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: 8521151
• 负责人: TIMOTHY Kaehler STARR
• 金额: $ 22.7万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2014-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8521151
• 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

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.

项目摘要/摘要 在此提案中，Starr博士将使用基于转座的诱变来筛选 新型癌症基因。将通过 改变其在细胞系中的水平，并测量对增殖，凋亡，菌落形成和 信号通路。 SB系统已用于驱动小鼠的肿瘤发生，然后识别 驱动白血病，肉瘤，肝细胞癌和新颖的遗传突变和新颖的遗传突变 结直肠癌。但是，一些组织类型已证明对SB诱变难治性。成功 这些组织中的基因筛选，Starr博士对SB系统提出了几种修改。 斯塔尔博士在担任博士后研究员任职期间一直在开发SB系统 在明尼苏达大学的共济会癌症中心（MCC）。帮助Starr博士过渡到 独立研究职业，他组建了一个由专家组成的咨询委员会 不同领域对于他的研究和职业发展至关重要。除了直接支持 Starr博士由他的导师和咨询委员会提供，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，高通量测序和小鼠 遗传学实验室能够产生转基因，敲除和敲除小鼠。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。 Starr博士的长期目标是在学术环境中确保担任独立研究人员的职位 他计划致力于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了大量 遗传缺陷有助于肿瘤生长，但仅表征了一个小子。此外， 在单个肿瘤类型中，单个肿瘤之间存在很大的异质性。该提议中的研究 解决癌症研究领域的关键需求，以开发出更好的方法来识别子集 遗传变化实际上正在推动肿瘤的生长。在特定的目标1和2中，几个新颖的候选人 斯塔尔博士在结直肠癌（WAC，TCF12和RSPO2）和肺部鉴定的癌症驱动基因 将研究癌症（AKAP13，CUL3和MAP4K3），以确定其在人类癌症中的具体作用。这 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，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提出了对SB系统的几个改进 和转座子，以发现新颖的肺癌基因。这些研究的结果可用于开发 更个性化的癌症治疗方法。

项目成果

Corrigendum to "Single cell sequencing reveals heterogeneity within ovarian cancer epithelium and cancer associated stromal cells" [Gyncol. Oncol. 144 (2017) 598-606].

“单细胞测序揭示卵巢癌上皮和癌症相关基质细胞内的异质性”的勘误 [Gyncol。

• DOI: 10.1016/j.ygyno.2018.07.015
• 发表时间: 2018
• 期刊: Gynecologic oncology
• 影响因子: 4.7
• 作者: Winterhoff,BorisJ;Maile,Makayla;Mitra,AmitKumar;Sebe,Attila;Bazzaro,Martina;Geller,MelissaA;Abrahante,JuanE;Klein,Molly;Hellweg,Raffaele;Mullany,SallyA;Beckman,Kenneth;Daniel,Jerry;Starr,TimothyK
• 通讯作者: Starr,TimothyK

Identification of Sleeping Beauty transposon insertions in solid tumors using linker-mediated PCR.

使用接头介导的 PCR 鉴定实体瘤中睡美人转座子的插入。

• DOI: 10.3791/50156
• 发表时间: 2013
• 期刊: Journal of visualized experiments : JoVE
• 作者: Janik,CallieL;Starr,TimothyK
• 通讯作者: Starr,TimothyK