Development of a high throughput gene trap vetor

描述（由申请人提供）： 目前提出的工作将有助于建立随机序列 在C57/B6胚胎干细胞中的标记插入文库中， 可以广泛地分发携带单个、已知、集成事件的数据， 科学界。为此提出了四个具体目标 objective.首先，我们将改进高通量序列标签采集 我们发明的用于ES细胞基因捕获载体的技术 自动化的格式。其次，我们将产生ES细胞基因捕获载体， 携带序列标签获取所需的序列元件 技术和重组基因序列元件（FRT位点），这将允许 序列在给定的整合位点被修饰。作为这一目标的一部分， 允许将替代序列元件“递送”到细胞的载体， 将建立和测试整合网站。第三，我们会建立一个 早期传代C57 BLI 6 JSvEvTac ES细胞系的储存库，其保留了 即使在许多嵌合小鼠中， 段落。另外，用于自动分离ES细胞克隆的方法 会得到考验最后，一个示范库的5,000个序列标签的索引 将产生ES细胞克隆。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 包含100，000个或更多事件的标签插入库，其中每个事件的位置 插入位点是已知的。

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.