A Universal Mouse Line to Assess Tumor Clonality

描述（由申请人提供）：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-失活的原理：父源或母源X-染色体中基因的随机失活发生在胚胎发生的非常早期，并且此后稳定遗传。因此，区分杂合子雌性中X连锁多态性等位基因的能力可用于追踪。然而，由于成体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。最近在几种组织中证实，较大的X-灭活斑片大小会混淆肿瘤克隆性的评估，使结果偏向单克隆性。一个理想的检测方法应该不依赖于细胞谱系，能够同时使用雄性和雌性细胞，并且易于执行。我们设计了一种方法在小鼠中实现这一点。我们的方法是基于在成人组织中产生随机双等位基因红色或绿色荧光表型的能力，因此完全独立于斑块大小问题。编码绿色荧光蛋白（GFP）单元和随后的反义方向的红色荧光蛋白（RFP）的盒被位于相反方向的LoxP位点包围。报告菌株在强CMV启动子下普遍表达GFP，在每种细胞类型中产生绿色荧光。当LoxP位点以相反方向定位时，CRE重组酶能够“翻转”DNA片段。因此，当报告菌株与组织特异性诱导型CRE杂交时，RFP被适当地定位用于表达，并且发生从绿色到红色荧光的转换，既瞬时（诱导型）又在特定细胞类型中发生。因此，可以在待分析的肿瘤细胞类型中特异性地产生随机双等位基因分布（绿色或红色细胞）。由于这种随机分布是在成人中产生的，因此可以评估肿瘤克隆性，而不受贴片大小的任何干扰。因此，该细胞系可用于重新评估可能存在组织特异性启动子的任何肿瘤模型中的克隆性。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种新方法。虽然克隆性评估是该提议的目标，但我们设想这种新的报告小鼠品系可能成为未来多种应用的强大资源（即，线追踪、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.