A Universal Mouse Line to Assess Tumor Clonality

描述(申请人提供)：肿瘤克隆性是任何致癌理论的核心。目前评估克隆性的方法是基于X失活原理的：父系或母系来源的X染色体中的基因随机失活在胚胎发育的早期就发生了，并在此后稳定地遗传。因此，在杂合子雌性中区分X连锁多态等位基因的能力可以用于追踪。然而，由于成体组织中的许多单个干细胞的后代被聚集在一起形成“补丁”，因此得出随机分布的假设是不可靠的。正如最近在几个组织中证实的那样，大的X-失活斑块大小将混淆对肿瘤克隆性的评估，使结果偏向单克隆性。理想的检测方法应该独立于细胞谱系，能够同时使用男性和女性，并且易于操作。我们已经设计了一种在老鼠身上完成这一任务的方法。我们的方法是基于在成人组织中创建随机双等位红色或绿色荧光表型的能力，因此完全独立于斑块大小问题。编码绿色荧光蛋白(GFP)单位的盒后跟随反义取向的红色荧光蛋白(RFP)被位于相反方向的loxP位点包围。该报告菌株在CMV强启动子作用下普遍表达GFP，在每种细胞类型中都能发出绿色荧光。当loxP位点位于相反方向时，Cre重组酶能够“翻转”一段DNA。因此，当报告菌株与组织特异的可诱导形式的Cre杂交时，RFP被适当地定位为表达，并发生从绿色到红色的荧光切换，无论是瞬时的(可诱导的)还是在特定的细胞类型中。因此，可以在要分析的肿瘤细胞类型中特定地产生随机双等位基因分布(绿色或红血球)。由于这种随机分布是在成人中产生的，因此可以在不受斑块大小干扰的情况下评估肿瘤克隆性。因此，这条线可以被用来重新评估潜在的任何肿瘤模型中的克隆性，其中有组织特异性启动子可用。在这项提案中，我们将利用我们最近在实验室开发的甲状腺肿瘤进展模型来验证这一新方法。虽然克隆性评估是这项建议的目标，但我们预计这种新的报告鼠品系可能成为未来多种应用(即线条追踪、肿瘤-间质/内皮细胞相互作用、监测远端转移等)的强大资源。因此，我们认为这条线将会引起其他NIH研究所(如NIDDK、NIEHS、NCI)的兴趣，并将对许多研究领域做出重大贡献。

DESCRIPTION (provided by applicant): Tumor clonality is central to any theory of carcinogenesis. Current methods to assess clonality are based on the principle of X-inactivation: random inactivation of genes in either the paternally or maternally derived X-chromosome occurs very early in embryogenesis and it is stably inherited thereafter. Thus, the ability to distinguish between X-linked polymorphic alleles in heterozygote females can be used for tracing. However, since many of the progeny of single stem cells in adult tissues are grouped together forming "patches", the assumption that a random distribution is attained is not reliable. A large X-inactivation patch size will confound the assessment of tumor clonality, biasing the results towards monoclonality, as recently confirmed in several tissues. An ideal assay should then be independent of cell lineage, able to use both males and females, and easy to perform. We have devised a method to accomplish this in the mouse. Our method is based on the ability to create a random biallelic red or green fluorescent phenotype, in an adult tissue, and therefore completely independent of patch size issues. A cassette encoding a green fluorescent protein (GFP) unit followed by a red fluorescent protein (RFP) in an antisense orientation is surrounded by LoxP sites positioned in opposite orientation. The reporter strain ubiquitously expresses GFP under the strong CMV promoter, giving green fluorescence in every cell type. CRE recombinase is able to "flip" a piece of DNA when LoxP sites are positioned in opposite orientation. Therefore, when the reporter strain is crossed with a tissue-specific inducible form of CRE, RFP is appropriately positioned for expression, and a switch from green to red fluorescence occurs, both transiently (inducible) and in a specific cell type. Thus, a random biallelic distribution can be generated (either green or red cells) specifically in the tumor cell type to be analyzed. Since this random distribution is generated in the adult, tumor clonality can be assessed without any interference from patch size. Thus, this line can be exploited to reassess clonality in potentially any tumor model, where a tissue-specific promoter is available. In this proposal, we will validate this new approach utilizing a thyroid tumor progression model that we have recently developed in the laboratory. Although clonality assessment is the goal of this proposal, we envision this new reporter mouse strain might become a powerful resource for multiple applications in the future (i.e., line tracing, tumor-stromal/endothelial interactions, monitoring distal metastasis, etc). Therefore, we consider this line will be of interest to other NIH institutes (e.g. NIDDK, NIEHS, NCI) and that will contribute significantly to many areas of research.