Stem/progenitor cell-specific enhanced green fluorescent protein expression driven by the endogenous Mcm2 promoter.

Previous studies have demonstrated expression of the minichromosome maintenance protein Mcm2 in cells that remain competent to divide, including stem/progenitor cells of the subventricular zone (SVZ) within the brain. Here, a transgenic mouse line in which the Mcm2 gene drives expression of enhanced green fluorescent protein (EGFP) was constructed by insertion of an internal ribosomal entry site (IRES)-EGFP cassette into the last exon of the gene, 3' to the stop codon. In these mice, expression of EGFP is observed in the SVZ and several other tissues with high proliferative activity, including the spleen, intestine, hair follicles, and bone marrow. These observations suggest that EGFP fluorescence in this mouse line provides an index of the proliferative capacity of different tissues. Immunohistological analysis demonstrates a direct concordance between expression of EGFP and Mcm2, consistent with a transcriptional level downregulation of Mcm2 expression in postmitotic cells. To test the utility of EGFP expression for recovery of live cells retaining the capacity to divide, EGFP-expressing and -nonexpressing cells from bone marrow and brain were isolated from an adult Mcm2(IRES-EGFP) mouse by fluorescence-activated cell sorting and assayed for clonal growth. The EGFP-positive fraction contained the entire clonogenic population of the bone marrow and greater than 90% of neurosphere-forming cells from the brain. Brain-derived clonogenic cells were shown to remain competent to differentiate towards all three neural lineages. These studies demonstrate that the Mcm2(IRES-EGFP) transgenic line constructed here can be used for recovery of proliferation competent cells from different tissue types.

Analysis of fluorescent protein expressing cells by flow cytometry.

The process for transfection of cells with expression and gene-trap vectors expressing fluorescent reporter proteins is described. The measurement and sorting of discrete populations of transfected cells is also described and illustrated. Of particular importance, the maintenance of stability may be important and a simple strategy to monitor this has been developed. Finally, an effective method for improving the ability to measure low-level fluorescence from autofluorescence is described.