Development of a high throughput gene trap vetor

说明(由申请人提供)：该计划的长远目标 目前提出的工作将有助于建立一个随机序列 C57/B6胚胎干细胞中的标记插入文库 携带已知的个人集成事件可以广泛分发到 科学界。为此，提出了四个具体目标 目标。首先，我们将提炼一种高通量的序列标签采集 我们发明的技术，用于ES细胞基因捕获载体 以一种自动格式。其次，我们将生成ES细胞基因陷阱载体 携带序列标签获取所需的序列元素 技术和重组序列元件(FRT位点)将允许 要修改的给定整合位置的序列。作为这一目标的一部分， 允许将可选的序列元素“传递”到 整合网站将建设和测试。第三，我们将建立 早期传代C57BLI6JSvEvTac ES细胞系的储存库 在嵌合小鼠中贡献生殖系的能力，即使在多次 段落。此外，用于自动分离ES细胞克隆的方法 将会受到考验。最后，建立了包含5,000个序列标签的示范库 将产生ES细胞克隆。在这里开发的方法和试剂 将完全足以允许快速建立随机序列 标签插入库包含100,000个或更多事件，其中每个事件的位置 插入位置是已知的。

DESCRIPTION (provided by applicant): The long-term objective towards which the current proposed work will contribute is the establishment of a random sequence tagged insertion library in C57/B6 embryonic stem cells from which cells carrying individual, known, integration events could be widely distribution to the scientific community. Four specific aims are proposed towards this objective. First, we will refine a high throughput sequence tag acquisition technology, which we have invented, for use in an ES cell gene-trapping vector in an automated format. Second, we will generate an ES cell gene trap vector carrying the sequence elements required for the sequence tag acquisition technology and recombinogenic sequence elements (FRT sites) that will allow the sequence at a given integration site to be modified. As part of this aim, vectors that allow "delivery" of alternative sequence elements to the integration site will be constructed and tested. Third, we will establish a repository of early passage C57BLI6JSvEvTac ES cell lines that retain the ability to contribute to the germ line in chimeric mice even after many passages. Additionally, methods for the automated isolation of ES cell clones will be tested. Finally, a demonstration library of 5,000 sequence tag indexed ES cell clones will be generated. The methodologies and reagents developed here will be fully sufficient to allow the rapid establishment of a random sequence tag insertion library of 100,000 or more events in which the location of each insertion sites is known.