Cancer gene discovery using Sleeping Beauty transposon-mediated mutagenesis

项目总结/摘要 在这项提案中，Starr博士将使用基于睡美人（SB）转座子的诱变来筛选 新的癌症基因将通过以下方法分析已鉴定基因的蛋白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、集落形成的影响， 信号通路SB系统已用于驱动小鼠中的肿瘤发生，并随后鉴定 已知的和新的基因突变，驱动白血病，肉瘤，肝细胞癌， 结肠直肠癌然而，一些组织类型已被证明对SB诱变是难治的。成功 筛选这些组织中的基因斯塔尔博士对SB系统提出了几项修改。 斯塔尔博士在担任博士后期间， 在明尼苏达大学共济会癌症中心（MCC）。帮助斯塔尔博士过渡到 独立的研究生涯，他组建了一个咨询委员会，由几个专家组成， 不同的领域对他的研究和职业发展的成功至关重要。除了直接支持 在他的导师和咨询委员会的帮助下，Starr博士将使用MCC中可用的共享资源。 其中包括生物成像、组织获取、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、敲除和敲入小鼠。候选人打算 继续他的博士后奖学金两年，同时致力于本研究计划的目标1和2。 博士斯塔尔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奉献给理解导致癌症的遗传机制。 记录人类肿瘤基因组内遗传变化的研究证实， 遗传缺陷有助于肿瘤生长，但只有一小部分已被鉴定。此外，委员会认为， 在单一肿瘤类型中，个体肿瘤之间存在很大的异质性。本提案中的研究 解决了癌症研究领域中的一个关键需求，即开发更好的方法来识别癌症的亚群。 基因的改变实际上是在驱动肿瘤的生长。在具体目标1和2中， Starr博士在结肠直肠癌筛查中发现的癌症驱动基因（Wac，Tcf 12和Rspo 2）和肺癌 癌症（Akap 13，Cul 3和Map 4k 3）的研究将确定它们在人类癌症中的特定作用。的 这些蛋白质的水平将在正常和转化的人类细胞系中改变，以确定它们的 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的调节的影响。 目的3提出了几个改进SB系统，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一种 更个性化的癌症治疗方法。

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.