Cell line Core

项目总结 这个项目将研究哺乳动物DNA聚合酶θ(POLθ)，这是DNA修复的决定酶 聚合酶Theta介导的末端连接(TMEJ)导致双链断裂。这是核心C(“细胞系” 工程核心“)，这是一个项目的一部分，名为”聚合酶theta，基因组不稳定性，和 癌症“。Core C将负责提供天然和基因工程细胞系，以支持所有 以细胞为基础的分析在计划项目的项目1、2和4中执行。 哺乳动物细胞系试剂的鉴定对于实验室的严密性和重复性至关重要 实验。通过建立和实施标准化的细胞系验证方案，高效的基因组 操作、冷冻保存和传播，核心将确保细胞系试剂和 将错误观察和解释的可能性降至最低。 在目标1，“本计划项目中使用的哺乳动物细胞系模型的认证”中，我们将实现 一种标准化和全面的哺乳动物细胞系鉴定和鉴定方案 在整个计划项目中使用的型号。 在目标2，“基于CRISPR的细胞系基因组工程服务计划”中，我们将提供 在设计和执行定制细胞系基因组工程任务方面的专家服务，以支持 计划项目目标。在这一目标中使用的Cas9蛋白将由Core B提供。 在目标3“转基因在哺乳动物细胞系中的可控和稳定表达”中，我们将利用 用于产生表达多种基因的细胞系的多西环素可诱导的构成转座子载体 诱变和/或标记的感兴趣的转基因。 核心C服务将在计划项目内高度协调，并将促进严谨和 高度综合的研究活动的可重复性和成本效益，目的是了解 DNA聚合酶Q在肿瘤中的作用和靶向性。

PROJECT SUMMARY This project will investigate mammalian DNA polymerase θ (Pol θ), the defining enzyme for repair of DNA double-strand breaks by polymerase theta-mediated end joining (TMEJ). This is Core C (“Cell Line Engineering Core”) which is part of a Program Project titled, “Polymerase theta, genome instability, and cancer”. Core C will be responsible for providing native and genetically engineered cell lines to support all of the cell-based assays performed in Projects 1, 2, and 4 of the Program Project. The authentication of mammalian cell line reagents is vital for rigor and reproducibility of laboratory experiments. By establishing and implementing standardized protocols for cell line validation, efficient genome manipulation, cryopreservation, and dissemination, the Core will ensure quality control of cell line reagents and minimize the likelihood of erroneous observations and interpretations. In Aim 1, “Credentialing of mammalian cell line models utilized in this Program Project”, we will implement a standardized and comprehensive protocol for authentication and characterization of mammalian cell line models used across the Program Project. In Aim 2, “CRISPR-based cell line genome engineering services for the Program Project”, we will provide expert services in the design and execution of customized cell line genome engineering tasks in support of Program Project goals. Cas9 protein utilized in this Aim will be provided by Core B. In Aim 3, “Controlled and stable expression of transgenes in mammalian cell lines”, we will utilize doxycycline-inducible and constitutive transposon vectors for the generation of cell lines that express a variety of mutagenized and/or tagged transgenes of interest. Core C services will be highly coordinated within the Program Project, and will facilitate rigor and reproducibility, as well as cost-effectiveness, of highly integrated research activities aimed at understanding the role and targetability of DNA Polymerase q in cancer.