Regulatory Genomics: BAC-GFP Library of Control Genes

调控基因组学：BAC-GFP 对照基因文库

• 批准号: 7198055
• 负责人: ERIC H DAVIDSON
• 金额: $ 25.49万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7198055
• 关键词: Appendix; Code; Communities; Complementary DNA; Data; Development; Embryo; Exons; Facility Construction Funding Category; Gene Expression; Gene Transfer; Genes; Genome; Genomics; Green Fluorescent Proteins; Internet; Laboratories; Larva; Letters; Libraries; Maps; Methods; Molecular Profiling; Pattern; Production; Proteins; Protocols documentation; Recombinants; Regulator Genes; Reporter; Research Personnel; Resources; Scientist; Sea Urchins; Site; Standards of Weights and Measures; Strongylocentrotus purpuratus; System; Techniques; Testing; Transcription factor genes; cDNA Library; egg; expression vector; genetic regulatory protein; homologous recombination; interest; programs; tool; transcription factor

DESCRIPTION (provided by applicant): We propose to develop a communal resource of BAC GFP expression vectors of all transcription factors expressed significantly in the embryo of the sea urchin, Strongylocentrotus purpuratus, the genomic sequence of which will go on line shortly. Critically, as shown by our laboratory, BAC GFP recombinants can be injected into eggs and with high efficiency, and they express accurately, capturing the entire cis-regulatory systems intact. This greatly accelerates detailed cis-regulatory analyses. These recombinants can also be used to introduce exogenous regulatory gene products in temporally and spatially restricted patterns within the embryo and larva. Both of these applications will greatly enhance analysis of how the genomic control systems which direct expression of regulatory genes prescribe early development. Specifically, we propose to isolate BAC recombinants for all relevant transcription factors (-100-200) in which the gene of interest is centrally located within the clone, and then to use standard homologous recombination techniques to replace the first exon of the endogenous gene with the coding sequence of the GFP reporter. An ongoing project within our laboratory has already established the expression profiles of most of the pertinent genes. We will then employ our methods for high throughput production of BAC-GFP recombinants in exon l of each gene, and build and authenticate each construct. GFP expression directed by each BAC recombinant will be analyzed to confirm faithful recapitulation of endogenous gene expression. We will also make BAC recombinants for any other gene encoding any kind of protein that scientists in the community may request. All information needed by other investigators to facilitate access and promote the use of these tools will be made available on the Sea Urchin Genome web site that is currently administrated by our laboratory. Numerous letters from outside investigators attesting interest in this facility are included as an Appendix to this application.

描述（由申请人提供）：我们建议开发所有转录因子的BAC GFP表达载体的公共资源，这些因子在海胆的胚胎，purvuratus的胚胎中显着表达，其基因组序列将很快在线上。至关重要的是，如我们的实验室所示，可以将BAC GFP重组者注入鸡蛋和高效率中，并且可以准确地表达，从而捕获整个顺式调节系统完整。这大大加速了详细的顺式调节分析。这些重组者也可以用于在胚胎和幼虫内的时间​​和空间限制模式中引入外源调节基因产物。这两种应用都将大大增强对调节基因直接表达早期发育的基因组控制系统的分析。具体而言，我们建议分离所有相关转录因子（-100-200）的BAC重组因素，其中感兴趣的基因位于克隆中，然后使用标准的同源重组技术代替GFP Ectorter的编码序列来代替内源基因的第一个外显子。我们实验室中的一个正在进行的项目已经确定了大多数相关基因的表达谱。然后，我们将采用我们的方法来在每个基因的外显子中进行高吞吐量生产，并构建和身份验证每个构建体。将分析由每种BAC重组者指示的GFP表达，以确认内源基因表达的忠实概述。我们还将为任何编码社区中科学家可能要求的任何其他基因进行BAC重组剂。其他调查人员所需的所有信息可以促进访问和促进这些工具的使用，将在我们实验室目前管理的海胆基因组网站上提供。外部调查人员的许多信件证明对该设施的兴趣作为此应用程序的附录。