A system for spatiotemporal gene inactivation

时空基因失活系统

• 批准号: 8325922
• 负责人: WENBIAO CHEN
• 金额: $ 32.05万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8325922
• 关键词: Address; Affect; Alleles; Benign; Cells; Collection; Communities; Data; Dependence; Deposition; Development; Disease; Excision; Exons; Figs - dietary; Gene Silencing; Gene Targeting; Genes; Genetic; Genetic Recombination; Human; International; Methods; Modeling; Mutagenesis; Mutation; Orthologous Gene; Pattern; Physiology; Principal Investigator; Production; Publishing; RNA; RNA Splicing; Reading Frames; Research Personnel; Resources; Sagittaria; Signal Transduction; Site; Somatic Cell; Specificity; Staging; System; Tamoxifen; Time; Tissues; Transgenic Organisms; Zebrafish; base; gene function; gene replacement; human disease; interest; promoter; public health relevance; recombinase; research study; spatiotemporal

DESCRIPTION (provided by Principal Investigator): This application aims to provide zebrafish researchers with conditional mutations for determining stage- and tissue-specific gene function. The zebrafish has become a popular model for functional analysis of genes. Although there are several gene inactivation methods in zebrafish, they all abolish gene function in all cells at all time, often concealing later or less pronounced functions. Determining temporal and spatial specific gene function requires conditional alleles that inactivate genes precisely in the stage and tissue of interest, usually by a site-specific recombinase. In zebrafish, however, conditional alleles are not currently available and transgenic lines with stage- and tissue-specific expression of a site-specific recombinase are very rare. This application aims to fill these voids and generate conditional alleles and transgenic recombinase lines for stage- and tissue-specific recombination in somatic cells. Our strategy for generating conditional mutations is to use gene trap mutagenesis. This approach takes advantage of the dependence of gene trap mutations on a strong 3' terminal exon in the right orientation and stable inversion of the gene trap using recombinase-catalyzed flip and excision (FlEx). We have constructed an invertible, bidirectional gene trap cassette with asymmetric mutagenicity and have used it to generate gene trap mutations. We have demonstrated that Cre and Flp can efficiently invert the gene trap cassette and switch it between mutagenic and non-mutagenic states. To make use of the conditional allele, we have generated tissue-specific Cre and tamoxifen-dependent Cre lines. We propose to expand the production of conditional alleles and transgenic recombinase lines as a community resource. Aim 1 is to generate a public collection of annotated conditional alleles. We will identify 500 annotated gene trap insertion lines containing a conditional cassette and deposit them in ZIRC for public distribution. For each insertion, we will determine the integration site and the affected gene, as well as document the expression pattern at 2 stages. We will analyze 3 selected insertions that are allelic to published mutations to further confirm utilities of the alleles. Aim 2 is to generate a collection of recombinase-expressing lines for stage- and tissue-specific recombination. We will generate a transgenic line for stage-specific recombination using Tg(hsp70l:CreERT2) and Tg(hsp70l:ERT2CreERT2) constructs. We will generate Cre- or tamoxifen-inducible Cre-expressing lines using characterized promoters, as well as targeted integration at gene trap sites with highly tissue-specific expression. The specificity of these lines will be characterized and 20 selected lines will be deposited at ZIRC. The application addresses several of the stated objectives of PAR 08- 139 and should broaden the use of zebrafish in understanding genetic basis of human diseases. PUBLIC HEALTH RELEVANCE: To better define gene function, we propose to establish a system for spatiotemporal specific gene inactivation and targeted gene replacement. Components of the system will be deposited at the Zebrafish International Resource Center for public distribution. Because most zebrafish genes have a human ortholog, the system will help understand genetic bases of human physiology and disease.

描述（由主要研究者提供）：该应用程序旨在为斑马鱼研究人员提供条件突变，用于确定阶段和组织特异性基因功能。斑马鱼已经成为基因功能分析的一种流行模型。虽然在斑马鱼中有几种基因失活方法，但它们都在所有细胞中始终消除基因功能，通常隐藏后期或不太明显的功能。确定时间和空间特异性基因功能需要条件等位基因，这些条件等位基因通常通过位点特异性重组酶在感兴趣的阶段和组织中精确地定位基因。然而，在斑马鱼中，条件等位基因目前不可用，并且具有位点特异性重组酶的阶段特异性和组织特异性表达的转基因品系非常罕见。本申请旨在填补这些空白，并产生条件等位基因和转基因重组酶系，用于体细胞中的阶段和组织特异性重组。我们产生条件突变的策略是使用基因陷阱诱变。该方法利用了基因陷阱突变对正确方向上的强3'末端外显子的依赖性以及使用重组酶催化的翻转和切除（FlEx）的基因陷阱的稳定倒位。我们已经构建了一个可逆的，双向的基因陷阱盒与不对称致突变性，并已使用它来产生基因陷阱突变。我们已经证明，Cre和Flp可以有效地反转基因陷阱盒，并在致突变和非致突变状态之间切换。为了利用条件等位基因，我们产生了组织特异性Cre和他莫昔芬依赖性Cre系。我们建议扩大条件等位基因和转基因重组酶系作为社区资源的生产。目标1是产生注释的条件等位基因的公共集合。我们将鉴定500个含有条件盒的注释基因陷阱插入系，并将它们存款在ZIRC中用于公开分发。对于每个插入，我们将确定整合位点和受影响的基因，并记录2个阶段的表达模式。我们将分析与已发表突变等位的3个选定插入，以进一步确认等位基因的效用。目的2是产生用于阶段和组织特异性重组的重组酶表达系的集合。我们将使用Tg（hsp 70 l：CreERT 2）和Tg（hsp 70 l：ERT 2CreERT 2）构建体产生用于阶段特异性重组的转基因系。我们将产生Cre或他莫昔芬诱导的Cre表达线使用特征的启动子，以及有针对性的整合在基因陷阱网站具有高度组织特异性的表达。将对这些品系的特异性进行鉴定，并将20个选定的品系存放在ZIRC。该申请解决了PAR 08- 139的几个既定目标，并应扩大斑马鱼在理解人类疾病遗传基础方面的应用。 公共卫生相关性：为了更好地定义基因的功能，我们建议建立一个系统的时空特异性基因失活和靶向基因替换。该系统的组成部分将存放在斑马鱼国际资源中心，供公众分发。由于大多数斑马鱼基因都有人类直系同源物，该系统将有助于了解人类生理和疾病的遗传基础。