Identification of Colon Cancer Genes Via Retrotransposon Mutagenesis

通过逆转录转座子诱变鉴定结肠癌基因

• 批准号: 7558568
• 负责人: Blair Bernard Madison
• 金额: $ 3.09万
• 依托单位: TRANSPOSAGEN BIOPHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7558568
• 关键词: Accounting; Cancer Etiology; Cancer Gene Mutation; Cells; Colon Carcinoma; Colorectal Cancer; DNA; DNA Damage; Development; Enhancers; Epigenetic Process; Gene Expression; Genes; Genetic; Genetic Markers; Genomics; Harvest; Insertional Mutagenesis; Interruption; Intestines; Lesion; Ligation; Location; Malignant Neoplasms; Mediating; Methods; Modification; Multipotent Stem Cells; Mus; Mutagenesis; Mutate; Mutation; Numbers; Oncogenes; Oncogenic; Pathway interactions; Polymerase Chain Reaction; Polyps; Property; Proto-Oncogenes; RNA; RNA Splicing; Race; Retrotransposition; Retrotransposon; Signal Transduction; Stem cells; Structure of intestinal gland; System; Thinking; Tissues; Transgenes; Transgenic Mice; Transgenic Organisms; Tumor Suppressor Proteins; adenoma; carcinogenesis; gain of function; intestinal epithelium; loss of function; mouse model; novel; polyposis; progenitor; promoter; tool; transgene expression; villin

DESCRIPTION (provided by applicant): Mutations caused by DNA damage in the cell is a major cause of cancer. Familial mutations in colorectal cancer (CRC) account for less than 3% of all CRC cases, while other key tumor suppressors or proto-oncogenes remain unidentified. For sporadic CRC, pinpointing the causative mutation(s) within a cancer containing many mutations and epigenetic modifications is also incredibly difficult. Thus, it is likely that many genetic modifiers and causative mutations remain unidentified. Recent developments of mutagenesis strategies in the mouse now make it possible to rapidly identify causative mutations and modifiers involved in carcinogenesis. The objectives of this proposal are (1) to develop a transgenic mouse model of colorectal cancer through random insertional mutagenesis in the intestinal epithelium, and (2) to identify novel cancer modifier genes within a mouse model of CRC. Genes to be identified include novel cancer genes and pathways, modifiers of APC/Wnt signaling, and synergistic or cooperative mutations. This proposal details a novel forward genetics approach using a transgenic L1 retrotransposon mutagenesis system. Two mutagenic L1 cassettes will be used in this proposal: one containing a small bidirectional splice acceptor gene trap for interruption of gene expression (LOF module), and one containing a constitutive promoter and splice donor for over-expression of possible oncogenes (GOF module). Both transgene cassettes will employ the mouse Vil1 (Villin) promoter. This promoter is unique in its ability to drive specific high-level copy-number dependent expression throughout the entire intestinal epithelium: in stem cells, progenitors, and differentiated cells. Polyps from a transgenic mouse can be harvested and mutations readily identified by ligation-mediated PCR of genomic DNA or by 3' RACE of RNA. Relevance: This proposal describes a method employing powerful genetic tools developed in the mouse for specifically targeting the intestinal epithelium, the tissue where colorectal cancer originates. This approach can potentially identify hundreds of cancer gene mutations in a single mouse, as opposed to traditional methods where only one gene is mutated in a single mouse. Thus, one can rapidly identify many mutations that have the ability to cause colorectal cancer. This may uncover new mechanisms for how cancer initiates.

描述（由申请人提供）：细胞中DNA损伤引起的突变是癌症的主要原因。结直肠癌（CRC）中的家族性突变占所有CRC病例的不到3%，而其他关键肿瘤抑制因子或原癌基因仍未确定。对于散发性CRC，在含有许多突变和表观遗传修饰的癌症中精确定位致病突变也非常困难。因此，很可能许多遗传修饰剂和致病突变仍然未被识别。最近的发展，在小鼠中的诱变策略，现在有可能迅速确定致癌突变和修饰剂参与致癌。本提案的目的是（1）通过肠上皮中的随机插入诱变来开发结直肠癌的转基因小鼠模型，以及（2）在CRC小鼠模型中鉴定新的癌症修饰基因。待鉴定的基因包括新的癌症基因和途径、APC/Wnt信号传导的修饰剂以及协同或合作突变。该建议详细介绍了一种新的正向遗传学方法，使用转基因L1反转录转座子诱变系统。本提案中将使用两种致突变L1盒：一种含有用于中断基因表达的小型双向剪接受体基因陷阱（LOF模块），另一种含有用于过表达可能的致癌基因的组成型启动子和剪接供体（GOF模块）。两种转基因盒都将采用小鼠Vil 1（Villin）启动子。该启动子是独特的，它能够驱动整个肠上皮细胞中的特定高水平拷贝数依赖性表达：在干细胞，祖细胞和分化细胞中。可以收获来自转基因小鼠的息肉，并且通过基因组DNA的连接介导的PCR或通过RNA的3' RACE容易地鉴定突变。相关性：该提案描述了一种采用在小鼠中开发的强大遗传工具的方法，用于特异性靶向肠上皮，结肠直肠癌起源的组织。这种方法可以在一只小鼠中识别出数百种癌症基因突变，而传统方法在一只小鼠中只突变一种基因。因此，人们可以快速识别许多能够导致结直肠癌的突变。这可能会揭示癌症如何启动的新机制。