Transgenic Mouse Construction
A transgenic mouse line in which expression of the tamoxifen-inducible Cre recombinase gene CreERT2  is directed by the Mcm2 gene was constructed using a knock-in approach. The starting point for this construct was pMcm2-IRES-EGFP, in which an IRES-EGFP cassette is inserted into the 3′-untranslated region of the Mcm2 gene. Following targeted recombination into the endogenous Mcm2 gene, this construct has previously been shown to express EGFP in stem/progenitor cells of a variety of tissues . Nonetheless, in the present study, correctly targeted W4 ES cells lines carrying a parallel construct in which the CreERT2 gene was substituted for EGFP failed to express Cre recombinase transcripts as assayed by either Northern blot analysis or reverse transcription PCR assays. Sequencing of 3′rapid amplification of cDNA ends products suggested that the reason for the lack of CreERT2 recombinase expression was the presence of a cryptic polyadenylation site located at position 2,920–2,925 in the 3′-untranslated sequence of the Mcm2 message (Fig. 1B; data not shown). Although use of this polyadenylation site is not detected in Mcm2-IRES-EGFP ES cells or mice , it becomes the predominant polyadenylation site when the CreERT2 gene is substituted for EGFP (data not shown). This is despite the fact that the substitution occurs at a position approximately 1 kbp 3′ to the polyadenylation site and the intervening sequences are identical. To prevent the use of this site, the sequence between the BstEII site at 2,802 and a BamHI site, located 17 bp 3′ to the XbaI site in Figure 1, was deleted in the Mcm2-ΔpA-IRES-CreERT2 construct. This modification resulted in expression of CreERT2 transcripts in correctly targeted W4 ES cells (Fig. 1F). Transgenic mice carrying the Mcm2-ΔpA-IRES-CreERT2 transgene were generated by blastocyst injection and are referred to below as Mcm2IRES-CreERT2 mice.
Bigenic Mcm2IRES-CreERT2/wt × Z/EG Reporter Mice Allow Tamoxifen-Inducible EGFP Expression in Stem/Progenitor Cells of Multiple Tissues
To determine whether CreERT2 expression from the Mcm2 promoter would allow genetic manipulation within the stem/progenitor cells of adult tissues, heterozygous Mcm2IRES-CreERT2/wt mice were crossed with mouse lines that carry Cre-dependent EGFP (Z/EG mice ) or in some cases lacZ (not shown) reporters. Prior studies have shown that, when Z/EG mice were crossed with mice expressing CreERT2 from a constitutive promoter, tamoxifen-dependent Cre-mediated EGFP expression occurs in a variety of different embryonic and adult somatic tissues where the frequency of recombination typically ranged from 5% to 10% . In the present study, expression of EGFP in various tissues was monitored in bigenic Mcm2IRES-CreERT2/wt × Z/EG mice that had been treated with tamoxifen followed by a 15-day chase in the absence of tamoxifen (Fig. 2). EGFP expression was observed in most tissues in these mice in a pattern that is consistent with expression in a subset of stem/progenitor cells and their differentiated progeny. The proportion of cells expressing EGFP in different tissues varied, consistent with the distribution of stem/progenitor cells within the different tissues. It can be estimated that ∼2%–10% of the stem/progenitor cells within most tissues underwent Cre-mediated recombination depending on the concentration of tamoxifen and route of administration. For example, in Figure 2G, 2I, and 2J, approximately 1/5 of the small intestine crypts exhibit EGFP expression. Each crypt contains between 6 and 8 intestinal stem cells . Furthermore, the length of time between tamoxifen-induced recombination and the point at which the mice were assayed was sufficient that all of the EGFP-expressing cells present in the small intestine must be derived from an EGFP-expressing stem cell. Hence, it can be estimated that approximately 2%–3% of the stem cells have undergone Cre-mediated recombination. Consistent with this estimate, approximately 3% of clonogenically isolated neurospheres (prepared as described in ), derived from bigenic Mcm2IRES-CreERT2/wt × Z/EG mice treated with tamoxifen as above, expressed EGFP (e.g., Fig. 2D). No EGFP expression was observed in any tissue of control animals carrying both Mcm2IRES-CreERT2 and the Cre-dependent reporter in the absence of tamoxifen (e.g., Fig. 2H, small intestine). These data demonstrate that the Mcm2IRES-CreERT2 transgene provides a useful means of conditionally modifying the genomes of a subset of adult somatic stem cells in a wide variety of tissues.
