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 underex plored aspect of the pathogenesis of diseases with focal origins. Here, we illustrate an approach for conducting such analyses based on mosaic pattern s of Cre recombinase expression in the adult mouse intestinal epithelium. T ransgenic mice were generated that express Cre in the stem cell niche of cr ypts located in specified regions of their intestine. Some of these mice we re engineered to allow for doxycycline-inducible Cre expression. Recombinat ion in all pedigrees was mosaic: Cre-expressing crypts that supported recom bination in all of their active multipotent stem cells were located adjacen t to "control" crypts that did not express Cre at detectable levels. Cre-me diated recombination of a flexed LacZ reporter provided direct evidence tha t adult small-intestinal crypts contain more than one active multipotent st em 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 epithel ial cells from crypts with or without recombination for subsequent gene exp ression profiling. Stained sections of intestine were used to create electr onic image templates to guide laser capture microdissection (LCM) of adjace nt frozen sections. This navigated form of LCM overcomes problems with mRNA degradation encountered when cells are marked directly by immunohistochemi cal methods. Combining Cre-engineered genetic mosaic mice with navigated-LC M will allow biology and pathobiology to be explored at the junction betwee n normal and perturbed cellular cohorts.