Regulatory Genomics: BAC-GFP Library of Control Genes

描述(申请人提供)：我们建议开发一个共同的BAC GFP表达载体资源，所有在海胆胚胎中显著表达的转录因子GFP表达载体，其基因组序列将很快上线。重要的是，正如我们的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，并且它们的表达准确，完整地捕获了整个顺式调控系统。这大大加快了详细的顺势监管分析。这些重组子还可以用于在胚胎和幼虫中以时间和空间受限的模式导入外源调控基因产物。这两个应用都将极大地加强对指导调控基因表达的基因组控制系统如何规定早期发育的分析。具体地说，我们建议分离所有相关转录因子(-100-200)的BAC重组体，其中目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，我们将使用我们的方法高通量地生产每个基因外显子L上的BAC-GFP重组体，并构建和验证每个构建体。将对每个BAC重组体指导的GFP表达进行分析，以确认内源基因表达的真实概括。我们还将根据社区科学家的要求，为任何其他编码任何类型蛋白质的基因制造BAC重组体。其他调查人员为便利获取和促进使用这些工具所需的所有信息将在目前由我们实验室管理的海胆基因组网站上提供。来自外部调查人员的大量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.