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First published online September 28, 2006
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2006-0032v1
25/1/132    most recent
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Submitted on January 17, 2006
Accepted on September 19, 2006

Tissue-Specific Stem Cells

Stem/Progenitor Cell Specific EGFP Expression Driven by the Endogenous Mcm2 Promoter

Alexander Y. Maslov 1, Kimberly J. Bailey 1, Lawrence M. Mielnicki 1, Amy L. Freeland 1, Xiaolei Sun 2, William C. Burhans 3, Steven C. Pruitt 1*

1 Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York
2 Department of Surgical Oncology, Roswell Park Cancer Institute, Buffalo, New York
3 Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York

* To whom correspondence should be addressed. E-mail: steven.pruitt{at}roswellpark.org.


  Abstract

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 EGFP was constructed by insertion of an 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 down-regulation of Mcm2 expression in post-mitotic cells. To test the utility of EGFP expression for recovery of live cells retaining the capacity to divide, EGFP expressing and non-expressing cells from bone marrow and brain were isolated from an adult Mcm2{wedge}IRES-EGFP mouse by FACS 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{wedge}IRES-EGFP transgenic line constructed here can be used for recovery of proliferation competent cells from different tissue types.

Key Words. stem cells, progenitor cells, EGFP, Mcm2, transgenic mouse




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S. C. Pruitt, K. J. Bailey, and A. Freeland
Reduced Mcm2 Expression Results in Severe Stem/Progenitor Cell Deficiency and Cancer
Stem Cells, December 1, 2007; 25(12): 3121 - 3132.
[Abstract] [Full Text] [PDF]


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