Visualizing Clonal Expansion in Living Mice

描述（由申请人提供）：驱动癌前细胞克隆扩张的力量，以及通常阻止它的屏障，很少被理解。这个探索性奖助金计划为研究这个问题开发关键资源。对于皮肤癌，已知最早的病变是携带p53突变的角质细胞克隆。我们发现这些突变细胞的克隆扩增需要持续的UVB，但不需要额外的突变。邻近的干细胞区室通常作为克隆扩增的屏障，但UVB允许p53突变的角质形成细胞突破这一屏障。通过创建携带荧光P53-mRFP1融合蛋白的敲入小鼠，我们将能够首次实时动态观察p53突变克隆的起源，扩增和回归。第二种方法可能适用于人类皮肤，通过局部应用紧密结合P53的荧光肽来可视化克隆。这些方法将在第二年用于直接观察突变细胞从一个干细胞室到另一个干细胞室的量化跳跃，研究克隆与癌前病变的前体-产物关系，并确定克隆扩增是否可以使用局部RNAi进行遗传修饰。在未来，我们会问：克隆扩增能产生新的干细胞吗？克隆是否通过光化性角化发展为癌？UVA能否通过减少细胞间通讯来促进克隆扩增？克隆会通过缩放来回归吗？抗退化克隆来自干细胞吗？在可见病变消退后，罕见的突变细胞是否持续存在？皮肤筛选RNAi文库能揭示影响克隆扩增的基因吗？因此，这些技术进步将开辟克隆扩展领域。

DESCRIPTION (provided by applicant): The forces that drive clonal expansion of a precancerous cell, and the barriers that normally prevent it, are little understood. This Exploratory Grant project develops key resources for studying this problem. For skin cancer, the earliest known lesions are keratinocyte clones carrying p53 mutations. We find that clonal expansion of these mutant cells requires sustained UVB, but not additional mutations. Neighboring stem cell compartments normally act as a barrier to clonal expansion, but UVB allows p53-mutant keratinocytes to breach this barrier. By creating a knock-in mouse carrying a fluorescent P53-mRFP1 fusion protein, we will be able, for the first time, to dynamically observe the origin, expansion, and regression of p53-mutated clones in real time. A second approach, potentially applicable to human skin, visualizes clones by topically applying fluorescent peptides that tightly bind P53. These methods will be used in the second year to directly observe quantized jumping of mutant cells from one stem cell compartment to another, investigate the precursor-product relation of clones to precancers, and determine whether clonal expansion can be modified genetically using topical RNAi. In the future, we will ask: Does clonal expansion create new stem cells? Does a clone progress through actinic keratosis to carcinoma? Can UVA drive clonal expansion by reducing intercellular communication? Do clones regress by squaming? Do the regression-resistant clones arise from stem cells? Do rare mutant cells persist long after visible lesions have regressed? Can skin screening of RNAi libraries reveal genes affecting clone expansion? Thus, these technical advances should open up the field of clonal expansion.