Figure Figure 2.. Tamoxifen-dependent enhanced green fluorescence protein (EGFP) expression in Mcm2-IRES-CreERT2 × Z/EG reporter mice. Six-week-old mice heterozygous for both Mcm2-IRES-CreERT2 and a Cre-dependent EGFP reporter (Z/EG)  were treated with five injections of 4-hydroxy-tamoxifen over a period of 10 days (except [H], which is from a mouse with the same genotype but not receiving tamoxifen). Fifteen days following the final tamoxifen treatment, mice were assessed for EGFP expression by fluorescence stereomicroscopy (A–C, G–K), compound microscopy (E, F), or inverted microscopy (D). (A): Tongue. (B): Hair follicles from the tail. (C): Ventricle (outlined with white dots). (D): Neurosphere derived from the brain of a tamoxifen-treated mouse following 10 days of growth in vitro. (E, F): Bone marrow at lower and higher magnifications respectively. (G, H): External views of small intestines from tamoxifen-treated and untreated mice, respectively. (I, J): Small intestine villi photographed from the side or from the top. (K): Colonic crypts photographed from the top.
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Mcm2IRES-CreERT2/IRES-CreERT2 Mice Are Viable and Develop Normally but Die Prematurely from Cancers
In prior studies, integration of IRES-EGFP into the 3′-untranslated region of the Mcm2 gene was shown not to significantly affect Mcm2 expression levels . Mice carrying the Mcm2-IRES-EGFP transgene could be bred to homozygosity and with no phenotypic effect and normal longevity. In contrast, homozygous Mcm2IRES-CreERT2/IRES-CreERT2 mice exhibit a very different phenotype. These mice are born at the expected frequency and develop normally through early adulthood. However, beginning at approximately 9 weeks of age, animals become moribund, and most do not survive beyond 12 weeks; the longest-surviving animal to date was 21 weeks old (Fig. 3A).
Figure Figure 3.. The Mcm2-IRES-CreERT2 allele results in hypomorphic Mcm2 expression and results in high rates of cancer in homozygous mice. (A): Survival of wt (n = 14), wt/Cre (n = 14), and Cre/Cre (n = 14) transgenic mice as a function of age in weeks. (B): Frequency of micronuclei in reticulocytes of wt (n = 3) and Cre/Cre (n = 3) mice. (C): Western blot analysis of protein extracts from wt-, wt/Cre-, and Cre/Cre-derived MEF cells for Mcm2, Mcm7, and β-actin as indicated. (D): Densitometric quantification of Mcm2 and Mcm7 expression normalized against β-actin levels. Abbreviations: Cre/Cre, Mcm2IRES-CreERT2/IRES-CreERT2; wt/Cre, Mcm2IRES-CreERT2/wt; wt, wild-type.
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The constellation of symptoms that accompany morbidity include a hunched appearance, rapid shallow respiration, generalized muscle weakness, limited movement, loss of adipose tissue, frailty, and (in some cases) modest hair loss or slight graying. Most animals die approximately 2–3 weeks after the appearance of respiratory symptoms. Necropsy of symptomatic homozygous animals carried on the 129/Sv genetic background revealed that most had extensive thymomas that filled the thoracic cavity (likely the cause of death). Many of the animals also exhibited an enlarged spleen, and several also exhibited polyps in both the small intestine and colon. No abnormalities in mammary glands of female mice were found.
