Regulatory Genomics: BAC-GFP Library of Control Genes

描述（由申请人提供）：我们建议开发一个在海胆（Strongylocentrotus purpuratus）胚胎中显著表达的所有转录因子的BAC GFP表达载体的公共资源，其基因组序列将很快上线。关键的是，正如我们实验室所显示的那样，BAC GFP重组体可以高效地注射到卵子中，并且它们表达准确，完整地捕获了整个顺式调控系统。这大大加快了详细的顺式监管分析。这些重组也可用于在胚胎和幼虫中以时间和空间限制的模式引入外源调控基因产物。这两种应用都将极大地增强对基因组控制系统如何指导调控基因表达的分析。具体来说，我们建议分离所有相关转录因子（-100-200）的BAC重组，其中感兴趣的基因位于克隆的中心位置，然后使用标准的同源重组技术将内源基因的第一个外显子替换为GFP报告基因的编码序列。我们实验室正在进行的一个项目已经建立了大多数相关基因的表达谱。然后，我们将采用我们的方法在每个基因的外显子1上高通量生产BAC-GFP重组，并构建和验证每个构建。将分析每个BAC重组基因引导的GFP表达，以确认内源性基因表达的忠实再现。我们还将根据社区科学家的要求，为编码任何种类蛋白质的任何其他基因制作BAC重组。其他研究人员为方便访问和推广使用这些工具所需的所有信息将在海胆基因组网站上提供，该网站目前由我们实验室管理。来自外部调查人员的许多信件证明对该设施感兴趣，作为本申请的附录。

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.