A system for spatiotemporal gene inactivation

时空基因失活系统

• 批准号: 8541838
• 负责人: WENBIAO CHEN
• 金额: $ 30.92万
• 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/8541838
• 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）：该应用旨在为斑马鱼研究人员提供有条件的突变，以确定分期和组织特异性基因功能。斑马鱼已成为基因功能分析的流行模型。尽管斑马鱼中有几种基因灭活方法，但它们始终都废除了所有细胞中的基因功能，通常会隐藏以后或不太明显的功能。确定时间和空间特异性基因功能需要有条件的等位基因，这些等位基因通常是通过位点特异性重组酶在感兴趣的阶段和组织中灭活基因的。然而，在斑马鱼中，有条件的等位基因目前尚不可用，并且具有特定位点特异性重组酶的分期和组织特异性表达的转基因线非常罕见。该应用旨在填补这些空隙并产生条件等位基因和转基因重组酶系，以用于体细胞中的分期和组织特异性重组。我们产生条件突变的策略是使用基因陷阱诱变。这种方法利用了使用重组酶催化的翻转和切除（FLEX）在右方向上的基因陷阱突变对强度3'末端外显子的依赖性。我们已经构建了具有不对称诱变性的可逆，双向基因陷阱盒，并使用它来产生基因陷阱突变。我们已经证明了CRE和FLP可以有效地倒转基因陷阱盒，并将其切换在诱变和非毒素状态之间。为了利用条件等位基因，我们已经产生了组织特异性的Cre和他莫昔芬依赖性的CRE系。我们建议扩大条件等位基因和转基因重组酶系的生产作为社区资源。目的1是生成带注释的条件等位基因的公共集合。我们将确定500个带有条件盒的注释基因陷阱插入线，并将其存放在Zirc中以进行公共分布。对于每个插入，我们将确定积分位点和受影响的基因，并在2个阶段记录表达模式。我们将分析3个选定的插入，这些插入与已发表的突变相位，以进一步确认等位基因的实用性。 AIM 2是生成表达重组酶的系列，以进行分期和组织特异性重组。我们将使用TG（HSP70L：CREERT2）和TG（HSP70L：ERT2CREERT2）构建体生成用于阶段特异性重组的转基因线。我们将使用表征启动子以及具有高度组织特异性表达的基因陷阱位点的靶向整合来生成CRE或他莫昔芬诱导的表达CRE线。这些线的特异性将被表征，并将20条线沉积在锆石上。该申请涉及第08-139款的几个既定目标，并应扩大斑马鱼在理解人类疾病的遗传基础中的使用。 公共卫生相关性：为了更好地定义基因功能，我们建议建立一个用于时空特异性基因失活和靶向基因置换的系统。该系统的组件将存放在斑马鱼国际公共分销中心。由于大多数斑马鱼基因都有人类直系同源物，因此该系统将有助于了解人类生理和疾病的遗传基础。