A system for spatiotemporal gene inactivation

描述(由首席调查员提供)：此应用程序旨在为斑马鱼研究人员提供条件突变，以确定特定阶段和组织特定的基因功能。斑马鱼已经成为基因功能分析的流行模型。虽然斑马鱼有几种基因失活方法，但它们在任何时候都会取消所有细胞的基因功能，往往掩盖了后来或不太明显的功能。确定时间和空间特定的基因功能需要条件等位基因，这些条件等位基因通常通过特定部位的重组酶准确地使感兴趣的阶段和组织中的基因失活。然而，在斑马鱼中，目前还没有条件等位基因，而且具有特定部位重组酶阶段和组织特异性表达的转基因品系非常罕见。这项应用旨在填补这些空白，并产生条件等位基因和转基因重组酶系，用于在体细胞中进行阶段和组织特异性重组。我们产生条件性突变的策略是使用基因诱变。该方法利用了基因陷阱突变对3‘端强外显子正确定位的依赖性，并利用重组酶催化的翻转和切除(FLEX)稳定地逆转了基因陷阱。我们已经构建了一个可逆的、具有不对称诱变性的双向基因捕捉盒，并用它来产生基因陷阱突变。我们已经证明，Cre和FLP可以有效地逆转基因捕捉盒，并将其在诱变和非诱变状态之间切换。为了利用条件等位基因，我们产生了组织特异性的Cre和他莫昔芬依赖的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。目标1是生成带注释的条件等位基因的公共集合。我们将识别500个含有条件盒的注释基因陷阱插入线，并将它们存放在Zirc中供公众分发。对于每个插入，我们将确定整合位置和受影响的基因，并记录两个阶段的表达模式。我们将分析3个与已发表的突变等位的插入片段，以进一步确认等位基因的用途。目标2是建立一组重组酶表达株，用于阶段和组织特异性重组。我们将使用TG(hsp701：CreERT2)和TG(hsp701：ERT2CreERT2)构建一个用于阶段特异性重组的转基因系。我们将使用特征性启动子，以及在具有高度组织特异性表达的基因陷阱位置进行靶向整合，建立Cre或他莫昔芬可诱导的Cre表达系。将对这些品系的特异性进行表征，并将在ZIRC存放20个精选品系。该申请解决了PAR 08-139规定的几个目标，并应扩大斑马鱼在了解人类疾病的遗传基础方面的用途。 公共卫生相关性：为了更好地定义基因功能，我们建议建立一个时空特异性基因失活和靶向基因替换的系统。该系统的组件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与人类同源，该系统将有助于了解人类生理和疾病的遗传基础。

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.