Tag: animal research

Sanger Science

Isolating cause and effect with sensitive mice

10 December 2014
By Natasha Karp

A study assessing the sensitivity of different experiment designs. B1: One Batch, B2: Two Batch, B3: Three Batch, MG: MultiBatch and R: Random. The power of the experiment is significantly increased when the experiment is organising into multiple batches. Credit: DOI: 10.1371/journal.pone.0111239

A study assessing the sensitivity of different experiment designs. B1: One Batch, B2: Two Batch, B3: Three Batch, MG: MultiBatch and R: Random.
The power of the experiment is significantly increased when the experiment is organising into multiple batches.
Credit: DOI: 10.1371/journal.pone.0111239

It’s tricky designing experiments to make sure you have isolated the particular thing you are testing so you make the right conclusion. Working with mice adds another dimension. Mice are responsive creatures, their survival requires that they constantly adapt to their environment. Often it’s difficult to know if the differences you are seeing in your experiment are interesting results or due to subtle variations in the environment the mice live in.

You could try the classic design where you standardise everything and only allow the therapy you are testing to vary. However, this introduces an artificial situation where the variation is so low, that you can see a treatment effect but it can be very specific to that environment [see references 1 and 2]. You could measure everything that varies and include that in the mathematic model. This becomes too complex and there isn’t enough data to fit. You need a design that encompasses variation and an associated mathematical model that works well enough to reliably detect the effects.

Studies involving mice are critical to the research effort to understand and treat human disease. With animals we have an ethical responsibility to ensure the experiments are reliable to ensure the use of the animals is appropriate and reduce future use of animals.

At the Wellcome Trust Sanger Institute, we are working to understand how genes function in the body by systematically recording the characteristics (phenotypes) of knockout animals where a gene has been switched off. The mice are characterised via a phenotyping pipeline and this includes things such as bone density or cholesterol levels.

This is a large scale project with an international consortium that has ambitions to knockout all known genes. This means we have a lot of control data, but due to operational constraints we don’t have controls collected on every day we have mice phenotyped. Even with our highly standardised environment, whether a behavioural or physiological screen, we can see the environment leading to fluctuations in the measurements.

My research focuses on how to design the experiments and analyse the data to answer the question being asked. We have developed a new approach to analysing the data [see reference 3] and then investigated when the method is reliable [see reference 4]. We have found that we can improve the accuracy of our experiments by phenotyping mice for a knockout line in multiple batches (where a batch is data collected for a day). By phenotyping smaller groups of mice on different days, we can separate fluctuation from environmental variation from the treatment effect and we can have a higher confident that a differences will be reproducible.

It is critical for these projects to unravel how to efficiently design the experiment and analyse the data. The findings from these large scale studies can also inform all animal experiments by reminding us of the critical things to manage and think about to ensure the experiments deliver.

Natasha Karp is a senior biostatistician who supports the International Mouse Phenotyping Consortium.

References

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Credit: Wellcome Library, London
Sanger Science

Odour, ardour and anger

04 September 2014
By Ximena Ibarra-Soria

Odorants and pheromones that provide information about the context and instruct how a mouse should react in a particular situation. Credit: Wellcome-Library, London

Odorants and pheromones that provide information about the context and instruct how a mouse should react in a particular situation. Credit: Wellcome-Library, London

Males and females behave very differently, especially when it comes to mice. For example, if a female and a male find each other, they will show courtship behaviours and will try to mate; but if it is a male that comes across another male, they will fight and try to establish the territory as their own.

So, how is it that males and females can react so differently to the same situation?

Mice, and many other animals, use their sense of smell to identify other animals around them. It is odorants and pheromones that provide information about the context and instruct how a mouse should react in a particular situation. These are detected by receptor genes present in neurons that are in the nose.

