将用于产生包括50个斑马鱼基因中的功能丧失和功能减少突变的等位基因系列。目标包括参与胚胎模式的基因，特别强调那些被假设控制发育中的后脑的模式和分割的基因。为了证明这种方法对斑马鱼群体更广泛利益的普遍适用性，还将在斑马鱼基因中鉴定突变，这些突变是目前在斑马鱼中可用的三代正向遗传筛选或反义方法无法获得的。这些基因包括母体效应基因、在成人神经系统中起作用的基因和假定的肿瘤抑制基因。该项目的长期目标是优化斑马鱼TILLING突变检测的效率，使用TILLING产生的突变来了解斑马鱼神经模式的遗传基础，最重要的是确定这种方法作为斑马鱼反向遗传学通用方法的可行性。