Tissue-Specific Mutagenesis in Rat as an In Vivo Tool for Colon Cancer Gene Disco

描述（由申请人提供）：本次SBIR申请的结果将是建立结直肠癌（CRC）大鼠模型，该模型可用于发现结肠癌治疗的新靶点。将利用转座根公司的新技术在大鼠体内引入体细胞突变来生产结直肠癌大鼠。已知许多种系和体细胞突变有助于遗传性和散发性结直肠癌的发生和进展，但确定与人类结直肠癌相关的致病分子和遗传缺陷已被证明是困难的。现有的小鼠结直肠癌模型不能准确地模拟人类疾病。大鼠更接近地概括了人类结直肠癌的分子、细胞和病理特征。与小鼠不同，大鼠的息肉会发展为腺癌，相当于人类T1期的CRC。使用已知的人类致癌物在正常大鼠中诱导的结肠癌会扩散到肝脏，这表明与小鼠不同，大鼠可能是CRC转移的有价值的模型。大鼠的体型也使其更容易接受内窥镜检查，从而使息肉监测和实时评估单个腺瘤中发生的体细胞突变，从而可以更准确地区分门卫突变和癌变后期的突变。在该申请的第一阶段，我们将创建并表征三种转基因大鼠：1)使用VilCre转基因显示肠道特异性表达Cre重组酶的大鼠；2)在普遍存在的ROSA26启动子的指导下表达转座酶（负责转座子动员的酶）基因的大鼠。将使用液氧-停止-液氧盒来限制转座酶的表达；3)含有基因诱捕供体转座子的转基因大鼠。在通过杂交培育的三转基因大鼠中，Cre的表达将触发转座酶的表达，转座酶随后将调动基因陷阱转座子并将其随机插入整个基因组。重要的是，由于Cre重组酶的组织特异性表达，这些突变只会发生在肠道中。我们的基因陷阱将能够创建空等位基因（功能丧失）和过表达转录本（功能获得）。序列特异性标签用于定位突变，与其他随机诱变方法（如化学或辐射策略）相比，用于快速识别肿瘤抑制因子和致癌基因的一个特别突出的优势。在后期的II期工作中，我们将对三转基因大鼠的CRC表型和克隆插入位点进行全表型分析，以验证该模型的有效性。通过我们在肠道中无偏置转座子诱变方法建立的结直肠癌大鼠模型可能会在结肠癌模型中发现与人类结直肠癌更相关的新癌症基因。

DESCRIPTION (provided by applicant): The outcome of this SBIR application will be a rat model of colorectal cancer (CRC), which can be used to discover new targets for colon cancer therapy. CRC rats will be produced using Transposagen's novel technology for introducing somatic mutations in rats. A number of germline and somatic mutations are known to contribute to the initiation and progression of both heritable and sporadic CRC, but identifying the causative molecular and genetic defects involved in human CRC has proven difficult. Existing mouse models for CRC do not accurately model human disease. Rats more closely recapitulate the molecular, cellular, and pathologic characteristics of human CRC. Polyps in the rat, unlike in the mouse, progress to adenocarcinomas that are equivalent to stage T1 CRC in humans. Colon cancer induced in normal rats using known human carcinogens will spread to the liver, indicating that the rat, unlike the mouse, may serve as a valuable model for CRC metastasis. The physical size of the rat also makes it much more amenable to endoscopy, thus enabling polyp monitoring and real-time assessment of somatic mutations taking place in a single adenoma, so that gate-keeper mutations can be more accurately discriminated from mutations involved in later stages of carcinogenesis. In Phase I of this application, we will create and characterize three lines of transgenic rats: 1) rats exhibiting intestine-specific expression of a Cre recombinase using a VilCre transgene; 2) rats expressing a transposase (the enzyme responsible for transposon mobilization) gene under the direction of the ubiquitous ROSA26 promoter. A lox-stop-lox cassette will be used to restrict the expression of the transposase; 3) transgenic rats containing a gene trap donor transposon. Cre expression in tri-transgenic rats, created by cross-breeding, will trigger expression of the transposase, which will then mobilize the gene trap transposon and insert it randomly throughout the genome. Importantly, these mutations will occur in the intestine only, because of the tissue-specific expression of the Cre recombinase. Our gene-trap will be capable of creating both null alleles (loss-of-function) and over-expressed transcripts (gain-of-function). A sequence-specific tag is used for mapping mutations, an especially salient advantage compared to other random mutagenesis methods (such as chemical or radiation strategies) for rapid identification of tumor suppressors and oncogenes. In later Phase II work, we will perform a full phenotypic analysis of the CRC phenotype of tri-transgenic rats and clone insertion sites to verify the efficacy of the model. The CRC rat model developed by our method of unbiased transposon mutagenesis in the intestine will likely identify novel cancer genes in a model of colon cancer that is more relevant to human CRC.