«Mendel’s laws of inheritance cannot be overridden»

Research institutions keep more animals than they use in experiments. Daniel Breustedt, Team Leader in the Scientific Operations/Comparative Medicine department at Novartis’ NIBR, explains why this cannot be completely avoided and what options are available for reducing animal use.

Interview with Dr. Daniel Breutstedt

Part of the process of developing new and innovative treatments involves testing the APIs in animals. This is to test their efficacy and tolerability in preclinical research. But more animals are bred than are actually needed. Why?

There are various reasons. The most important is ensuring the scientific reliability of the research involving animals. Very often, particular research issues require animals with particular genetic features. As part of the process of breeding animals with these specific features, other animals are inevitably born.

You talk about genetic features. What role do the laws of inheritance play in the fact that more animals are born than are actually used in the experiment?

Breeding genetically modified lines – and that’s what I’m talking about here – is subject to the laws of biology. Gregor Mendel used peas to find out how inheritance works as long ago as the second half of the 19th century. And Mendel’s laws apply to humans, mice and essentially all living things. They cannot be overridden. That’s why animals that do not have the genetic features needed for a particular experiment are inevitably also born.

Can you give an example to illustrate that?

Even if we’re breeding a mouse line with only one genetic modification, for example, we get animals that don’t have the feature we want as well as ones that do. That’s pretty much prescribed by Mendel’s laws . However, the biological processes we’re investigating are becoming more complex and that means the animal and mouse models we use are becoming more complex too. Today around 50 percent of our animals have one genetic modification, around 30 percent have two and about 20 percent have three or more.

Let me give you an example to explain. Let’s assume that we have to combine three genetic features – A, B and C – in one mouse line for an experiment. Feature A is a genetic on/off switch and is found in mouse line A. Feature B, in mouse line B, is a target gene that can be disabled and is associated with a disease, for example. Feature C in mouse line C is a fluorescent dye that can be activated. Breeding the ABC mouse line needed for the experiment is a complex task because it is impossible to combine all three features in one breeding step. We can only ever pair a male mouse with feature A with a female mouse with feature B. Their offspring will then have features A and B. The next step is then to pair an AB mouse with a mouse with feature C. This multiple-stage breeding process means that more animals are born than are used in the experiment.

What happens to the animals that are not used in an experiment?

Surplus animals from complex breeding processes are used as sentinels, for example. That means they are used to monitor the health of research colonies. Sentinels live in their own cages and have regular contact with other animals’ bedding and/or used cage components.That means that pathogens may potentially be present.

A vet examines the sentinels regularly, which gives us a way of periodically checking the colony’s hygiene status. Apart from that, we also offer the animals that have not acquired the genetic modification required for the experiment to our researchers for other research or training purposes – say if a new surgical method such as inserting a microchip needs to be learnt.

Are there ways of minimizing the number of animals you breed and is that something you’re working towards?

That’s our ambition. Careful planning and smart breeding strategies can decrease the number of animals both in simple cases with just one genetic modification and in more complex cases too. We’ve centralised animal breeding for that purpose. Today, experienced breeding and genetics specialists are the link between researchers and animal carers. Their task is to plan, review and implement complicated breeding strategies. Implement means having the necessary number of animals of the appropriate age and with the correct genotype – the specific genetic features, in other words – available for a specific experiment at a specific point in time. Using smart breeding schemes, we can reduce the number of animals that are bred quite substantially. However, it is still important to understand that we can never completely avoid surplus animals because of the biological laws at play.

The right number of appropriate animals. That sounds immensely challenging. What’s the reason for this level of precision?

It’s connected to the statistical robustness of the experiment. Quite simply, biological processes produce variety even in animals that are genetically identical. That means that two identical animals do not have to behave exactly the same in an experiment. To obtain robust and meaningful results, there is therefore a statistically necessary number of animals, which is accurately determined by a biostatistician.

Why is the animals’ age so important?

Their age range is relevant because the effect that we want to measure – what we call the phenotype – develops and expresses itself over time. That’s why it’s important to have a comparable age group. If an eight-week-old animal behaves differently from a twelve-week-old one, the comparability of the data is limited and the robustness and reliability of the experiment is not guaranteed. I would like to use two examples to illustrate a second factor connected with the animals’ age. Alzheimer’s is a disease that tends to manifest at an advanced age. Accordingly, transgenic mouse models use older mice in this case. By contrast, immunological research uses younger animals because their immune systems work and respond better than those of older animals.

How relevant is the animals’ homogeneity to the quality and robustness of the experiments?

We generally aim for the greatest possible group homogeneity. In biological and medical research in particular, some of the changes we have to observe and measure in the processes we investigate are tiny. To be able to do that, we have to reduce the amount of “background noise”. That means making sure that both the intrinsic factors – in other words homogeneity – and extrinsic factors are constant. The extrinsic factors include, for example, the way the animals are handled by their carers, the temperature in the animal facility, ambient noise and vibrations, the cycle of lightness and darkness and the smell. The intrinsic factors, by which I mean what we call homogeneity within the animals, depend on factors such as the animals’ age.

Furthermore, there are some experiments where we have to look closely at the animals’ family tree because the animals used in the experiment and the animals used as controls have to be siblings from the same litter. The animals’ intestinal flora can also be a factor. For example, two identical animals or two animals from an identical line but from two different institutions normally have slightly different intestinal flora and this can be detected in some experiments.

What technologies can you employ to reduce the number of animals used in research?

We have animals in conservation breeding that are used to continue a particular strain because researchers do not want to lose animals with genetic modifications or other features. One reduction option here is cryoconservation. This involves freezing embryos in liquid nitrogen at a temperature below minus 195 degrees Celsius. If the strain has to be started afresh at some point, the embryos can beinserted into a surrogate mother. There is also the relatively new technology ofCRISP/Cas9. This enables us to edit genes so that we can give animals new genetic modifications faster and more accurately. Doing so skips a few breeding steps or enables us to insert several desired properties into the genome of the original animals in a mouse line at once. Like any new technology CRISP/Cas9 also presents new challenges, and we must keep a close eye on these if we intend to take an open approach to this new technology. The key thing for me is reducing the number of animal experiments by adopting smart breeding schemes and holding regular dialogue on the subject with all stakeholders, including other institutes, so that we can keep research animal breeding as efficient as possible.