The breeding scheme for crossing the lacZ
-plasmid reporter construct into the Ames background is shown in . While Ames dwarf mice have a heterogeneous genetic background, the lacZ
-plasmid animals were in C57BL/6J, with the transgene cluster at two chromosomal locations, one on chromosome 3 and the other on chromosome 4. We initially decided to cross the Ames dwarf mutation as far as possible into the lacZ
-C57BL/6J background. This would have had the advantage of simultaneously homogenizing the Ames background into mostly C57BL/6J, thereby reducing heterogeneity, and generating homozygosity for the lacZ
loci, thus increasing the number of lacZ
-plasmids that can be recovered for mutation analysis. However, with an increased contribution of the C57BL/6J background, mortality of homozygous dwarf mice between weaning and adulthood was found to rise appreciably. Although unknown to us at the time, similar results for Ames dwarf mice were obtained by Nasonkin et al. who found that a fraction of pups with a higher contribution of the C57BL/6J background display lethargy and die precipitously between weaning and adulthood (Nasonkin et al., 2004
). For this reason we decided to cross the F5 Prop1df
back to the original heterozygous dwarf animals (Prop1df/Prop1+
). This reduction in C57BL/6J background proved to be sufficient to reduce the early mortality to a normal level.
Using this strategy we established four cohorts: (1) ad lib
wild-type; (2) CR wild-type, (3) ad lib
dwarf and (4) CR dwarf. The survival of animals under all four conditions was determined in parallel (). As expected, calorie restriction extended the longevity of wild-type mice compared to that in ad lib
wild-type mice (P=0.002), and ad lib
Ames dwarf mice outlived ad lib
wild-type animals (p=6.35e-06). However, we did not observe the further increase in longevity for the Ames dwarf mice under CR as reported previously (Bartke et al., 2001
). Indeed, CR appeared to obliterate the gain in life-span of the dwarf cohort! Although we were careful to gradually calorie restrict the dwarf mice (20 % for the first month and then the 40 % reduction of food intake that is commonly applied for CR by the animal facility in San Antonio) we did see excess mortality in the CR dwarf cohort. Reasoning that 40% CR might be too severe for these fragile dwarf mice we reduced this to 30% after 19 months. However, even after censoring the cohorts for mortality before that age, we did not see any appreciable improvement of the survival curve. Indeed, the earliest age at which censoring deaths improved survival of the CR dwarf cohort was 34 months. It was only at that late age that the survival curve began to parallel the ones for the ad lib
dwarf and CR wild type cohorts (results not shown).
Survival plots of Ames dwarf (DF) and wild-type (WT) mice fed ad lib (AL) or calorie restricted (CR).
In view of the high costs associated with this type of study we limited our comparison of spontaneous mutation frequency at the lacZ
locus to two, relatively early time points. For each cohort, between 5 and 8 mice were sacrificed at 7 and 15 months of age. Tissues were frozen and DNA extracted following a schedule in which always all four conditions were handled side-by-side. For example, liver DNAs for one time point were extracted simultaneously for all four conditions. All samples were coded and mutant frequency determinations performed blindly. For mutant frequency determinations we selected liver, kidney and small intestine on the basis of the robust age-related increases previously observed in these organs (Dollé et al., 2006
; Dollé et al., 1997
; Dollé et al., 2000
Spontaneous mutant frequencies for liver, kidney and small intestine of the lacZ
-Ames dwarf hybrids at the two ages are shown in . All raw data are available from Supplementary Table 1
–Supplementary Table 5
. The lacZ
mutant frequency data were subjected to statistical analysis by comparing ratios of mutant frequencies in dwarf versus wild type by diet, CR versus ad lib
by genotype () and ad lib
dwarf versus CR wild type, CR dwarf versus combined CR wild type and ad lib
dwarf, and ad lib
wild type versus combined CR wild type and ad lib
Figure 3 Mutant frequency at the lacZ reporter locus of Ames dwarf mice and their wild-type littermate controls at 7-month old (white bars) and 15-month old (black bars) animals after caloric restriction or fed ad lib. Error bars represent 95% confidence intervals. (more ...)
Table 1 Ratio of mutant frequency observed in Ames dwarf mice to mutant frequency observed in wild-type mice by dietary condition, tissue and age; and ratio of mutant frequency observed in CR mice to mutant frequency observed in Ad lib mice by genotype, tissue (more ...)
