involvement in other events such as increased nuclear import or enhanced chromatin loading. With respect to the early lymphoma susceptibility phenotype in Mcm4Chaos3/Chaos3 Mcm2Gt/+ mice, it is unclear whether the type of tumor is dictated primarily by the particular Mcm depletion (in this case MCM2, thus resembling Mcm2IRES-CreERT2/IRES-CreERT2 animals), the genetic background, or the age of particular cancer onset (if animals die of thymic lymphoma at an early age, they will be unable to manifest later-arising mammary tumors). The compound mutant mice used for the aging aspects of this study were bred to at least the N3 generation in strain C3H. Mcm4Chaos3/Chaos3 mice congenic in this background are predis- posed exclusively to mammary tumors, whereas lymphomas were observed in mutants of mixed background [17]. Presently, we favor the idea that genetic background and age of tumor type onset are primary determinants of the cancers that arise in the mice we have studied thus far. Genetic background has also been reported to influence tumor latency in MCM2-deficient mice [21]. The MCM2-7 pan-reduction in Chaos3 cells is consistent with other studies involving mutation or knockdown of a single MCM in mammalian cells [16,20,29,37]. In these examples of parallel MCM decreases, the general assumption is that there is hexamer destabilization or impaired MCM chromatin loading followed by degradation of monomers. However, we found that the protein decreases are related to decreased mRNA levels. These large (,40%) decreases do not appear to be attributable to transcrip- tional alterations from cell cycle disruptions (these cells have a small elevation in the G2/M population), but rather occur at the post-transcriptional level (unpublished observations). Since we also found that MEFs carrying only 1 functional Mcm2 allele caused ,20% decreases of Mcm3-7 mRNAs, it is possible that mRNA downregulation drove MCM reductions in these other model systems. However, the mechanism for coordinated mRNA regulation, and what triggers it, is a mystery that we are currently investigating. Our data contribute to a growing body of data that replication stress, which can occur via perturbations of the DNA replication machinery, plays a significant role in driving cancer [38–41]. While the Mcm4Chaos3 mutation is an unique case, the deleterious consequences of MCM reductions suggest that genetically-based variability in DNA replication factors can have physiological consequences. Such variability in functions or levels may be caused by Mendelian mutations or multigenic allele interactions. Mutations affecting transcriptional activity of one or more Mcms, which might occur in non-coding cis-linked sequences or unlinked transcription factors, could have such effects. This has implications for cancer genome resequencing projects, whereby such mutations would not be obviously associated with MCM expression. The allelic collection we generated, when used alone or in combination with each other or Mcm4Chaos3/Chaos3 mice, allow the generation of mouse models with a graded range of MCM levels. These should be valuable for investigations into the impact of replication stress on animal development, cancer formation, and cellular homeostasis. Materials and Methods MEF culture and proliferation assays MEFs from 12.5- to 14.5-dpc embryos were cultured in DMEM+10% FBS, 2 mM GlutaMAX, and penicillin-streptomy- cin (100 units/ml). Assays were conducted on cells at early passages (up to P3). For cell proliferation assays, 56104 cells were seeded per well of a 6 well plate. They were then cultured and harvested at the indicated time points to perform cell counts. Quantitative Mcm Perturbation in Mice iPS induction from MEFs using lentiviral vectors Doxycycline inducible lentiviral vectors [42] were prepared by co-transecting viral packaging plasmids psPAX2 and and pMD2.G along with vectors encoding rtTA, Oct4, Sox2, Klf4, or c-Myc (plasmids were obtained from Addgene.org, serial numbers 12259, 12260, 20323, 20322, 20324, and 20326) into 293T cells using TransIT-Lt1 transfection reagent (Mirus). Viral supernatants were collected at 48 and 72 hours, and concentrated using a 30kd NMWL centrifugal concentrator. MEFs from 13.5d embryos, up to P3, were seeded to gelatin coated tissue plates at a density of 6.756103 cells/cm2 and allowed to attach in standard MEF media for 24 hours before infection with lentiviral vectors. After 24 hours incubation the culture media was changed to KO-DMEM supplemented with 15% KO serum replacement (Gibco), recombinant LIF, 2 mg/mL doxycycline (Sigma), 100 mm MEM non-essential amino acids solution, 2mM GlutaMax, 100 units/ mL penicillin and 100 mg/mL streptomycin (Gibco). The induction media was refreshed daily for 13 days until the cells were passaged to 100 mm plates prepared with irradiated feeders. Cells were cultured for an additional 10 days in the induction media in the absence of doxycyline before iPS colony counting, cell counts, and flow cytometry. For flow cytometric quantification of iPS cells derived from reprogramming of MEFs, ,16106 cells were trypsinized for 10 minutes, then washed twice with cold PBS. They were gently but completely resuspended in 1ml of 4% paraformaldehyde in PBS at room temperature for 30 minutes. The fixed cells were pelleted by centrifugation at 5006G for 2 minutes and washed twice with 10 ml TBS-TX (0.1% Triton X-100) buffer. For antibody staining, the cells were blocked with 1ml TBS-TX buffer with 1% BSA for 15 min at room temperature, then stained with primary ‘‘stemness’’ antibodies (monoclonal anti-SSEA1, Milli- pore; rabbit polyclonal anti-LIN28, Abcam) for 60 min, washed twice, then secondary antibody was applied for 60 minutes. Immunolabeled cells were analyzed by flow cytometry using a 488nm laser. Secondary antibodies were goat anti-mouse IgG- FITC (South Biotech) and goat anti-rabbit IgG-594 (Molecular Probes). Cells were considered to be iPS cells if they were LIN28/ SSEA1 positive. Calibration of the flow cytometer and gates were set using untransfected MEFs as negative controls, and v6.4 ES cells as positive controls. For quantification by colony formation, plates containing the passaged reprogrammed cells were examined microscopically at 206, and 4 fields were scored and averaged. Colonies were considered as iPS clones based on morphological criteria: well defined border, three-dimensionality, and tight packing of cells. Flow cytometric analyses of micronuclei and iPS cells Micronucleus assays, which include CD71 staining, were performed essentially as described [43]. Nuclei isolation and immunofluorescence staining MEFs were plated at 46106 cells/150 mm culture dish for 60 hr, trypsinized, then resuspended in 1ml PBS. To the suspension was added TX-NE (320 mM sucrose, 7.5 mM MgCl2, 10 mM HEPES, 1% Triton X-100, and a protease inhibitor cocktail). The cells were gently vortexed for 10 seconds and incubated on ice for 30 min. Dounce homogenization was unnecessary. Nuclei were then pelleted by centrifugation at 5006G for 2 min and washed twice with 10 ml TX-NE, then resuspended in 1ml TX-NE. Nuclei yield and integrity was monitored microscopically with trypan blue staining. The nuclei were fixed by adding 15ml cold methanol for 60 min on ice. The fixed nuclei were pelleted by centrifugation at 5006G for 2 min, PLoS Genetics | www.plosgenetics.org 13 September 2010 | Volume 6 | Issue 9 | e1001110