Mouse Models of Early Intestinal Neoplasia

描述（由申请人提供）：人类癌症含有多种突变，并且不确定哪些突变是必要的和充分的，哪些是被动乘客突变，以及是否存在对转化至关重要的其他尚未发现的突变。一种更好地理解特定突变作用的前瞻性方法是在正常细胞中设计这些突变。我们建议通过零星地和体细胞地创造具有确定基因型的“突变”干细胞来工程小鼠肿瘤。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floxed肿瘤抑制基因（TSGs）的失活将随机发生在孤立的，广泛分散的单细胞中。非功能性框外（An+1）种系Cre等位基因将在DNA错配修复缺陷背景中被激活（An+1至An），随后重组floxed基因以产生明确定义的突变基因型。当前An+1转基因Cre等位基因将靶向遍在表达的ROSA 26基因座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细胞中。我们的研究将检查肠道中偶发的Apc、Pten或Trp 53失活。因为不确定转化需要多少突变，所以将包括floxed B-半乳糖苷酶等位基因，使得可以鉴定“突变”细胞，而不管所得表型。某些TSG可以调节干细胞存活，并且即使单个TSG的损失可能不会赋予肿瘤表型，干细胞存活也可能增加，导致在其他正常外观的肠中出现更多数量的蓝色染色“突变”细胞。多步骤肿瘤进展意味着癌症在一系列突变后出现。这些研究将前瞻性地重现这一过程，并确定获得一个或多个伴随基因改变的正常哺乳动物组织中单个干细胞的命运。赋予肿瘤表型的某些突变的成功或失败将挑战或确认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.