Table 2 Ratio of mutant frequency observed in Ad lib Ames dwarf mice to mutant frequency observed in CR wild-type mice by tissue and age; ratio of mutant frequency observed in CR Ames dwarf mice to mutant frequency observed in combined CR wild-type mice and (more ...)
As expected, all three organs experienced an increase of the spontaneous mutant frequency between 7 and 15 months (although for kidney this was not statistically significant). With the exception of small intestine at 7 months, there was a significantly lower mutant frequency in the dwarf background as compared to the ad lib wild-type (), as indicated by the ratio of mutant frequencies. Of note, while it was clear that overall there was a significant protective effect of the Ames dwarf mutation against spontaneous mutations in the small intestine, 7 month-old Ames dwarf animals showed a higher mutant frequency (14.10 × 10−5) than their wild-type littermate controls (10.55 × 10−5). In our experience, the spontaneous mutant frequency at the lacZ locus in small intestine at this age level is generally around 15 × 10−5. After re-confirming the genotypes we decided to re-extract DNA from the remaining tissues and repeat the experiment. Since essentially the same results were obtained we conclude that these observations were part of the natural variation, which is not unusual for an end point such as mutation frequency. It is also not impossible that animal-to-animal variation is higher for animals in a mixed genetic background, although that was not obvious in this case (compare and ).
Mutant frequency at the lacZ reporter locus of C57BL/6J mice at 7-, 15- and 25-months of age after caloric restriction (white bars) or fed ad lib (black bars). Error bars represent 95% confidence intervals.
For the CR diet, there was a lower mutant frequency in Ames dwarf mice compared to wild-type in kidney and small intestine at 7 months and small intestine at 15 months (indicated by ratios less than 1.00; , left columns). This can also be concluded from the ratios CR versus ad lib in the dwarf. In addition to the Ames dwarf mutation, CR in this mixed background significantly reduced spontaneous mutant frequency in kidney at 7 months and small intestine at 7 and 15 months (; right columns). In wild-type mice, CR reduced mutant frequency compared to ad lib in all tissues and at both ages except for small intestine at 7 months ().
In all tissues and at both ages, there was no significant difference in mutant frequency between ad lib Ames dwarf mice and CR wild-type mice (ratios not significantly different from 1.00; , left columns). For comparison with other groups we then combined the ad lib Ames dwarf mice and CR wild-type mice. The mutant frequency in CR Ames dwarf mice was significantly lower than in the combined ad lib Ames dwarf and CR wild-type mice in kidney and small intestine at 7 months and in small intestine at 15 months. Mutant frequency was higher in ad lib wild type than in combined ad lib Ames dwarf and CR wild-type mice in all tissues at both ages except small intestine at 7 months ().
Except for the results for small intestine at 7 months, the mutant frequency results are consistent across the different tissues and the two ages. Except for CR Ames dwarf mice, the longevity and mutant frequency results overall are consistent; ad lib wild type mice have the highest mutant frequency and the poorest survival while ad lib Ames dwarf and CR wild type have similar mutant frequencies and survival.
Since the observed CR effect was not as strong as the effect of the dwarf mutation, we decided to separately test for CR in a lacZ-C57BL/6 cohort, which would allow us to compare the effects of caloric restriction alone in a homogeneous genetic background. In this approach, animals were housed under caloric restriction or ad lib conditions and samples were collected at 7, 15 and 25 months of age. Mutant frequencies at the lacZ reporter locus of C57BL/6J mice by age and dietary condition are shown in for liver and small intestine. Mutant frequency increased with age in both liver (p=0.0001, ) and small intestine (p=0.0016; ). In liver, CR mice had lower mutant frequencies than ad lib mice at all ages but this difference approached statistical significance only at 15 months (). In small intestine, the mutant frequency in calorie-restricted mice was lower than ad lib controls at every age but the difference was statistically significant only at 25 months of age (). Hence, the results from this separate cohort essentially confirmed the results obtained for CR with the Ames dwarf mutant cohort.
Ratio of mutant frequency observed in CR C57BL/6 mice to mutant frequency observed in Ad lib C57BL/6 mice by tissue and age