In a previous post we explained why we had opted to work with outbred—genetically diverse—mice, narrowing down our options to either the UM-HET3 or Diversity Outbred (DO) stocks. Our first pilot involved purchasing a small batch of DO mice (20 males, 20 females) to see whether we could work with these mice in practice. Now the data is in: Our choice for Rejuvenome is the UM-HET3 stock.

When we bought the pilot DOs back in November 2021 they were very young, just a month old. DO mice tend to display more aggression as they mature so we needed to wait. We chose six months as the threshold for “sufficiently mature”, and it was only very recently when they became old enough. The DO mice were ordered from The Jackson Laboratory (JAX) who did warn us of potential aggression problems, suggesting that we should not mix different litters of mice, and be prepared to single-house particularly aggressive individuals, especially after 6 weeks of age. Additionally they recommended that we avoid the use of igloos so that mice don’t get territorial about them. Other groups recommended that we use larger than standard cages, closely monitor for aggression, and remove aggressive individuals immediately. But in a vivarium with established practices and equipment, these measures are not always pragmatic, especially for large cohorts.

We started housing DOs at a density of five per cage (these are standard size Tecniplast cages). But by six months of age, 35% (7 out of 20) of the male mice are single housed because of aggression. This is a small sample, but it matches data we obtained from other groups working with DOs. A similar proportion are housed in pairs, with the rest housed in larger groups. We expect aggression to go down with age and indeed other groups have been able to socially house more than half of their DOs by the time mice are 15 months old.

The target density for Rejuvenome is five animals per cage, which we have been able to meet for female DOs but not males. We could have chosen to study just females to work around the aggression problems, but we wanted to have a 1:1 sex split for our mice, ruling out this design. We do have to mention that there are studies only in female DOs: Nazmul Huda et al. (2020), Zhang et al. (2022) or Ouellette et al. (2022). Kurtz et al. (2020) and Chen et al. (2022) in particular single out aggression as the reason why the study includes only female DOs. Some of these studies on DOs tend to focus heavily on the genetics of aging, or the interactions between interventions and genotype. For that purpose, the diversity of the DOs is a great asset, and prioritizing genetic diversity is a reasonable choice. For us, the purpose of diversity is not so much to examine the effects of specific genetic variants on longevity or how well an individual responds to an intervention, but rather to ensure the effects we find are robust and not tied to one specific strain of mice.

We considered other factors for whether to keep a DO arm in our first cohort: having some DO data could help other groups doing work on DOs. But we expect they will also face the same difficulties we found, and so studies with large samples of aging interventions won’t be likely to use this stock, making our own DO data less useful, at least if we include males. Besides aggression, DOs also take longer to undergo anesthesia compared to B6. This means there is less time to perform assays: less of a concern for smaller experiments, but for us having enough time for our extensive phenotyping plans is of key importance.

JAX will soon have available pre-aged HET3 (26-52 week old in May 2023, and 90+ week olds in May 2024). This could help us accelerate our timelines as we will be able to purchase pre-aged mice instead of waiting until they are old enough for us to try an intervention. This is not possible at the moment for the DOs.

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