A Universal Mouse Line to Assess Tumor Clonality

用于评估肿瘤克隆性的通用小鼠系

• 批准号: 7106415
• 负责人: DANIEL L ALTSCHULER
• 金额: $ 20.13万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-03 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7106415
• 关键词: Universal; Mouse; Line; Assess; Tumor

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.

描述（由申请人提供）：肿瘤克隆性是任何癌变理论的核心。当前评估克隆性的方法基于X灭活的原理：在胚胎发生的早期，在父母或母体衍生的X染色体中，基因的随机失活是在胚胎发生的早期发生的，此后稳定地遗传了。因此，可以使用杂合雌性中X连锁多态性等位基因区分X连锁的多态性等位基因的能力。但是，由于成人组织中的许多单个干细胞的后代被分组在一起形成“斑块”，因此获得随机分布的假设是不可靠的。大型X灭活斑的大小将混淆肿瘤克隆性的评估，从而使结果偏向单克隆性，正如最近在几个组织中所证实的那样。然后，理想的测定应独立于细胞谱系，能够同时使用男性和女性，并且易于执行。我们已经设计了一种在鼠标中完成此操作的方法。我们的方法基于在成人组织中创建随机的双重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，然后在反义方向上的红色荧光蛋白（RFP）的盒式盒子被置于相反方向的LOXP位点所包围。记者菌株在强CMV启动子下普遍表达GFP，在每种细胞类型中都能产生绿色荧光。当LOXP位点位于相反的方向时，CRE重组酶能够“翻转”一块DNA。因此，当报道菌株与组织特异性诱导形式的CRE交叉时，将RFP适当放置以表达表达，并且在绿色到红色荧光的转换都会瞬时（可诱导）和特定的细胞类型。因此，可以在要分析的肿瘤细胞类型中特异性地生成随机的双重分布（绿色或红细胞）。由于这种随机分布是在成年人中产生的，因此可以评估肿瘤克隆性，而不会受到斑块大小的任何干扰。因此，可以利用这条线在任何肿瘤模型中重新评估克隆性，在任何肿瘤模型中都有可用的肿瘤模型。在此提案中，我们将利用我们最近在实验室中开发的甲状腺肿瘤进展模型来验证这种新方法。尽管克隆性评估是该提案的目标，但我们设想这种新的记者小鼠菌株可能会成为将来多个应用的​​强大资源（即线跟踪，肿瘤 - 层/肿瘤/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机构（例如Niddk，Niehs，NCI），这将对许多研究领域产生重大贡献。