Cell line Core

项目概要 该项目将研究哺乳动物 DNA 聚合酶 θ (Pol θ)，它是 DNA 修复的定义酶 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”）是名为“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 该计划项目的项目 1、2 和 4 中进行的基于细胞的测定。 哺乳动物细胞系试剂的认证对于实验室的严谨性和可重复性至关重要 实验。通过建立和实施细胞系验证的标准化协议，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的质量控制 最大限度地减少错误观察和解释的可能性。 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，我们将实施 用于哺乳动物细胞系认证和表征的标准化和综合方案 整个计划项目中使用的模型。 在目标2“项目项目基于CRISPR的细胞系基因组工程服务”中，我们将提供 设计和执行定制细胞系基因组工程任务的专家服务，以支持 计划 项目目标。该目标中使用的 Cas9 蛋白将由 Core B 提供。 在目标 3“哺乳动物细胞系中转基因的受控和稳定表达”中，我们将利用 多西环素诱导型和组成型转座子载体，用于生成表达多种表达的细胞系 感兴趣的诱变和/或标记转基因。 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，旨在了解 DNA 聚合酶 q 在癌症中的作用和靶向性。

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.