Homozygous Mcm2IRES-CreERT2/IRES-CreERT2 Mice Exhibit Reduced Levels of Mcm2 and Modest Levels of Genomic Instability
Northern blot analysis suggested that the level of the Mcm2IRES-CreERT2 transcript (∼7 kb) was reduced relative to that of Mcm2 (∼3.3 kb). However, the large difference in transcript size prevented a quantitative comparison. To define Mcm2 expression levels, Western blot analysis was performed on Mcm2wt/wt, Mcm2wt/IRES-CreERT2, and Mcm2IRES-CreERT2/IRES-CreERT2 embryos (not shown) and passage 3 MEFs (derived from E14.5 embryos; Fig. 3C, 3D). These studies showed that Mcm2 protein levels were reduced in McmIRES-CreERT2 heterozygotes and homozygotes to 62% or 35% of wild-type levels. Despite the reduced level of Mcm2 expression, no difference in the size of Mcm2wt/IRES-CreERT2 or Mcm2IRES-CreERT2/IRES-CreERT2 embryos was observed relative to wild-type embryos. In addition, the passage time for MEFs derived from Mcm2wt/IRES-CreERT2 or Mcm2IRES-CreERT2/IRES-CreERT2 embryos was similar to that of MEFs derived from wild-type embryos through passage 5. Finally, expression of the related protein Mcm7, which is a component of the heterohexamer prereplication complex [14, –16], was assayed, showing a modest reduction to 79% or 73% of wild-type levels in MEFs from heterozygotes and homozygotes, respectively.
To determine whether the reduced expression of Mcm2 was affecting chromosomal stability, a reticulocyte micronucleus assay was used . Comparison of wild-type and Mcm2IRES-CreERT2/IRES-CreERT2 mice demonstrated an approximately 2.5-fold increase in the number of micronuclei present in peripheral blood erythrocytes (Fig. 3B).
Stem/Proliferative Cells Are Deficient in Multiple Tissues of Homozygous Mcm2IRES-CreERT2/IRES-CreERT2 Mice
Despite the observation that most Mcm2IRES-CreERT2/IRES-CreERT2 mice exhibit tumors and that this is generally the cause of death, the mice showed a spectrum of additional phenotypes characteristic of age-related dysfunction. One potential explanation for these additional phenotypes is that reduced Mcm2 expression has a general effect on proliferating cells within multiple tissues. To determine the effect of Mcm2 deficiency on somatic stem cells and proliferative progenitors, three tissues, the subventricular zone (SVZ) of the brain, skeletal muscle, and small intestine, were examined.
To assess cell proliferation within the SVZ, wild-type and Mcm2IRES-CreERT2/IRES-CreERT2 mice were administered IdU for a period of 3 days and CldU 2 hours prior to examination. Histological sections were prepared and stained for IdU, CldU, and Mcm2. In prior studies, we [8, 11] and others [17, , , , –22] have shown that this combination of double-nucleoside analog labeling in conjunction with staining for Mcm2 expression provides an effective means of estimating the rate of cycling of the proliferative progenitors and distinguishing slowly dividing stem cells from more rapidly dividing proliferative progenitors. Most proliferative progenitors cycle at a rate of approximately once every 12.7 hours  and, in wild-type mice, are labeled with IdU over the 3-day labeling period. Those proliferative progenitors that are in S-phase during the 2-hour period prior to sacrifice will be labeled with CldU, providing a measure of the rate of cycling of these cells. Cells that have not cycled over a 3-day period but continue to express Mcm2 are putative stem cells, as evidenced by (a) the ability of such cells to re-enter the cell cycle in long-term labeling experiments , (b) the lack of an effect of AraC treatment on this population of cells and the ability of these cells to repopulate the SVZ following AraC removal , (c) expression of Musashi  and Sox2  in the Mcm2+/IdU− cells in situ, and (d) the observation that neurosphere-forming cells reside in the EGFP-positive fraction of cells derived from Mcm2IRES-EGFP mice .
