Mouse Models of Early Intestinal Neoplasia

描述（由申请人提供）：人类癌症包含多种突变，不确定哪些突变是必要和充分的，哪些是被动的乘客突变，以及是否有其他尚未发现的突变对转化至关重要。更好地理解特定突变的作用的一种前瞻性方法是在正常细胞中设计这些突变。我们建议在小鼠中通过零星地和体细胞地创造具有特定基因型的“突变”干细胞来改造肿瘤。与大多数小鼠模型不同，我们的方法将更好地模拟散发性肿瘤发生，因为cre介导的松散肿瘤抑制基因（TSGs）失活将随机发生在分离的、广泛分散的单细胞中。在DNA错配修复缺陷的背景下，非功能性外框（An+l）种系cre等位基因将被激活（An+l到An），并随后重组固定基因以产生明确的突变基因型。当前An+ 1转基因Cre等位基因将针对普遍表达的ROSA26位点，以更好地控制其激活。与散发性肿瘤发生类似，随机和体细胞突变将发生在被正常细胞包围的单个细胞中。我们的研究将检查肠道中散发性Apc、Pten或Trp53失活。由于不确定转化需要多少个突变，因此将包含一个固定的b -半乳糖苷酶等位基因，以便无论结果的表型如何，都可以识别“突变”细胞。某些TSG可能会调节干细胞的存活，即使单个TSG的丢失可能不会导致肿瘤表型，干细胞存活可能会增加，导致在其他正常的肠道中出现更多蓝色染色的“突变”细胞。多步肿瘤进展意味着癌症发生在一系列突变之后。这些研究将有希望重现这一过程，并确定正常哺乳动物组织中单个干细胞获得一个或多个伴随基因改变的命运。某些赋予肿瘤表型的突变的成功或失败将挑战或证实典型的肿瘤进展模型。

DESCRIPTION (provided by applicant): Human cancers contain multiple mutations and it is uncertain which mutations are necessary and sufficient, which are passive passenger mutations, and whether there are other yet undiscovered mutations crucial for transformation. One prospective approach to better understand the roles of specific mutations is to engineer these mutations in normal cells. We propose to engineer tumors in mice by sporadically and somatically creating "mutant" stem cells with defined genotypes. Unlike most mice models, our approach will better mimic sporadic tumorigenesis because Cre-mediated inactivation of floxed tumor suppressor genes (TSGs) will stochastically occur in isolated, widely scattered single cells. A nonfunctional out-of-frame (An+l) germline Cre-allele will become activated (An+l to An) in a DNA mismatch repair deficient background and subsequently recombine floxed genes to create well-defined mutant genotypes. A current An+l transgenic Cre allele will be targeted to the ubiquitously expressed ROSA26 locus to better control its activation. Similar to sporadic tumorigenesis, stochastic and somatic mutations will occur in single cells surrounded by normal cells. Our studies will examine sporadic Apc, Pten, or Trp53 inactivation in the intestines. Because it is uncertain how many mutations are required for transformation, a floxed B-galactosidase allele will be included so that "mutant" cells may be identified regardless of resultant phenotype. Certain TSGs may modulate stem cell survival, and even though loss of a single TSG may not confer a neoplastic phenotype, stem cell survival may be increased, resulting in greater numbers of blue staining "mutant" cells in otherwise normal appearing intestines. Multistep tumor progression implies that cancers arise after a series of mutations. These studies will prospectively recreate this process and define the fates of single stem cells in normal mammalian tissues that acquire one or more concomitant gene alterations. The success or failure of certain mutations to confer neoplastic phenotypes will challenge or confirm canonical tumor progression models.