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.
Maslov
Alexander Y
AY
Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York, New York 14263, USA.
Bailey
Kimberly J
KJ
Mielnicki
Lawrence M
LM
Freeland
Amy L
AL
Sun
Xiaolei
X
Burhans
William C
WC
Pruitt
Steven C
SC
D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000502:physiology; D005091:Exons; D017930:Genes, Reporter; D016678:Genome; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D064111:Minichromosome Maintenance Complex Component 2; D009687:Nuclear Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D013234:Stem Cells / Q000502:physiology*; D014162:Transfection
D009687:Nuclear Proteins; D049452:Green Fluorescent Proteins; D064111:Minichromosome Maintenance Complex Component 2