Identification of Colon Cancer Genes Via Retrotransposon Mutagenesis

描述(申请人提供)：由细胞中的DNA损伤引起的突变是癌症的主要原因。结直肠癌(CRC)的家族性突变占所有CRC病例的不到3%，而其他关键的肿瘤抑制基因或原癌基因仍未确定。对于散发性结直肠癌来说，精确定位包含许多突变和表观遗传修饰的癌症中的致病突变(S)也是极其困难的。因此，很可能许多基因修饰物和致病突变仍未确定。小鼠诱变策略的最新进展使快速鉴定致癌突变和致癌修饰物成为可能。该方案的目的是(1)通过肠上皮随机插入突变建立结直肠癌转基因小鼠模型，(2)在结直肠癌小鼠模型中寻找新的肿瘤修饰基因。待鉴定的基因包括新的癌症基因和途径，APC/Wnt信号的修饰物，以及协同或协同突变。这项建议详细介绍了一种使用转基因L1反转录转座子突变系统的新的正向遗传学方法。在本方案中将使用两个诱变L1盒：一个包含用于基因表达中断的双向剪接受体小基因陷阱(LOF模块)，另一个包含用于可能的癌基因过度表达的组成型启动子和剪接供体(GOF模块)。两个转基因盒都将使用小鼠Vil1(Villin)启动子。这种启动子的独特之处在于它能够在整个肠道上皮细胞中驱动特定的高水平拷贝数依赖的表达：在干细胞、祖细胞和分化细胞中。可以从转基因小鼠身上获取息肉，并通过连接介导的基因组DNA聚合酶链式反应或3‘RACE的RNA很容易地识别突变。相关性：这项建议描述了一种方法，该方法利用在小鼠身上开发的强大的遗传工具，专门针对肠上皮，结直肠癌起源的组织。这种方法可以潜在地在一只小鼠身上识别数百个癌症基因突变，而不是传统的方法，即在一只小鼠身上只有一个基因突变。因此，人们可以快速识别出许多有能力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。

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.