Organogenesis from dissociated cells: generation of mature cycling hair follicles from skin-derived cells.

Hair follicle formation and cycling involve extensive and continuous interactions between epithelial and mesenchymal components. A system for rapidly and reproducibly generating hair follicles from dissociated epithelial and mesenchymal cells is described here. The system serves both as a tool for measuring the trichogenic property of cells and as a tool for studying the mechanisms that dissociated cells use to assemble an organ. In this system, hair follicles develop when dissociated cells, isolated from newborn mouse skin, are injected into adult mouse truncal skin. This morphogenetic process involves the aggregation of epithelial cells to form clusters that are sculpted by apoptosis to generate "infundibular cysts". From the "infundibular cysts", hair germs form centrifugally followed by follicular buds and then pegs that grow asymmetrically to differentiate into cycling mature pilosebaceous structures. Marker studies correlate the molecular differentiation of these follicles with in situ systems. This study suggests that the earliest phase of a developing epithelial-mesenchymal system--even from dissociated cell preparations--requires an epithelial platform.

High-throughput engineering of the mouse genome coupled with high-resolution expression analysis.

One of the most effective approaches for determining gene function involves engineering mice with mutations or deletions in endogenous genes of interest. Historically, this approach has been limited by the difficulty and time required to generate such mice. We describe the development of a high-throughput and largely automated process, termed VelociGene, that uses targeting vectors based on bacterial artificial chromosomes (BACs). VelociGene permits genetic alteration with nucleotide precision, is not limited by the size of desired deletions, does not depend on isogenicity or on positive-negative selection, and can precisely replace the gene of interest with a reporter that allows for high-resolution localization of target-gene expression. We describe custom genetic alterations for hundreds of genes, corresponding to about 0.5-1.0% of the entire genome. We also provide dozens of informative expression patterns involving cells in the nervous system, immune system, vasculature, skeleton, fat and other tissues.