Tilling the Zebrafish Genome: A Reverse Genetic Approach

描述(申请人提供)：斑马鱼已经成为越来越多对了解脊椎动物发育、疾病和进化机制感兴趣的研究人员选择的模型系统。虽然对化学诱变所致突变的正向遗传筛查已经发现了许多斑马鱼基因的功能，但到目前为止还没有开发出既便宜又高通量的反向遗传技术。本申请建议开发一种方法来识别N-乙基-N-亚硝脲(ENU)诱导的斑马鱼感兴趣基因的突变。这种方法被称为TILLING，它使用一种细胞酶，在单个碱基不匹配的位置切割DNA，以检测ENU在PCR扩增的基因组DNA片段中诱导的突变。初步数据表明，该方法可以有效地识别ENU诱导的斑马鱼基因组DNA突变，每碱基对2.03×10“突变，或每493kb一个突变。为了确定这种方法在更大范围内应用于斑马鱼的可行性，将构建一个由10,000条ENU诱变鱼组成的文库，这些鱼以冷冻精子和基因组DNA的形式保存，原则上可以筛选任何感兴趣的基因的突变。该文库将用于产生包括50个斑马鱼基因功能缺失和功能缺失突变的等位基因序列。目标包括参与胚胎构型的基因，特别强调那些被假设控制发育中的后脑的构图和分割的基因。为了证明这种方法对斑马鱼群落更广泛利益的普遍适用性，还将确定斑马鱼基因中的突变，这些突变是目前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，以及可能的肿瘤抑制基因。该项目的长期目标是通过在斑马鱼中进行耕作来优化突变检测的效率，利用耕作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的一般方法的可行性。

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.