Quantitative Mcm Perturbation in Mice Figure 2. Mcm gene trap alleles and associated mRNA levels, peripheral blood micronuclei, and cancer frequency. (A) Genomic structures of mutated Mcm genes. Indicated is the intron/exon structure of each gene (not to scale), the locations of the gene trap insertions, and qPCR primer locations. (B) qRT-PCR analyses of MEF mRNA from gene trap heterozygotes. Charted are the percent levels of the indicated RNAs in mutant compared to WT (considered to be 100%). For all but Mcm7, the data were obtained from at least 3 MEF cultures from different embryos. The Mcm7 data represents the average of three replicates from 1 MEF culture, hence there are no error bars. Otherwise, error bars show SEM. (C) Micronucleus levels in Mcm gene trap-bearing male mice. At least 5 animals were analyzed for each single gene trap mutant allele. The ‘‘2GT’’ (two gene trap) group contains: 4 mice doubly heterozygous for Mcm2Gt and Mcm3Gt (‘‘Mcm2/3’’), 4 Mcm2/4 mice, and 4 Mcm3/4 mice. The 3GT group contains: 4 Mcm2/3/4 mice, 1 Mcm2/3/6 mouse, 1 Mcm2/4/6 mouse, and 3 Mcm3/4/6 mice. The 4-5GT group contains: 3 Mcm2/3/4/6 mice, 1 Mcm2/ 3/6/7 mouse, and 2 Mcm2/3/4/6/7 mouse. SEM bars are shown. doi:10.1371/journal.pgen.1001110.g002 concentrations. However, our genetic experiments uncovered one notable exception: hemizygosity for Mcm3 did not cause any severe haploinsufficiency phenotypes (increased lethality and decreased weight) as did Mcm2/6/7 in the Mcm4Chaos3/Chaos3 background, or Mcm4Gt in trans to Mcm4Chaos3 (Figure 3A; Figure S2). Since extreme reductions of MCM3 in cultured human cells caused GIN and cell cycle arrest [16], the absence of synthetic effects with McmChaos3 led us to hypothesize that either mice are more tolerant to lower levels of this particular MCM, or that MCM3 is present in a stoichiometric excess compared to the other MCMs, at least in a subset of cell types. To explore these issues we performed additional phenotype analyses, and also sought to uncover potential effects of MCM3 reduction by reducing other MCMs simultaneously. Strikingly, rather than exacerbating the synthetic lethality in Mcm4Chaos3/Chaos3 Mcm2Gt/+ mice, Mcm3Gt heterozygosity signifi- cantly rescued their viability to 72.5% from 29.7% (Figure 5A and Fig S3). Not only was viability rescued, but also growth (weight) of Figure 3. Synthetic lethality and growth retardation between Mcm4Chaos3 and Mcm2, Mcm6 and Mcm7. (A) Graphed are viability data from crosses presented in Figure S2, which includes statistics. Unless otherwise indicated, the values represent expected proportions of indicated genotypes that were present at wean. (B) Weights of surviving animals are graphed over time. SEM bars are shown. doi:10.1371/journal.pgen.1001110.g003 PLoS Genetics | www.plosgenetics.org 6 September 2010 | Volume 6 | Issue 9 | e1001110