GENE LIBRARY RESOURCE FOR SEA URCHIN S. PURPURATUS

这一应用是为了支持向海胆发育分子和细胞生物学研究社区分发高密度阵列过滤器(宏阵列)，其中包含代表胚胎发育的每个阶段、几种单个细胞类型和各种成体组织的大型cDNA文库。我们还将创建和分发带有社区特别感兴趣的基因的微阵列。宏微阵列都将使用加州理工大学的Genetix机器人生成。该文库已全部构建完成，并已作为赤眼莲基因组计划的一部分进行排列。通过申请书中的信件证明，已经开始在试点范围内向选定的实验室分发Macroarray过滤器，反应非常热烈。我们将定期进行质量控制测试，我们打算维持的服务将包括回收和运输大阵列用户要求识别的任何克隆。排列好的图书馆将保存在我实验室的冷冻库中。另一项重要的额外目标是管理一个不断更新的、可供公众查阅的网站数据库，其中有关阵列中克隆人身份的信息是从使用这些克隆的所有实验室收集的。拟议工作的辅助研究和开发部分包括完善一种强大的大阵列差异消减杂交新方法；测量微阵列的杂交动力学，以确定在不同实验情况下有用的灵敏度参数；开发用于微阵列和大阵列的探针，以代表海胆胚胎中的任何特定调控状态；以及完成定制的独特处理系统，用于使用复杂探针对大阵列屏幕进行定量差异分析。

This application is to support the distribution to the sea urchin developmental molecular and cell biology research community of high density arrayed filters (macroarrays) containing large cDNA libraries, that represent every stage of embryogenesis, several individual cell types, and various adult tissues. We will also create and distribute microarrays bearing genes of specific interest to the community. Both macro- and microarrays will be generated using the Genetix robot at Caltech. The cDNA libraries have already all been constructed and arrayed as part of the Strongylocentrotus puipurntus Genome Project. Macroarray filter distribution to selected laboratories has been initiated on a pilot scale, with extremely enthusiastic responses, as attested by letters within the Application. Quality control testing will be routinely carried out, and the service we intend to maintain will include recovery and shipping of any clones identified by users of the macroarrays on request. The arrayed libraries wffl be maintained in freezer banks in my laboratory. An important additional objective is the management of a continuously updated, publicly available web site data base in which information as to the identity of clones in the arrays is accumulated from all laboratories utilizing them. The ancillary research and development component of the proposed work includes the perfection of a powerful new methodology for differential subtractive hybridization on the macroarrays; measurement of hybridization kinetics on microarrays to establish useful parameters of sensitivity under diverse experimental situations; development of probes for use on both micro-and macroarrays that represent any given regulatory state in the sea urchin embryo; and completion of a custom made unique processing system for quantitative differential analysis of macroarray screens using complex probes.