Immunohistological images are shown for wt and Mcm2IRES-CreERT2/IRES-CreERT2 SVZs in Figure 4A–4F. The density of nuclei within the SVZs of Mcm2IRES-CreERT2/IRES-CreERT2 mice was substantially reduced relative to wild-type. Furthermore, the number of cells incorporating either CldU or IdU was reduced to approximately one-third the number in wild-type mice (Fig. 4G) such that the proportion of IdU+ cells that also express CldU was similar or slightly higher in Mcm2IRES-CreERT2/IRES-CreERT2 mice relative to wild-type (Fig. 4H). This observation suggests that despite the reduced intensity of Mcm2 staining over the nuclei from Mcm2IRES-CreERT2/IRES-CreERT2 mice (Fig. 4C [wild-type] compared with Fig. 4F [mutant]), the rate of division of the proliferative progenitors in the mutant mice was at least as high as in wild-type mice. In parallel with the reduction in nucleoside analog incorporation, the number of Mcm2+/IdU− cells was also reduced such that there were approximately one-third as many Mcm2+/IdU− cells in Mcm2IRES-CreERT2/IRES-CreERT2 mice as in wild-type mice (Fig. 4I). Finally, the frequency of neurosphere formation in vitro was determined using a clonogenic assay . Consistent with immunohistological studies, the number of neurospheres resulting from Mcm2IRES-CreERT2/IRES-CreERT2 mice was approximately one-third of that from wild-type mice (Fig. 4J). Despite this reduction, no effect on the size of the neurospheres was evident between wild-type and Mcm2IRES-CreERT2/IRES-CreERT2 mice (not shown). Together, these studies demonstrate that neurogenesis within the SVZ of Mcm2IRES-CreERT2/IRES-CreERT2 is significantly reduced, and similar to the case for aged mice , this reduction is a consequence of a reduced number of neural stem cells rather than a reduced rate of cycling of the proliferative progenitors.
Figure Figure 4.. Effect of hypomorphic Mcm2 expression on subventricular zone (SVZ) neurogenesis. (A–F): Immunofluorescent-stained paraffin sections from the same region of the SVZ of wt (A–C) or Mcm2IRES-CreERT2/IRES-CreERT2(D–F) mice. (A, D): IdU (red) and 4,6-diamidino-2-phenylindole (blue); (B, E): IdU (red) and CldU (green); (C, F): Mcm2 (green) and IdU (red). (G): Quantification of the number of CldU+ and IdU+ cells present over an interval of 0.5 mm along the SVZ as indicated. (H): Percentage of IdU-labeled cells that are also labeled with CldU for wt (black columns) or Mcm2IRES-CreERT2/IRES-CreERT2 (gray columns) mice. (I): number of Mcm2+ cells and number of Mcm2+ cells that failed to stain for IdU (Mcm2+/IdU−) over an interval of 0.5 mm along the SVZ for wt (black columns) and Mcm2IRES-CreERT2/IRES-CreERT2 (gray columns) mice. (J): Number of neurospheres forming in a clonogenic assay from the brains of wt (black columns, n = 3) or Mcm2IRES-CreERT2/IRES-CreERT2 (gray columns, n = 3) mice. Abbreviations: cre, Mcm2IRES-CreERT2/IRES-CreERT2; wt, wild-type.
