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Cell line Core

细胞系核心

基本信息

批准号：
10468635
负责人：
Gaorav P Gupta
金额：
$ 15.58万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10468635
关键词：
Aliquot Alleles Biological Assay CRISPR/Cas technology Cell Line Cells Clone Cells Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Communities Credentialing Cryopreservation Custom DNA Double Strand Break DNA Repair Enzymes DNA-Directed DNA Polymerase Doxycycline Engineering Ensure Enzymes Experimental Designs Future Gene Targeting Generations Genes Genetic Engineering Genome Genome engineering Genomic Instability Genomics Genotype Goals Human In Vitro Karyotype Karyotype determination procedure Knock-in Knock-out Ligands Malignant Neoplasms Mammalian Cell Manuscripts Mediating Methods Modeling Monitor Mus Mycoplasma Polymerase Process Proteins Protocols documentation Publications Quality Control Reagent Reproducibility Research Research Activity Research Personnel Role Secure Services Standardization Structure of thyroid parafollicular cell System Testing Time Transgenes Validation base cost effectiveness design digital empowered experimental study genetically modified cells homologous recombination in vivo interest laboratory experiment multidisciplinary overexpression parity programs protein expression quality assurance repaired repository screening service programs success synergism transcriptome sequencing transgene delivery transgene expression vector

项目摘要

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.

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 Core C 将负责提供天然和基因工程细胞系来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。