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 (核心董事：克里斯·沃德) Core 1将使用TALEN和CRISPR为对PKD感兴趣的用户提供基因靶向，这是 诱导双链DNA断裂，这些断裂是通过容易出错的非同源末端连接修复的 (NHEJ)途径。NHEJ经常导致较短的删除或插入，因此在 如果以编码区为靶点，则敲除基因功能。核心将与用户合作，确定该地区 要针对的基因和要使用的菌株。核心还将提供低形态的PKD1小鼠，如果 研究人员希望快速评估杂合子TALEN或CRISPR诱导的促囊活性 在一个温和的囊肿化的‘敏感的’背景上为零。核心还将进行特定的定制更改(基因组 编辑或敲入突变)，并将为具有 与PC1/PC2复合体的物理相互作用有关。Core还将以细胞系为目标 (包括ES细胞)产生具有突变的匹配系，用于生理性包囊形成分析和 电生理学，并将串联亲和标签(TAP)标签引入候选人的开放阅读框架 允许候选和相互作用的蛋白质在生理条件下从细胞中分离出来的基因 条件，没有过度表达。基因定位核心将与密歇根大学的现有核心合作 堪萨斯医学中心，包括转基因和基因靶向机构和基因组 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.