Cell line Core

细胞系核心

• 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
• 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

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.

项目摘要 本计画将探讨哺乳动物DNA聚合酶θ（Pol θ），DNA修复的定义酵素 通过聚合酶θ介导的末端连接（TMEJ）的双链断裂。这是核心C（“细胞系 工程核心”），这是题为“聚合酶θ，基因组不稳定性， 癌症”。核心C将负责提供天然和基因工程细胞系，以支持所有 在项目1、2和4中进行的基于细胞的试验。 哺乳动物细胞系试剂的认证对于实验室的严谨性和重现性至关重要 实验通过建立和实施细胞系验证的标准化方案， 操作、冷冻保存和传播，核心将确保细胞系试剂的质量控制， 尽量减少错误的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 用于鉴定和表征哺乳动物细胞系的标准化和全面的方案 在整个计划项目中使用的模型。 在目标2“基于CRISPR的细胞系基因组工程服务计划项目”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， 计划项目目标。用于该目的的Cas9蛋白将由核心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产生表达多种 诱变的和/或标记的目的转基因。 核心C服务将在计划项目内高度协调，并将促进严格性和 重复性，以及成本效益，高度综合的研究活动，旨在了解 DNA聚合酶q在癌症中的作用和靶向性。