Genetic mosaic analysis based on Cre recombinase and navigated laser capture microdissection

Defining molecular interactions that occur at the interface between “normal” and “abnormal” cell populations represents an important but often underexplored aspect of the pathogenesis of diseases with focal origins. Here, we illustrate an approach for conducting such analyses based on mosaic patterns of Cre recombinase expression in the adult mouse intestinal epithelium. Transgenic mice were generated that express Cre in the stem cell niche of crypts located in specified regions of their intestine. Some of these mice were engineered to allow for doxycycline-inducible Cre expression. Recombination in all pedigrees was mosaic: Cre-expressing crypts that supported recombination in all of their active multipotent stem cells were located adjacent to “control” crypts that did not express Cre at detectable levels. Cre-mediated recombination of a floxed LacZ reporter provided direct evidence that adult small-intestinal crypts contain more than one active multipotent stem cell, and that these cells can be retained in both small-intestinal and colonic crypts for at least 80 d. A method was developed to recover epithelial cells from crypts with or without recombination for subsequent gene expression profiling. Stained sections of intestine were used to create electronic image templates to guide laser capture microdissection (LCM) of adjacent frozen sections. This navigated form of LCM overcomes problems with mRNA degradation encountered when cells are marked directly by immunohistochemical methods. Combining Cre-engineered genetic mosaic mice with navigated-LCM will allow biology and pathobiology to be explored at the junction between normal and perturbed cellular cohorts.

界定发生于"正常"与"异常"细胞群交界区域的分子互作，是局灶起源性疾病发病机制中一项重要却常被忽视的研究维度。本研究基于成年小鼠肠上皮组织中Cre重组酶（Cre recombinase）的嵌合表达模式，展示了开展此类分析的实验策略。我们构建了在肠道特定区域肠隐窝的干细胞微环境中表达Cre的转基因小鼠，其中部分小鼠经过基因工程改造，可实现多西环素诱导的Cre表达。所有实验谱系的重组均呈嵌合状态：可在全部活性多能干细胞中介导重组的Cre阳性隐窝，与检测不到Cre表达的"对照"隐窝相邻分布。通过floxed LacZ报告基因的Cre介导重组实验，我们获得直接证据：成年小鼠小肠隐窝中存在不止一种活性多能干细胞，且这类细胞可在小肠及结肠隐窝中至少存留80天。我们开发了一种分离重组阳性与重组阴性隐窝上皮细胞的方法，用于后续的基因表达谱分析。我们通过染色的肠道组织切片构建电子图像模板，以引导相邻冰冻切片的激光捕获显微切割（laser capture microdissection, LCM）。这种导航式激光捕获显微切割技术，可解决通过免疫组织化学方法直接标记细胞时所面临的mRNA降解难题。将Cre工程化基因嵌合小鼠与导航式激光捕获显微切割技术相结合，将为探究正常与扰动细胞群交界区域的生物学及病理生物学特征提供可行的研究路径。