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

基本信息

批准号：
8320975
负责人：
TIMOTHY Kaehler STARR
金额：
$ 24.15万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-01 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8320975
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 Tumor Tissue Universities Work abstracting anticancer research base cancer therapy career career development clinically relevant colorectal cancer screening gain of function mutation gene discovery gene function improved leukemia lung cancer screening mouse genome mouse model novel overexpression research and development research study sarcoma success therapeutic target tumor tumor growth tumorigenesis

项目摘要

Project Summary/Abstract 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.

项目概要/摘要在这项提案中，斯塔尔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。将通过以下方法分析已识别基因的蛋白质产物在人类癌症中的作用：改变它们在细胞系中的水平并测量对增殖、凋亡、集落形成和信号通路。 SB 系统已被用于驱动小鼠肿瘤发生并随后识别导致白血病、肉瘤、肝细胞癌和结直肠癌。然而，某些组织类型已被证明对 SB 诱变具有抵抗力。才能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 博士在担任博士后研究员期间对 SB 系统的开发做出了重要贡献在明尼苏达大学共济会癌症中心 (MCC)。为了帮助斯塔尔博士过渡到在独立研究生涯中，他组建了一个由多个领域的专家组成的咨询委员会对他的研究和职业发展的成功至关重要的不同领域。除了直接支持在他的导师和咨询委员会的帮助下，斯塔尔博士将使用 MCC 中提供的共享资源。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、高通量测序和小鼠遗传学实验室能够产生转基因、基因敲除和基因敲入小鼠。候选人打算继续他的博士后研究两年，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，其中他计划将自己的职业生涯致力于了解癌症的遗传机制。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，但只有一小部分的特征得到了表征。此外，在单一肿瘤类型内，各个肿瘤之间存在很大的异质性。本提案中的研究满足癌症研究领域的迫切需求，开发更好的方法来识别子集实际上驱动肿瘤生长的基因变化。在具体目标 1 和 2 中，几个新颖的候选者 Starr 博士在结直肠癌（Wac、Tcf12 和 Rspo2）和肺癌筛查中发现的癌症驱动基因将研究癌症（Akap13、Cul3 和 Map4k3）以确定它们在人类癌症中的具体作用。这这些蛋白质的水平将在人类细胞系（正常细胞系和转化细胞系）中发生改变，以确定它们的对生长、细胞凋亡、在软琼脂中形成菌落的能力以及关键信号通路的调节的影响。 Aim 3 使用新开发的高活性转座酶对 SB 系统提出了多项改进和转座子，以发现新的肺癌基因。这些研究的结果可用于开发更加个性化的癌症治疗方法。