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）：在此提案中，Starr博士将使用基于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，菌落形成和信号传导途径的影响，将分析已鉴定基因的蛋白质产物在人类癌中的作用。 SB系统已用于驱动小鼠的肿瘤发生，随后鉴定出驱动白血病，肉瘤，肝细胞癌和结直肠癌的已知和新型基因突变。但是，一些组织类型已证明对SB诱变难治性。为了成功筛选这些组织中的基因，Starr博士提出了对SB系统的几种修改。 斯塔尔博士在明尼苏达大学共济会癌症中心（MCC）任职期间一直在开发SB系统。为了帮助Starr博士过渡到独立研究职业，他组建了一个咨询委员会，该咨询委员会由多个不同领域的专家组成，这对于他的研究和职业发展至关重要。除了他的导师和咨询委员会提供的直接支持外，Starr博士还将使用MCC中可用的共享资源。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，高通量测序以及能够产生转基因，敲除和敲门小鼠的小鼠遗传学实验室的核心设施。候选人打算在研究本研究建议的目标1和2时继续他的博士后奖学金两年。斯塔尔博士的长期目标是在学术环境中确保成为独立研究人员的职位，他计划将自己的职业生涯奉献，以了解驱动癌症的遗传机制。 记录人类肿瘤基因组中遗传变化的研究证实，大量的遗传缺陷会导致肿瘤生长，但仅表征了一个小的子集。此外，在单个肿瘤类型中，单个肿瘤之间存在很大的异质性。该提案中的研究涉及癌症研究领域的关键需求，以开发出更好的方法来确定实际上促进肿瘤生长的遗传变化的子集。在特定的目标1和2中，Starr博士在结直肠癌（WAC，TCF12和RSPO2）和肺癌（AKAP13，CUL3和MAP4K3）中确定的几个新型候选癌症驾驶基因将研究其在人类癌症中的特定作用。这些蛋白质的水平将在正常和转化的人类细胞系中改变，以确定它们对生长，凋亡，在软琼脂中形成菌落的能力以及关键信号通路的调节。 AIM 3提出了使用新开发的高活性转座酶和转座子来发现新型肺癌基因的SB系统的几种改进。这些研究的结果可用于开发一种更个性化的癌症治疗方法。 公共卫生相关性：该项目的目的是使用动物模型来了解肺癌和大肠癌的遗传基础。然后，使用该动物模型发现的遗传突变将在人类肺和结直肠癌组织和细胞系中进行功能测试。这些研究的结果将有助于我们决定如何更好地治疗这些常见的致命癌症。