Receptors work like a lock-and-key: when an odorant matches the shape of the receptor, it can bind and activate that neuron. Active neurons then send a signal to the brain where the animal interprets the meaning of the smell and instructs the appropriate behaviour. In order to be able to detect the trillions of odours that exist, the mouse genome contains more than 1,500 receptor genes.

These genes are present in the genomes of both male and female mice, so I wanted to understand if the great differences in behaviour could be explained by how the receptors are turned on, or expressed, in their noses. Surprisingly, I found that they all look almost identical. This suggests that the different responses between the sexes are not provoked by their capacity to detect distinct smells. Instead, the difference could be encoded in how the detected odour signals are processed in the brain.

The methodology used for this study was RNAseq, which is a very powerful technology that allows probing all the genes in the genome in a single experiment. This means that we can get a global picture of the tissue of interest, and analyse every gene that is turned on. Despite not finding any sexual differences in the expression of the receptors, being able to study the more than 1,500 receptor genes simultaneously revealed some interesting things. I found that a few unusual receptors are turned on at much higher levels than others. I am now working on understanding what it means to have so much more expression of a specific receptor gene, and if this impacts our sensitivity to certain smells.

Interestingly, when I compared the expression of the receptor genes between different animals, I found an identical pattern: the highly expressed receptors are always the same. In this case, all the animals tested have the same genome, and this may explain why the expression is conserved across them.

I am now studying whether changes in the genome or in the environment can alter the expression of these genes. All this information together will help us better understand how our sense of smell works, and why animals (and eventually humans) sense and react to the world differently.

Ximena Ibarra-Soria is currently a PhD student in the Genetics of Behaviour group where she works under the supervision of Darren Logan on understanding the expression of olfactory receptor genes and their relationship to perception and behaviour

References

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Sanger Science

Is it as plain as the nose on one’s face?

Bronze statue of Constantine I, in The Capitoline Museums, Rome. Credit: Darren Logan

13 September 2012

Written by Darren Logan

Have you ever considered whether we all sense the world the same way? My lab at the Sanger Institute studies how our genes influence how we perceive the environment around us, and how our brains makes sense of our senses.

When it comes to studying sensory genes, the nose is a veritable goldmine. We have many hundreds of genes that each encodes a unique chemical receptor. It is these receptors, found only in the nerves of our nose, which capture the rich blend of chemical odorants that diffuse up from our morning coffee. When an odorant is the right shape to slot into its matching receptor, much like a lock and key, a signal is transmitted along that nerve to the brain. It is the combination of these many signals that our brains interpret as that rich coffee flavour.

Some animals, like mice or dogs, are super-smellers and have over one thousand of these receptor genes. They use some of the additional ones to detect special types of body odours, called pheromones.  Since mice can’t talk and do most of their socializing in the dark of night, they use pheromones to identify and communicate with each other: social networking with smells, if you like.

Inquisitive young male mice are very attracted to the pheromones of a receptive female, for example, but avoid those from a dominant male. Because we can easily observe the natural behaviour of mice in response to pheromones, they make a great model for studying the receptors and the genes that encode them.

Just like humans, not all mice behave the same way. Some strains are more aggressive and others tame, some are much better at mating and others are superior parents.  My lab wanted to know whether differences in their pheromone receptor genes might be a reason for these behavioural differences. After all, if a mouse does not interpret a social signal properly, it will not be able to respond correctly.

We searched for copies of these receptors in the genomes of 17 different strains of mice, comparing over 6000 genes in total. We found that they were unusually variable, over two times more than the average gene. All strains were completely lacking some receptors and a few had gained extra ones. No two strains were alike. Though among this diversity there were a few receptor genes that seemed to be particularly important, as they were completely unchanged in almost all strains. We think these may perceive critical social signals necessary for survival and are now working to find out exactly what behaviours they influence.

We recently published this work in BMC Genomics and we think the implications are rather remarkable: each mouse clearly has a very different capacity to perceive social signals. Like mice, do you and I also perceive smells differently? While we were studying mouse receptors, some colleagues in Israel were looking into human odorant receptor genes. The results were very similar: they report that (unless we are related) our receptor inventories probably differ by a third.  So as Juliet told Romeo, “A rose by any other name would smell as sweet.” But we now know a rose, by any other nose, does not.

