A system for spatiotemporal gene inactivation

时空基因失活系统

• 批准号: 8725647
• 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/8725647
• 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.

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。斑马鱼已成为基因功能分析的流行模型。尽管斑马鱼有多种基因失活方法，但它们都始终废除所有细胞中的基因功能，常常隐藏后来或不太明显的功能。确定时间和空间特定基因功能需要条件等位基因，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灭活基因。然而，在斑马鱼中，目前还没有条件等位基因，并且具有位点特异性重组酶的阶段和组织特异性表达的转基因系非常罕见。该应用旨在填补这些空白并生成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) 实现基因陷阱的稳定反转。我们构建了一个具有不对称诱变性的可逆、双向基因陷阱盒，并用它来产生基因陷阱突变。我们已经证明Cre和Flp可以有效地反转基因陷阱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。我们将鉴定 500 个包含条件盒的带注释的基因陷阱插入线，并将它们存放在 ZIRC 中以供公共分发。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选定插入，以进一步确认等位基因的效用。目标 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(hsp70l:CreERT2) 和 Tg(hsp70l:ERT2CreERT2) 构建体生成用于阶段特异性重组的转基因系。我们将使用特征化的启动子以及在具有高度组织特异性表达的基因捕获位点进行靶向整合来生成 Cre 或他莫昔芬诱导型 Cre 表达系。这些品系的特异性将得到表征，20 个选定的品系将存放在 ZIRC。该应用程序解决了 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用途。

项目成果

Conditional gene-trap mutagenesis in zebrafish.

• DOI: 10.1007/978-1-62703-721-1_19
• 发表时间: 2014
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Maddison, Lisette A;Li, Mingyu;Chen, Wenbiao
• 通讯作者: Chen, Wenbiao

Multiplex conditional mutagenesis in zebrafish using the CRISPR/Cas system.

• DOI: 10.1016/bs.mcb.2016.04.018
• 发表时间: 2016
• 期刊: Methods in cell biology
• 作者: L. Yin;Lisette A Maddison;Wenbiao Chen

High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9.

• DOI: 10.1101/gr.186379.114
• 发表时间: 2015-07
• 期刊: Genome research
• 影响因子: 7
• 作者: Varshney GK;Pei W;LaFave MC;Idol J;Xu L;Gallardo V;Carrington B;Bishop K;Jones M;Li M;Harper U;Huang SC;Prakash A;Chen W;Sood R;Ledin J;Burgess SM
• 通讯作者: Burgess SM

Modeling Pancreatic Endocrine Cell Adaptation and Diabetes in the Zebrafish.

在斑马鱼中对胰腺内分泌细胞适应和糖尿病进行建模。

• DOI: 10.3389/fendo.2017.00009
• 发表时间: 2017
• 期刊: Frontiers in endocrinology
• 影响因子: 5.2
• 作者: Maddison LA;Chen W
• 通讯作者: Chen W

Therapeutic Silencing of Centromere Protein X Ameliorates Hyperglycemia in Zebrafish and Mouse Models of Type 2 Diabetes Mellitus

着丝粒蛋白 X 的治疗性沉默可改善 2 型糖尿病斑马鱼和小鼠模型中的高血糖症

• DOI: 10.3389/fgene.2019.00693
• 发表时间: 2019
• 期刊: Frontiers in Genetics
• 影响因子: 3.7
• 作者: Zang Liqing;Shimada Yasuhito;Nakayama Hiroko;Chen Wenbiao;Okamoto Ayaka;Koide Hiroyuki;Oku Naoto;Dewa Takehisa;Shiota Masayuki;Nishimura Norihiro
• 通讯作者: Nishimura Norihiro