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To examine the effect of Mcm2 deficiency on proliferative cells within the skeletal muscle, myoblasts were recovered from the tibialis anterior muscles of 8-week-old wild-type and Mcm2IRES-CreERT2/IRES-CreERT2 mice and plated to microtiter wells. Following 6 days in culture, the number of cells per well was determined and is plotted in Figure 5A. Approximately 1/10 as many cells were present in cultures derived from Mcm2IRES-CreERT2/IRES-CreERT2 mice relative to wild-type animals. To determine whether the difference resulted from a difference in cell cycle time, cells were reseeded at a constant number, and the number of cells per well was followed over a period of 6 days for eight cultures each of cells derived from mutant and wild-type animals. No difference in doubling time was observed, suggesting that the difference in plating efficiency in primary cultures resulted from the presence fewer myogenic cells in the muscles of Mcm2IRES-CreERT2/IRES-CreERT2 mice in vivo (Fig. 5B). Despite the similar growth rates, examination of H2AX foci, which accumulate at sites of double-strand breaks during DNA repair , demonstrates that there is a higher frequency of foci in cells derived from Mcm2IRES-CreERT2/IRES-CreERT2 mice than in cells derived from Mcm2wt/wt mice (Fig. 5C–5E).
Figure Figure 5.. Effect of hypomorphic Mcm2 expression on the number and growth rate of muscle satellite cells and the frequency of γ-H2AX foci. (A): Number of colonies forming in a clonogenic assay of cells derived from the anterior tibialis muscle of wt (black column) or Mcm2IRES-CreERT2/IRES-CreERT2 (gray column) mice. (B): Doubling rate of cells following secondary passage from colonies derived from anterior tibialis muscle of wt (diamonds, solid line) or Mcm2IRES-CreERT2/IRES-CreERT2 (squares, dashed line) mice. (C, D): Images of passage 2 muscle satellite cells stained for the presence of γ-H2AX foci (red) and 4,6-diamidino-2-phenylindole (blue); (C): wt; (D): Mcm2IRES-CreERT2/IRES-CreERT2. (E): Quantification of the frequency of γ-H2AX foci from four cultures derived from two different mice of each genotype (wt, black column; Mcm2IRES-CreERT2/IRES-CreERT2, gray column), where between 100 and 250 nuclei were assayed per sample. Abbreviation: wt, wild-type.
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Finally, the effect of Mcm2 deficiency on small intestinal crypts was examined. For these experiments, Mcm2wt/wt and Mcm2IRES-CreERT2/IRES-CreERT2 mice were administered IdU for a period of 2 days and injected with CldU 2 hours prior to sacrifice. Histological sections were prepared and assayed for IdU, CldU, and Mcm2 as before. The overall morphology of crypts and villi within the small intestine of Mcm2IRES-CreERT2/IRES-CreERT2 mice appeared disorganized relative to wild-type mice (compare Fig. 6A and 6B with Fig. 6D and 6E, particularly in the region near to the tops of the crypts). Nonetheless, the number of IdU- and CldU-labeled cells was similar between each of these genotypes, suggesting that the rate of cycling and overall rate of generation of cells was not affected in Mcm2IRES-CreERT2/IRES-CreERT2 mice. Furthermore, there was very little difference in the distribution of CldU-labeled cells within the crypts as a function of position from the base of the crypt (Fig. 6C, 6F). Mcm2 expression levels were reduced, and the region of the crypts over which Mcm2 was detectable was limited to more basal cell positions relative to wild-type or heterozygous mice (Fig. 6C, 6F). Nonetheless, the large majority of cell division occurred within even this reduced Mcm2 expression domain in wild-type and Mcm2IRES-CreERT2/IRES-CreERT2 mice.