Darren Logan joined the Wellcome Trust Sanger Institute faculty in 2010 in the Mouse and Zebrafish Programme more…

Review Articles:

Elizabeth H Wynn, Gabriela Sánchez-Andrade, Keren J Carss and Darren W Logan ‘Genomic variation in the vomeronasal receptor gene repertoires of inbred mice’

BMC Genomics 2012, 13:415 doi:10.1186/1471-2164-13-415

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Laboratory Mouse
Sanger Science

Handle with care: animal husbandry’s virtuous circle

3rd September 2012

Written by Jacqui White, leader of the Mouse Genetics Project Phenotyping Team.

Mice at the Wellcome Trust Sanger Institute Research Support Facility where researchers are committed to treating the animals in the most humane, caring and sensitive way possible.
Credit: Genome Research Ltd

Animal research is a vital way to explore the effects of genes on human health and disease. By studying how the genes work in an animal such as a mouse or zebrafish, we can understand how it works in people and, hopefully, find new avenues for treatment or even cure.

The research support facility is a modern animal facility, operated by our dedicated team of animal technicians, supporting the daily husbandry of animals in research. We are committed to treating our animals in the most humane, caring and respectful way possible. We seek to study the fewest animals we can and to ensure that their conditions and experiences are as comfortable as possible. This approach, known as the 3Rs – replacement, reduction, refinement – not only has the best interests of the animals at heart, it also improves the accuracy and reliability of our study results. The more accurate the results, the less likely it is that we will need to repeat a study.

We are always looking for ways to refine our animal care and are constantly reviewing how we interact with our animals. All forms of animal care (feeding, cleaning and measuring) have some effect on the animals but, until now, nobody knew how much these standard practices affect study results. To find out, we studied the effects of mouse handling techniques (such as moving and cleaning cages, and measuring core temperature and blood glucose levels) on levels of short-term and medium-term stress in mice. What we discovered is already helping us to improve our animal welfare and our research results, meaning that fewer mice need to be studied.

We found that simple animal husbandry and experimental procedures significantly influence mouse physiology and behaviour. Blood pressure, heart rate, movement, core temperature and blood glucose in mice were all elevated in response to being handled. Also, these responses were different between the sexes: for example female mice displayed more sustained cardiovascular responses and movement than males.

The results will help the scientific community to refine their practices and are helping us to design studies that are both more accurate and require fewer mice. By reducing stress as much as possible, we are improving animals’ welfare, minimising disturbance and improving the quality of our results by reducing biases.

For example, one of the key changes we have made is adjusting how often (and when) we interact with the mice. Mice become used to the smell of their bedding and they find being given clean bedding quite stressful. In fact, it can take up to two hours for them to completely acclimatise. Now that we know this, we always give our mice at least two hours to get used to any change before we take any measurements to avoid bias due to stress. Second, we schedule bedding changes to take place at the same time as taking measurements so that our mice are disturbed just once instead of twice. Third, we now only change bedding once every fortnight, instead of our previous practice of once a week, halving the level of disturbance.

Our discoveries are helping us to improve the accuracy of our findings and continue to lower the number of animals required for each study. We hope that our work will be taken on by other research institutions to help them continue the good work of the 3Rs.

Jacqui White is a principal scientific manager and leader of the Mouse Genetics Project Phenotyping Team at the Wellcome Trust Sanger Institute. Her research interests include more…

Review Article

Anna-Karin et al. Experimental and husbandry procedures as potential modifiers of the results of phenotyping tests. Physiology & Behavior Vol 106, Issue 5, 16 July 2012, Pages 602-611 http://www.sciencedirect.com/science/article/pii/S003193841200131X

Related Links

Mouse and Zebrafish Genetics