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

Abstract

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.mouse intestinal stem cells ͉ genetic mosaic analysis

Of mice and models: improved animal models for biomedical research

“…With the limitations presented by in utero mammalian development (compared to using in ovo developmental models), fate analysis of progenitors is currently technically challenging. We envisage recombinase-mediated cell marking of specific populations such as hematopoietic precursors or stem cells, neural progenitors or germ cells as a very powerful tool for following cell fates during development (156,160).…”

Minichromosome maintenance (Mcm) proteins, cyclin B1 and D1, phosphohistone H3 and in situ DNA replication for functional analysis of vulval intraepithelial neoplasia

“…Thus, it seems likely that combinations of these approaches will provide a useful synergism in the study of VIN. Mcm staining may also be useful in directing laser capture analysis of expressed transcripts encoding relevant viral and immunological factors in the ongoing biological analysis of VIN (Wong et al, 2000). Further work in long-term follow-up studies is required to establish the clinical relevance of these analyses.…”

The Pseudomonas aeruginosa generalized transducing phage φPA3 is a new member of the φKZ-like group of ‘jumbo’ phages, and infects model laboratory strains and clinical isolates from cystic fibrosis patients

“…Generalized transducing bacteriophages are useful genetic tools for strain engineering and for functional genomic analysis in strains that are not naturally competent, such as P. aeruginosa (Wong et al, 2000). Several P. aeruginosa generalized transducing bacteriophages have been described, though the transduction efficiency of these bacteriophages, in particular DMS3, UT1 and F116, can be very low (Budzik et al, 2004;Kidambi et al, 1994;Ripp et al, 1994).…”

“…The organism can live as biofilms, colonies or as planktonic cells (Tolker-Nielsen & Molin, 2004;Wagner & Iglewski, 2008). Due to its metabolic diversity and its importance as a human pathogen, P. aeruginosa has become an organism of intense study, so genetic tools for this organism have become increasingly important.Generalized transducing bacteriophages are useful genetic tools for strain engineering and for functional genomic analysis in strains that are not naturally competent, such as P. aeruginosa (Wong et al, 2000). Several P. aeruginosa generalized transducing bacteriophages have been described, though the transduction efficiency of these bacteriophages, in particular DMS3, UT1 and F116, can be very low (Budzik et al, 2004;Kidambi et al, 1994;Ripp et al, 1994).…”