A system for spatiotemporal gene inactivation

描述（由首席研究员提供）：该应用程序旨在为斑马鱼研究人员提供条件突变，以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流行模型。虽然在斑马鱼中有几种基因失活方法，但它们都是在所有细胞中同时取消基因功能，往往掩盖了后来或不太明显的功能。确定时间和空间特异性基因功能需要条件等位基因，这些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灭活基因。然而，在斑马鱼中，条件等位基因目前还不可用，具有阶段和组织特异性表达位点特异性重组酶的转基因系非常罕见。本应用程序旨在填补这些空白，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诱捕诱变。该方法利用了基因陷阱突变依赖于正确方向的强3'末端外显子，并利用重组酶催化的翻转和切除（FlEx）稳定地反转基因陷阱。我们构建了一个可逆的、双向的、不对称诱变的基因诱捕盒，并用它来产生基因诱捕突变。我们已经证明，Cre和Flp可以有效地反转基因陷阱盒，并在诱变和非诱变状态之间切换。为了利用条件等位基因，我们已经生成了组织特异性Cre和他莫昔芬依赖性Cre系。我们建议扩大条件等位基因的生产和转基因重组酶系作为一个社区资源。目标1是生成一个带注释的条件等位基因的公共集合。我们将鉴定500个带注释的基因陷阱插入系，其中包含一个条件磁带，并将它们放入ZIRC中公开分发。对于每次插入，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3个选择的与已发表突变等位的插入，以进一步确认这些等位基因的效用。目的2是生成一组表达重组酶的细胞系，用于阶段和组织特异性重组。我们将使用Tg（hsp701:CreERT2）和Tg（hsp701:ERT2CreERT2）构建一个针对特定阶段重组的转基因系。我们将使用特征启动子，以及在具有高度组织特异性表达的基因陷阱位点进行靶向整合，生成Cre或他莫昔芬诱导的Cre表达系。将对这些谱线的特异性进行表征，并选择20条谱线在锆石上沉积。该申请解决了PAR 08- 139的几个既定目标，并应扩大斑马鱼在了解人类疾病遗传基础方面的应用。

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.