Click each question to navigate down to its answer.
- Why is my colony no longer breeding?
- How can I assess the integrity and reproducibility of the genetic background of my stock?
- How can I assess the integrity and reproducibility of my Cre strain?
- How do I plan a colony? How many breeders do I need to set up?
- When should breeders be replaced?
- How do I know when to refresh my colony?
- I am interested in using a mouse strain that is not available from a commercial supplier. Where can I look to see if the strain has already been created?
- I am ordering a new mouse strain and will receive it as frozen sperm. Where can I find information on how to deal with the frozen sperm when it arrives?
- I need to breed my animals in several different litters. How do I design my experiment so I can analyse results from different litters together?
- I want to include both sexes in my study but am not specifically looking for sex differences. How can I design my experiment to use both sexes without doubling my animal numbers
For answers to more specific questions about colony management or breeding difficult strains please see our Breeding and colony management advice service.
There are several factors to consider when a colony stops breeding. Common problems are listed below.
Problem 1: Stock animals are older than normal breeding age for the strain, or matings were set up late.
Is the breeding stock older than the usual breeding age for the strain? For mice, the age at which they breed is often dependent on when they are first mated; for example, if mice are mated at eight weeks of age they may breed well until 18 weeks of age or older. However, if naive mice, who have never been mated before, are put together at 15 weeks, they may never start breeding.
- Are there male mice available that can be mated to inbred wild type females as a backcross? Often male mice have a longer breeding age and mating them with young females may generate enough pups to save the colony.*
- Alternately, if there is a risk the alleles will be lost entirely, it might be better to mate both male and female GA mice with wild type mice in order to generate some pups to re-establish the colony.*
- Sperm can be isolated from male mice. Even if they are past mating age, the sperm is often good enough for use in an in vitro fertilisation.
- Are there any sperm or embryos archived? Can these be used to rederive the strain?
*For the first two points caution should be taken when using wild type mice from a different source if the colony is usually a closed colony, as this may change the genetic background and affect the phenotype. This is not an issue for strains where the genetic background is standardised by frequent backcrossing.
Problem 2: Mice have been held as a closed colony for some time and may be subject to inbreeding depression.
If mice have been kept as a closed colony, for example where matings are always set up between mice from within the colony without crossing to wild type mice (usually from a genetically controlled inbred strain), they could be subject to inbreeding depression. This is where detrimental alleles that have occurred through spontaneous mutations (genetic drift) become homozygous because of the closed breeding scheme. Often the first problems are seen with fertility, fecundity or viability, as strains that have not been crossed frequently to a genetically controlled inbred strain may encounter reductions in litter size, general breeding ability or survival rates.
- Cross mice to a genetically controlled inbred strain. At commercial suppliers or in controlled breeding colonies, inbred mice are rederived from archived stock every five to ten generations, greatly reducing genetic drift and inbreeding depression. Backcrossing the GA strain to a genetically controlled inbred strain should reinvigorate the breeding.
- Are embryos or sperm available from a previous generation? These can be rederived to re-start the colony using gametes from before the reduction in breeding performance.
Even if this round of inbreeding depression can be ameliorated, continuing to keep the GA strain as a closed colony may lead to future inbreeding depression. To mitigate this in the future the colony should be crossed to a controlled inbred strain, using an appropriate cross design, every three to five generations.
The first step for assessing the genetic integrity should be to consult the breeding records. Are there complete records for the stock for every generation since the founder generation? If breeding has always been performed to a genetically controlled inbred strain, you should continue this strategy and screen occasional generations (every two to three) to ensure that no contamination has occurred (usually due to human error). However, if mice have been bred to colony mates or records are incomplete, you may need to assess the genetic integrity, for example by sequencing or checking for well-known SNPs that differentiate between inbred strains. Note that SNP panels may not be fully conclusive and in the case of an unknown or unclear genetic background it would be sensible to backcross onto a genetically controlled inbred strain, to ensure the strain is on a stable background for the future.
For further information on controlling genetic integrity and reproducibility of stock see 'How do I know when to refresh my colony?'.
One of the most important aspects of assessing a Cre strain is to ensure it is the exact Cre allele that is expected. It is not sufficient to genotype strains carrying Cre recombinase with a generic Cre genotyping assay. Assays should be developed that are allele-specific and able to detect the difference between different Cre strains and therefore avoid any mix ups or contamination.