Figure Figure 6.. Effect of hypomorphic Mcm2 expression on intestinal crypt morphology and proliferation. (A–F): Derived from an experiment in which wild-type or Mcm2IRES-CreERT2/IRES-CreERT2 mice were administered IdU in their drinking water for a period of 2 days and CldU by injection 2 hours prior to sacrifice. Immunofluorescence images of the same regions of wild-type (A, B) and Mcm2IRES-CreERT2/IRES-CreERT2(D, E) mice stained for CldU (green) and IdU (red) (A, D) or Mcm2 (red) (B, E). 4,6-Diamidino-2-phenylindole (DAPI) is shown in blue for each set. (C, F): Quantifications of the proportion of CldU, IdU, and Mcm2+ at different nuclear positions from the base of the crypts where 18 crypts were assessed for each point. (G–P): Derived from an experiment in which Mcm2IRES-CreERT2/wt × Z/EG and Mcm2IRES-CreERT2/IRES-CreERT2 × Z/EG mice were first administered tamoxifen at approximately 6 weeks of age, following a resting period of 1 month in the absence of any treatment; mice were then administered IdU in their drinking water for a period of 2 days and CldU by injection 2 hours prior to sacrifice. Small intestine whole mounts were examined by stereofluorescence microscopy for enhanced green fluorescence protein (EGFP) expression (G–J), where (G) is from an Mcm2IRES-CreERT2/wt × Z/EG mouse and (H–J) are from an Mcm2IRES-CreERT2/IRES-CreERT2 × Z/EG mouse. (K–P): Immunofluorescence images of crypts, where (K) is from an Mcm2IRES-CreERT2/wt × Z/EG mouse and (L–P) are from an Mcm2IRES-CreERT2/IRES-CreERT2 × Z/EG mouse. (L–O): From the same region; (P): from a different region. Stains were as follows: (K): EGFP (green) and DAPI (blue); (L): EGFP (green); (M): EGFP (green) and DAPI (blue); (N): CldU (green) and DAPI (blue); (O): IdU (red) and DAPI (blue); and (P): EGFP (green) and IdU (red). Arrows marked by an asterisk (K–P) indicate EGFP-positive cells within the crypt, and arrows without an asterisk (L–O) indicate EGFP-negative cells within the crypt that had incorporated CldU and IdU as shown in (N, O). (P): Composite of three serial sections showing EGFP expression (green) in cells near the top of the villi and in a single quiescent EGFP-positive cell at the base of the crypt despite the presence of IdU-labeled cells (red) throughout the crypt, demonstrating that stem cell quiescence is maintained in at least some crypts within Mcm2IRES-CreERT2/IRES-CreERT2 mice, similar to the situation in wild-type mice (K). Abbreviations: CldU, 5-chloro-2′-deoxyuridine; IdU, 5-iodo-2′-deoxyuridine.
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To further examine the consequences of Mcm2 deficiency in the small intestinal crypts, Mcm2wt/IRES-CreERT2 and Mcm2IRES-CreERT2/IRES-CreERT2 mice carrying a Cre-dependent EGFP reporter were generated using the Z/EG reporter line. Mice were treated with tamoxifen at 6 weeks of age and rested for 1 month prior to assaying for EGFP expression. This resting period is sufficient to allow cells in which recombination had occurred in proliferative progenitors to be lost from the tissue, ensuring that any EGFP-expressing cells are the progeny of an EGFP-expressing stem cell. Since Cre-dependent recombination is incomplete in these mice, in most crypts, EGFP expression occurs in only a single stem cell and its progeny. Comparison of EGFP-expressing crypts in mice that were heterozygous or homozygous for the Mcm2-IRES-CreERT2 allele by stereofluorescence microscopy revealed a difference in the proportion of marked cells within those crypts exhibiting EGFP expression. In heterozygous mice, only a subset of cells within a crypt expressed EGFP, and the progeny of these cells formed continuous EGFP-expressing runs that were as much as half of the length of the villus (Figs. 2G, 2I, 2J, 6G). In contrast, in Mcm2IRES-CreERT2/IRES-CreERT2 mice, when EGFP-expressing cells were present within the crypt, they generally constituted the majority of the cells (Fig. 6H, 6I, 6L, 6M). In cases where EGFP-expressing cells had migrated to the adjacent villi, runs of positive cells were typically shorter than in Mcm2IRES-CreERT2/wt mice but made up a larger fraction of the labeled villus (Fig. 6J). These observations are consistent with either a longer period of quiescence between stem cell divisions or the presence of fewer stem cells per crypt in Mcm2IRES-CreERT2/IRES-CreERT2 relative to Mcm2wt/IRES-CreERT2 mice.