Cancer gene discovery using Sleeping Beauty transposon-mediated mutagenesis

利用睡美人转座子介导的诱变发现癌症基因

• 批准号: 7959113
• 负责人: TIMOTHY Kaehler STARR
• 金额: $ 10.22万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959113
• 关键词: AKAP13 gene; Address; Advisory Committees; Agar; Animal Model; Apoptosis; Area; Automobile Driving; Bioinformatics; Biological; Biometry; CUL3 gene; Cancer Center; Cancer Etiology; Cell Line; Colon Carcinoma; Colorectal Cancer; Comparative Pathology; Complementary DNA; Core Facility; Coupled; Cytogenetics; Data; Development; Diagnostic; Disease; Epigenetic Process; Epithelial Cells; Event; Fellowship; Flow Cytometry; Gene Mutation; Genes; Genetic; Genetic Screening; Genome; Goals; Growth; Heterogeneity; Histocompatibility Testing; Human; Human Cell Line; Image; Individual; Insertion Mutation; Intestines; Knock-in Mouse; Knock-out; Laboratories; Location; Lung; Lung Neoplasms; Malignant Neoplasms; Malignant neoplasm of lung; Maps; Measures; Mediating; Mentors; Messenger RNA; Methods; Minnesota; Modeling; Modification; Molecular Target; Mus; Mutagenesis; Mutate; Mutation; Normal tissue morphology; Oncogenes; Oncogenic; PTEN gene; Phase; Phenotype; Positioning Attribute; Postdoctoral Fellow; Primary carcinoma of the liver cells; Proteins; RNA Interference; Refractory; Regulation; Research; Research Personnel; Research Proposals; Resistance; Resource Sharing; Role; Secure; Signal Pathway; Signal Transduction; Sleeping Beauty; Somatic Mutation; System; Techniques; Technology; Testing; Tissue Procurements; Tissues; Transgenic Organisms; Transposase; Universities; Work; anticancer research; base; cancer therapy; career; career development; clinically relevant; colorectal cancer screening; gain of function mutation; gene discovery; gene function; improved; leukemia; lung basal segment; lung cancer screening; mouse genome; mouse model; novel; overexpression; public health relevance; research study; sarcoma; success; therapeutic target; tumor; tumor growth; tumorigenesis

DESCRIPTION (provided by applicant): In this proposal Dr. Starr will use Sleeping Beauty (SB) transposon-based mutagenesis to screen for novel cancer genes. The protein products of identified genes will be analyzed for their role in human cancer by altering their levels in cell lines and measuring the effect on proliferation, apoptosis, colony formation, and signaling pathways. The SB system has been used to drive tumorigenesis in mice and subsequently identify both known and novel genetic mutations that drive leukemias, sarcomas, hepatocellular carcinomas, and colorectal cancers. Some tissue types, however, have proven refractory to SB mutagenesis. To successfully screen for genes in these tissues Dr. Starr has proposed several modifications to the SB system. Dr. Starr has been instrumental in developing the SB system during his tenure as a post-doctoral fellow in the Masonic Cancer Center (MCC) at the University of Minnesota. To help Dr. Starr transition to an independent research career, he has assembled an advisory committee consisting of experts in several different areas critical for the success of his research and career development. In addition to the direct support provided by his Mentor and advisory committee, Dr. Starr will use the shared resources available in the MCC. These include core facilities in biological imaging, tissue procurement, flow cytometry, comparative pathology, cytogenetics, bioinformatics/biostatistics, RNA interference, high-throughput sequencing, and a Mouse Genetics Laboratory capable of generating transgenic, knock-out, and knock-in mice. The candidate intends to continue his post-doctoral fellowship for two years while working on Aims 1 and 2 of this research proposal. Dr. Starr's long-term goal is to secure a position as an independent investigator in an academic setting where he plans on dedicating his career to understanding the genetic mechanisms driving cancer. Studies documenting genetic changes within human tumor genomes confirm that a large number of genetic defects contribute to tumor growth, yet only a small subset have been characterized. Furthermore, within a single tumor type there is great heterogeneity between individual tumors. The studies in this proposal address a critical need in the field of cancer research to develop better methods of identifying the subset of genetic changes that are actually driving tumor growth. In Specific Aims 1 and 2, several novel candidate cancer-driving genes identified by Dr. Starr in a screen for colorectal cancer (Wac, Tcf12, and Rspo2) and lung cancer (Akap13, Cul3, and Map4k3) will be studied to determine their specific role in human cancer. The levels of these proteins will be altered in human cell lines, both normal and transformed, to determine their effect on growth, apoptosis, the ability to form colonies in soft agar, and regulation of key signaling pathways. Aim 3 proposes several improvements to the SB system, using newly developed highly active transposases and transposons, to uncover novel lung cancer genes. The results of these studies can be used to develop a more personalized approach to cancer therapy. PUBLIC HEALTH RELEVANCE: The goal of this project is to use an animal model to understand the genetic basis of lung and colorectal cancer. The genetic mutations discovered using this animal model will then be functionally tested in human lung and colorectal cancer tissue and cell lines. The results of these studies will help us decide how to better treat these common, deadly cancers.

