A Universal Mouse Line to Assess Tumor Clonality

描述（由申请人提供）：肿瘤克隆是任何致癌理论的核心。目前评估克隆性的方法是基于x染色体失活的原理：父系或母系衍生的x染色体的基因随机失活发生在胚胎发生的很早期，并且此后稳定遗传。因此，在杂合子雌性中区分x连锁多态性等位基因的能力可以用于追踪。然而，由于成人组织中单个干细胞的许多后代聚集在一起形成“斑块”，因此获得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，较大的x -失活贴片大小会混淆肿瘤克隆的评估，使结果偏向单克隆。一个理想的实验应该独立于细胞谱系，能够使用男性和女性，并且易于执行。我们设计了一种方法来在老鼠身上实现这一点。我们的方法是基于在成人组织中创建随机双等位基因红色或绿色荧光表型的能力，因此完全独立于贴片大小问题。编码绿色荧光蛋白（GFP）单元和反义方向的红色荧光蛋白（RFP）的卡带被相反方向的LoxP位点包围。报告菌株在CMV强启动子下普遍表达GFP，在每种细胞类型中都发出绿色荧光。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性诱导形式的CRE杂交时，RFP被适当地定位于表达，并且从绿色荧光切换到红色荧光，这是短暂的（可诱导的）和特定细胞类型。因此，在待分析的肿瘤细胞类型中，可以产生随机的双等位基因分布（绿色或红色细胞）。由于这种随机分布是在成人中产生的，因此可以在不受贴片大小干扰的情况下评估肿瘤的克隆性。因此，这条细胞系可以被用来重新评估潜在的任何肿瘤模型的克隆性，其中组织特异性启动子是可用的。在本提案中，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验证这种新方法。虽然克隆性评估是本提案的目标，但我们设想这种新的报告小鼠品系可能在未来成为多种应用的强大资源（即系追踪，肿瘤-基质/内皮相互作用，监测远端转移等）。因此，我们认为这条线将引起其他NIH研究所（例如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.