Biomedical Research Core 1: Gene Targeting Core

项目摘要 生物医学研究核心1：基因靶向核心 (Core导演：Chris Ward Core 1将使用TALEN和CRISPR为对PKD感兴趣的用户提供基因靶向， 诱导双链DNA断裂，所述双链DNA断裂使用易错非同源末端连接修复 （NHEJ）途径。NHEJ通常导致短的缺失或插入，因此在治疗中是非常有效和有效的。 如果靶向编码区，则敲除基因功能。核心将与用户合作， 目标基因和所用菌株的序列。Core还将提供亚型Pkd 1小鼠，如果 研究者希望快速评估杂合TALEN或CRISPR诱导的TALEN的促囊肿活性， 在轻度囊性“致敏”背景下为空。核心还将进行特定的定制改变（基因组 编辑或敲入突变），并将为突变基因提供服务， 与PC 1/PC 2复合物的物理相互作用有关。核心还将针对细胞系 （包括ES细胞）产生具有用于生理性包囊形成测定的突变的匹配系， 电生理学，并且将串联亲和标签（TAP）标签引入候选物的开放阅读框架中。 基因，以允许候选蛋白和相互作用蛋白在生理条件下从细胞中分离。 条件下，没有过度表达。基因靶向核心将与现有的核心在大学的工作， 堪萨斯医学中心，包括转基因和基因靶向机构设施和基因组 测序设施。

Project Abstract Biomedical Research Core 1: Gene Targeting Core (Core Director: Chris Ward) Core 1 will offer gene targeting for users interested in PKD using both TALENs and CRISPRs, which induce double-stranded DNA breaks that are repaired using the error-prone non-homologous end joining (NHEJ) pathway. NHEJ often results in short deletions or insertions and thus are very efficient and effective in knocking out gene function if targeted to coding regions. The Core will work with users to pin-point the region of the gene to be targeted and the strain to be used. The Core will also supply hypomorphic Pkd1 mice if the investigator wishes to quickly assess the cyst-promoting activity of a heterozygous TALEN or CRISPR-induced null on a mildly cystic `sensitized' background. The Core will also make specific custom alterations (genome edits or knock-in mutations) in the Pkd1 or Pkd2 gene, and will provide services for mutating genes that have been implicated in physically interacting with the PC1/PC2 complex. The Core will also target cell lines (including ES cells) producing matched lines with mutations for physiological cyst forming assays and electrophysiology, and will introduce tandem affinity tags (TAP) tags into the open reading frame of candidate genes to allow the candidate and interacting proteins to be isolated from the cells under physiological conditions, without overexpression. The Gene Targeting Core will work with existing cores at the University of Kansas Medical Center, including the Transgenic and Gene-Targeting Institutional Facility and the Genome Sequencing Facility.