COOPERATIVE AGREEMENT: VIRGINIA MASON BAC LIBRARY RESO*

细菌人工染色体（BAC）文库已成为现代分子生物学研究的关键试剂，在比较基因组学和功能基因组学研究中尤为重要。 然而，由于其极高的成本，其建设所需的专门知识，以及其生成和分配所需的专门设备，很少有实验室能够定期生成这些资源。 因此，RFA建立资源实验室，专门从具有医学相关性的真核物种中生成BAC文库是一项势在必行的举措，我的实验室特别适合这一举措。 我们在分子进化和发育基因组学方面的研究兴趣决定了我们需要生成BAC文库，因此，我们已经从我们研究的各种脊索动物物种中构建了几个文库。我的研究小组最近搬到了弗吉尼亚梅森研究中心（西雅图），我们的新实验室已经建立了必要的基础设施，空间和机器人仪器，专门用于执行RFA规定的工作类型。 我们在本次提交中的主要目标是确保为感兴趣的真核物种生成高质量的BAC文库，并且这些文库可被科学界广泛访问。 我们建议作为一个中心，每年从六个拟南芥大小的基因组（物种）中产生高质量的10倍覆盖率BAC（等于或> 150 kb平均插入大小）。 我们将排列这些库，并制作必要的副本，分发给库的主要用户和其他实体，这些实体将作为中央资源分发者。 由于我实验室的生物学重点很重，而且我们的强项是BAC库的构建而不是分发，因此我们采用了一种模式，即我们主要作为资源生成器。 我们主张在这个RFA计划中产生的资源尽可能容易地被科学界获取（广泛分布），并在我们能够的范围内，我们将为社区生产数量有限的高密度过滤器，并作为我们产生的图书馆的档案网站。 除了文库的构建外，我们还对BAC构建中涉及的方案进行了改进，包括高分子量DNA的分离、部分限制性酶切、制备性脉冲场凝胶电泳（PFGE）和从凝胶中洗脱部分酶。我们还采取了后勤措施和程序，大大简化了整个BAC克隆过程。 为了与RFA的技术开发部分保持一致，已经进行了初步实验，以便从机械剪切的样品中克隆大的DNA片段，并从异质分子群体中特异性捕获大的基因组片段。 最后，我们希望与BAC科学顾问小组建立良好的工作关系，并与其他BAC图书馆资源中心建立关键和合作的串扰。

Bacterial artificial chromosome (BAC) libraries have become pivotal reagents for modern molecular biology research, and are particularly important for comparative and functional genomics. However, due to their extreme cost, the expertise required for their construction, and the specialized equipment necessary for their generation and distribution, very few laboratories have been capable of routinely generating these resources. This RFA to establish resource laboratories to specifically generate BAC libraries from eukaryotic species of medical relevance is thus an imperative initiative, and one for which my laboratory is especially well-suited. Our research interests in molecular evolution and developmental genomics have dictated our need for generating BAC libraries, and consequently, we have constructed several libraries from the various chordate species that we study. My research group has recently moved to the Virginia Mason Research Center (Seattle) and our new laboratory has been set up with the necessary infrastructure, space, and robotics instruments expressly for performing the type of work prescribed by the RFA. Our main goal in this submission is to assure that high quality BAC libraries are generated for the eukaryotic species of interest and that these libraries are widely accessible to the scientific community. We propose to serve as a center for the generation of high quality 10X coverage BAC (equal to or > 150 kb average insert size) from six mammalian-sized genomes (species) per year. We will array these libraries and make requisite replicas for distribution to major users of the library and other entities that will serve as central resource distributors. Because of the heavy biological focus in my laboratory and because our strength is BAC library construction and not distribution, we thus adopt a model whereby we primarily serve as resource-generators. We advocate that the resources generated in this RFA program be as easily accessible (widely distributed) to the scientific community as possible, and to the extent that we are able, we will produce a limited number of high-density filters for the community and serve as an archival site for the libraries that we generate. Insofar as library generation per se, we have improved on the protocols involved in BAC construction, including high molecular weight DNA isolation, partial restriction digestion, preparative pulsed field gel electrophoresis (PFGE), and elution of partial digests from gels. We have also adopted logistical measures and procedures that streamline the entire BAC cloning process considerably. In keeping with the technology development component of this RFA, preliminary experiments have been performed in order to clone large DNA fragments from mechanically sheared samples and for specifically capturing large genomic fragments from heterogeneous populations of molecules. Finally, we wish to establish a good working relationship with the BAC Scientific Advisory Panel and to establish critical and cooperative crosstalk with the other BAC library resource centers.