描述（由申请人提供）：在本提案中，Starr 博士将使用基于睡美人 (SB) 转座子的诱变来筛选新的癌症基因。通过改变细胞系中的水平并测量对增殖、凋亡、集落形成和信号通路的影响，分析已识别基因的蛋白质产物在人类癌症中的作用。 SB系统已被用于驱动小鼠肿瘤发生，并随后识别驱动白血病、肉瘤、肝细胞癌和结直肠癌的已知和新型基因突变。然而，某些组织类型已被证明对 SB 诱变具有抵抗力。为了成功筛选这些组织中的基因，Starr 博士提出了对 SB 系统的一些修改。 Starr 博士在明尼苏达大学共济会癌症中心 (MCC) 担任博士后期间，在 SB 系统的开发中发挥了重要作用。为了帮助斯塔尔博士过渡到独立研究生涯，他组建了一个咨询委员会，由几个不同领域的专家组成，这些领域对他的研究和职业发展的成功至关重要。除了导师和咨询委员会提供的直接支持外，Starr 博士还将利用 MCC 中的共享资源。其中包括生物成像、组织采购、流式细胞术、比较病理学、细胞遗传学、生物信息学/生物统计学、RNA干扰、高通量测序的核心设施，以及能够产生转基因、敲除和敲入小鼠的小鼠遗传学实验室。该候选人打算继续从事两年的博士后研究，同时致力于本研究计划的目标 1 和 2。斯塔尔博士的长期目标是在学术环境中获得独立调查员的职位，他计划将自己的职业生涯致力于了解驱动癌症的遗传机制。 记录人类肿瘤基因组内遗传变化的研究证实，大量遗传缺陷会导致肿瘤生长，但只有一小部分已被表征。此外，在单一肿瘤类型内，各个肿瘤之间存在很大的异质性。该提案中的研究解决了癌症研究领域的迫切需求，即开发更好的方法来识别实际上驱动肿瘤生长的基因变化子集。在具体目标 1 和 2 中，将研究 Starr 博士在结直肠癌（Wac、Tcf12 和 Rspo2）和肺癌（Akap13、Cul3 和 Map4k3）筛查中发现的几种新型候选癌症驱动基因，以确定它们在人类癌症中的具体作用。这些蛋白质的水平将在正常和转化的人类细胞系中发生变化，以确定它们对生长、细胞凋亡、在软琼脂中形成集落的能力以及关键信号通路调节的影响。 Aim 3提出了对SB系统的多项改进，使用新开发的高活性转座酶和转座子来发现新的肺癌基因。这些研究的结果可用于开发更加个性化的癌症治疗方法。 公共卫生相关性：该项目的目标是利用动物模型来了解肺癌和结直肠癌的遗传基础。使用该动物模型发现的基因突变将在人类肺癌和结直肠癌组织和细胞系中进行功能测试。这些研究的结果将帮助我们决定如何更好地治疗这些常见的致命癌症。