Intron-based Mutagenic Transposons for Zebrafish

基于内含子的斑马鱼诱变转座子

• 批准号: 7290028
• 负责人: Stephen Carl Ekker
• 金额: $ 33.98万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7290028
• 关键词: Alleles; Bacteria; Base Sequence; Behavioral; Behavioral Assay; Biological; Biological Assay; Biology; Blood Vessels; Caenorhabditis elegans; Cataloging; Catalogs; Collection; Communities; Computer Simulation; Databases; Defect; Development; Drosophila genus; Eligibility Determination; Embryonic Development; Functional RNA; Gene Silencing; Generations; Genes; Genetic; Genetic Models; Genetic Screening; Genome; Genomics; Goals; Growth and Development function; Imagery; Insertional Mutagenesis; Introns; Laboratories; Measures; Methods; MicroRNAs; Modeling; Molecular; Mus; Mutagenesis; Mutagens; Mutation; Nicotine; Organism; Pattern; Phenotype; Policies; Process; Proteins; Reagent; Research; Research Personnel; Role; Screening procedure; Series; Sleeping Beauty; Standards of Weights and Measures; Structure; Substance Addiction; System; Testing; Tissues; Vertebrates; Work; Writing; Yeasts; Zebrafish; addiction; base; design; desire; gene function; in vivo; interest; loss of function; novel; novel strategies; red fluorescent protein; response; sperm cell; tool; vector; web based interface

DESCRIPTION (provided by applicant): One of the major next genomic goals is the functional annotation of the over tens of thousands of protein- encoding, non-coding RNA and micro-RNA genes of unknown in vivo function. We have developed intronic- based mutagenesis for the zebrafish as an method for effective and regulated loss-of-function approach for this model vertebrate. We have developed gene-breaking and translational gene trap vectors as insertional mutagens as new approaches for the genome-wide assignment of function using forward (phenotype- based), reverse (sequence-based) and expression-based genetic approaches in this model vertebrate. We will achieve our goal to functionally annotate unknown genes in zebrafish by accomplishing the following specific aims: Develop and test GBTs specifically designed for forward genetic screens to isolate novel genes required for zebrafish development and substance addiction. We will develop and test our 3' gene trap vector as an insertional mutagen suitable for forward genetic screening in zebrafish. Biological assays for the identification of visibly apparent defects in embryogenesis, vascular development, and a novel behavioral assay for non-visible effects of mutations on the addiction process will be employed to measure the functional mutagenicity of this vector. Develop and test GBTs specifically designed for expression-based genetic screens for the isolation of genes with restricted expression patterns that are required for zebrafish development and substance addiction. We will develop and test our red fluorescent protein (RFP)-based translational gene trap vector for the isolation of genes with tissue-specific expression patterns. . Develop and test gene-break transposons for sequence-based genetics in zebrafish. We will generate a sequence-based database of insertional alleles in genes of high interest from GBTs. These lines will be catalogued in silico and stored as cryopreserved sperm for subsequent use by the zebrafish community for sequence-based searches, regulated mutagenesis studies, and as the starting point for null allele generation. The biological reagents developed over the course of this project will meet the goals of PAR-05-080 by providing the zebrafish community with tools that will allow the functional annotation of hundreds of protein- encoding and ncRNA genes necessary for development and growth of zebrafish as well as novel genetic loci involved with vascular biology, substance addiction and vertebrate development.

描述(申请人提供)：下一个主要的基因组目标之一是对数以万计的蛋白质编码、非编码RNA和微RNA基因进行功能注释，这些基因在体内的功能未知。我们已经为斑马鱼开发了基于内含子的突变，作为这种模式脊椎动物有效和受调控的功能丧失的方法。我们开发了基因断裂和翻译基因陷阱载体作为插入突变剂，作为在该模式脊椎动物中使用正向(基于表型)、反向(基于序列)和基于表达的遗传方法进行全基因组功能分配的新方法。我们将通过实现以下具体目标来实现对斑马鱼未知基因进行功能注释的目标：开发和测试专为正向遗传筛选而设计的GBTS，以分离斑马鱼发育和物质成瘾所需的新基因。我们将开发和测试我们的3‘基因陷阱载体，作为一种适合斑马鱼正向遗传筛选的插入诱变剂。用于识别胚胎发生、血管发育中明显缺陷的生物学测试，以及突变对成瘾过程不可见影响的新的行为测试，将被用来测量该载体的功能诱变性。开发和测试专门为基于表达的遗传筛选而设计的GBTS，以分离斑马鱼发育和物质成瘾所需的表达模式受限的基因。我们将开发和测试我们的基于红色荧光蛋白(RFP)的翻译基因陷阱载体，以分离具有组织特异性表达模式的基因。。开发和测试斑马鱼基于序列遗传学的基因断裂转座子。我们将从GBTS中建立一个基于序列的插入等位基因数据库。这些品系将被编目在电子计算机中，并作为冷冻保存的精子储存起来，供斑马鱼群落随后用于基于序列的搜索、受调控的突变研究，以及作为零等位基因产生的起点。在该项目过程中开发的生物试剂将实现PAR-05-080的目标，为斑马鱼群落提供工具，允许对斑马鱼发育和生长所需的数百个蛋白质编码和ncRNA基因以及与血管生物学、物质成瘾和脊椎动物发育有关的新遗传基因进行功能注释。