Intron-based Mutagenic Transposons for Zebrafish

描述（由申请人提供）：主要的下一个基因组目标之一是对数万种体内功能未知的蛋白质编码、非编码RNA和微小RNA基因进行功能注释。我们已经开发了基于内含子的斑马鱼突变作为一种有效的和受调控的功能丧失的方法，这种模式脊椎动物的方法。我们已经开发了基因断裂和翻译基因陷阱载体作为插入诱变剂，作为在该模型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我们的目标：开发和测试专门用于正向遗传筛选的GBT，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'基因陷阱载体作为适合于斑马鱼正向遗传筛选的插入诱变剂。将采用用于鉴定胚胎发生、血管发育中明显缺陷的生物学试验以及用于鉴定突变对成瘾过程的不可见影响的新型行为试验来测量该载体的功能致突变性。开发和测试专门设计用于基于表达的遗传筛选的GBT，以分离斑马鱼发育和物质成瘾所需的具有限制表达模式的基因。我们将开发和测试我们的红色荧光蛋白（RFP）为基础的翻译基因陷阱载体的分离与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。我们将产生一个基于序列的数据库的插入等位基因的基因的高度感兴趣的GBT。这些品系将在计算机中编目，并作为冷冻保存的精子储存，供斑马鱼群体随后用于基于序列的搜索、调控诱变研究，以及作为无效等位基因产生的起点。在本项目过程中开发的生物试剂将满足PAR-05-080的目标，为斑马鱼群体提供工具，允许对斑马鱼发育和生长所需的数百种蛋白质编码和ncRNA基因以及与血管生物学、物质成瘾和脊椎动物发育相关的新遗传基因座进行功能注释。

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.