Intron-based Mutagenic Transposons for Zebrafish

描述（由申请人提供）：基因组学的下一个主要目标之一是对数以万计的体内未知功能的蛋白质编码、非编码RNA和微RNA基因进行功能注释。我们已经为斑马鱼开发了基于内含子的诱变方法，作为一种有效和可调节的功能丧失方法。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对斑马鱼未知基因进行功能性注释的目标：开发和测试专门为前向遗传筛选设计的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'基因诱捕载体作为插入诱变原适用于斑马鱼的正向遗传筛选。用于鉴定胚胎发生、血管发育中明显缺陷的生物检测，以及用于检测成瘾过程中突变的不可见影响的新型行为检测，将用于测量该载体的功能性诱变性。开发和测试专门设计用于基于表达的遗传筛选的GBTs，以分离斑马鱼发育和物质成瘾所需的受限表达模式的基因。我们将开发和测试基于红色荧光蛋白（RFP）的翻译基因陷阱载体，用于分离具有组织特异性表达模式的基因。在斑马鱼中开发和测试基于序列遗传学的基因断裂转座子。我们将从gbt中产生一个基于序列的高兴趣基因插入等位基因数据库。这些细胞系将在计算机上进行编目，并作为冷冻保存的精子储存起来，供斑马鱼群落用于基于序列的搜索、调控诱变研究，以及作为零等位基因产生的起点。该项目开发的生物试剂将满足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种蛋白质编码和ncRNA基因进行功能注释，以及涉及血管生物学、物质成瘾和脊椎动物发育的新遗传位点。

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.