Inducible colon-specific transgenic mouse for cancer research

描述(申请人提供)：在过去的20年里，我们对结肠癌的分子病因学的了解有了极大的扩展。尽管如此，结肠癌在美国仍然是一个严重的健康问题。该领域取得进展的障碍之一是缺乏能够概括人类结肠癌病因的具有良好特征的动物模型。虽然我们已经从各种化学诱导或基因编程的啮齿动物模型中学到了许多癌症，但在这些模型中发展起来的癌症在潜伏期、肠道位置或分子特征方面往往与人类结肠癌有很大不同。以机制和预防为重点的结肠癌研究需要开发动物模型，允许精确计时、特定于结肠的肠道生物学修饰。我们提出的研究的目标是使用驱动大肠特异性表达碳酸氢酶1(CA1)基因的启动子来创建一只转基因小鼠，该转基因小鼠具有结肠上皮细胞特异性表达的Cre重组酶(Cre)-与仅对雌激素类似物他莫昔芬(CAC-ERT2)具有高亲和力的修饰的雌激素受体(ER)配体结合域(CAC-ERT2)。融合基因的ERT2部分赋予转基因紫杉胺诱导性，并允许对小鼠基因组中的等位基因进行时间控制的删除。一种转基因载体已经被生产出来，它将用于在普渡转基因小鼠核心设施生产小鼠。转基因表达水平和组织分布将通过聚合酶链式反应进行评估。将CAC-ERT2小鼠与ROSA26R指示性小鼠杂交后，将评估他莫昔芬对转基因功能的诱导作用。他莫昔芬诱导肿瘤形成的能力将在CAC-ERT2小鼠与带有一个或两个牙线状APC等位基因的小鼠杂交后进行评估。在葡聚糖硫酸钠(DSS)引起结肠炎的情况下，将进行进一步的研究。该项目完成后，CAC-ERT2小鼠将成为唯一的转基因模型，既将Cre的表达限制在大肠上皮细胞，又允许控制Cre何时起作用以删除漂浮的等位基因。这将允许结肠癌研究人员轻松结合基因突变(即与结肠癌病因学相关的多个丛生等位基因)，并在生命的任何阶段引发结肠癌。这将允许更仔细地评估致癌机制，并改进对成年小鼠的化学预防或治疗药物的测试。虽然我们感兴趣的是将该模型用于结肠癌，但该模型也将对对下肠炎症(即IBD，克罗恩病)感兴趣的研究人员有用。1

DESCRIPTION (provided by applicant): Over the past 20 years our understanding of the molecular etiology of colon cancer has expanded dramatically. Despite this, colon cancer remains a significant health problem in the US. One of the barriers to progress in the field is the lack of well-characterized animal models that recapitulate the etiology of human colon cancer. While we have learned a great deal from cancers resulting from various chemically induced- or genetically programmed-rodent models, the cancer that develops in these models is often significantly different from human colon cancer in terms of latency, intestinal location, or molecular signature. Mechanistic and prevention focused colon cancer research requires the development of animal models that permit precisely timed, colon-specific modification of intestinal biology. Our goal for the proposed research is to use the promoter that drives large intestine-specific expression of the carbonic anhydrase 1 (CA1) gene to create a transgenic mouse with colon-epithelial cell-specific expression of Cre recombinase (Cre)-fused to a modified estrogen receptor (ER) ligand binding domain with high affinity only for the estrogen analog tamoxifen (CAC- ERT2). The ERT2 portion of the fusion gene confers taxomifen-inducibility to the transgene and permits temporally controlled deletion of floxed alleles in the mouse genome. A transgene vector has been produced and it will be used to produce mice at the Purdue Transgenic Mouse Core Facility. Transgene expression level and tissue distribution will be assessed by PCR. Inducibility of transgene function by tamoxifen will be assessed after crossing the CAC-ERT2 mouse to the ROSA26R indicator mouse. The ability of tamoxifen to induce tumor formation will be assessed in CAC-ERT2 mice crossed to mice with one or two floxed APC alleles. Additional studies will be done in the presence of colonic inflammation induced by dextran sulfate sodium (DSS). Upon completion of the project, the CAC-ERT2 mouse will be the only transgenic model that both limits Cre expression to the epithelial cells of the large intestine and permits control over when Cre functions to delete floxed alleles. This will permit colon cancer researchers to easily combine genetic mutations (i.e. with multiple floxed alleles relevant to colon cancer etiology) and initiate colon cancer at any stage of life. This will permit more careful assessment of mechanisms of carcinogenesis and improved testing of chemopreventative or therapeutic agents in adult mice. While our interests are in using this model for colon cancer, this model will also be useful for researchers interested in inflammatory conditions of the lower bowel (i.e. IBD, Crohn's disease). 1