Identification of Colon Cancer Genes Via Retrotransposon Mutagenesis

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）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因子或原癌基因仍未确定。对于散发性结直肠癌，在包含许多突变和表观遗传修饰的癌症中精确定位致病突变也是非常困难的。因此，很可能许多基因修饰因子和致病突变仍未被确认。小鼠诱变策略的最新发展使得快速识别致癌过程中的致病突变和修饰因子成为可能。本课题的目的是：(1)通过肠上皮随机插入诱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信号的修饰因子，以及协同或协同突变。本提案详细介绍了一种利用转基因L1反转录转座子诱变系统的新型正向遗传学方法。本提案将使用两个诱变L1磁带：一个包含一个小的双向剪接受体基因陷阱，用于中断基因表达（LOF模块），另一个包含一个组成启动子和剪接供体，用于过度表达可能的致癌基因（GOF模块）。这两种转基因磁带都将使用小鼠Vil1 （Villin）启动子。该启动子在整个肠上皮细胞（干细胞、祖细胞和分化细胞）中驱动特异性高水平拷贝数依赖性表达的能力是独一无二的。通过基因组DNA的连接介导PCR或RNA的3' RACE，可以收获转基因小鼠的息肉，并且很容易识别突变。相关性：本提案描述了一种利用强大的遗传工具在小鼠中开发的方法，专门针对结肠直肠癌起源的组织肠上皮。这种方法有可能在一只老鼠身上识别出数百种癌症基因突变，而传统方法只能在一只老鼠身上识别出一种基因突变。因此，人们可以迅速识别出许多有能力导致结直肠癌的突变。这可能会揭示癌症发生的新机制。

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.