As with other GA strains, it is important to check colony and production records to verify information about the background strain (see How can I assess the integrity and reproducibility of the genetic background of my stock?). In the case of an unknown or unclear genetic background, it would be sensible to backcross onto a genetically controlled inbred strain to ensure the strain is on a stable background for the future.
There is often information about the expression of the Cre in the tissue of interest, for example, if the recombinase is intended to be expressed in dopamine neurons there is often clear evidence of brain expression, even down to a cellular level. However, expression of Cre in other tissues should not be ignored, as even brief expression during development can affect the integrity of the final experiment. Reporter strains can be used that express reporter genes coding for enzymes such as lacZ or fluorescent proteins such as GFP, following exposure to Cre. Crossing these reporter strains to your Cre strain and harvesting and imaging at different time points can give a clearer picture of the full extent of Cre expression across target and non-target tissues in a particular strain. If these strains are not available, a survey of all major body tissues for the recombined allele in the genomic sequence can indicate the level of ‘leakiness’ of the Cre.
Firstly, it is important to know the breeding characteristics of your GA strain and background strain in order to make the correct decisions for colony management. You should consider the following points:
- Mice from different backgrounds do not all breed in the same way. Some mice breed well if mated at 18 weeks of age. Some will not breed at all if mated after 12 weeks of age.
- Different strains also behave differently depending on the type of mating set up. Some strains breed effectively as trios, others need to be set up in pairs.
- Neonate mortality can also vary considerably between strains, ranging from 2% in some outbred strains to more than 20% in some commonly used inbred strains.
- Litter size and general fecundity also differ between strains and should be considered for the particular GA strain in question. It is worth bearing in mind that for some GA strains, the phenotype is different in males than in females (e.g. males might be infertile, females might be aggressive).
Note that some of these characteristics are also dependent on environmental factors so may not be the same between different facilities. If data on breeding characteristics is not available for a particular GA strain, a good starting point is to use the information for the relevant inbred strain (for examples of reproductive characteristics of common inbred strains see this table from The Jackson Laboratory). However, once breeding starts, data should be captured and assumptions updated. Once these details are known about the GA strain, a breeding strategy should then be developed.
Once the breeding characteristics have been established, the breeding strategy will need to be planned based on the purpose of the colony. If a colony is being held but no mice are being produced for experiments, we recommend holding as a backcross and using the intermittent breeding strategy (see Worked Example of Intermittent Breeding), which ensures the best genetic quality using the minimum number of animals.
If breeding is required to produce experimental cohorts information on the breeding characteristics of the GA strain (as detailed above) and the experimental requirements should be used to calculate breeding numbers.
Worked example of calculating breeding numbers for an experimental cohort
The experiment requires 10 homozygous females, 10 homozygous males, 10 wild type females and 10 wild type males.
It is recommended to breed this from a heterozygous intercross. This generates the homozygotes and wild types from the same matings and removes any potentially confounding factors that could occur if the homozygotes were bred separately to the wild types (e.g. maternal and paternal effects as a result of the genotype of the parent, maternal environment, feeding and social interactions prior to weaning, handling by different staff in different rooms etc.).
To calculate the number of matings required, account for the following factors:
- The probability of achieving a genotype – this may be based on Mendelian ratios but these ratios may not always be achieved, especially within small cohorts. Furthermore, the sex ratio will on average be 50:50 but will deviate by chance, especially with small cohorts.
- Neonatal mortality – this will be dependent on (sub)strain and facility.
- Average litter size – this will be dependent on (sub)strain and facility.
- Average breeding performance – often a percentage of females will not get pregnant within the required timeframe, this will be dependent on strain and facility.
The table below details the four-step calculation for the number of matings needed to generate the required number of animals for an experiment:
1. Account for the probability of generating the required genotype.
Number of animals of the required genotype needed for the experiment
Number of pups needed to produce the required genotype*
Number of viable pups required
If 20 homozygous animals are required and 1 out of 4 pups from a heterozygous intercross are homozygous on average, then 20 x 4 = 80 viable pups are required.
2. Account for neonatal mortality.
(Number of viable pups required
% survival at weaning)
Total number of pups required to be born
If 80 viable pups are required at weaning and the neonatal mortality of the strain is 18%, then (80 pups ÷ 82%) x 100 = 97.5 pups are required to be born in total.
3. Account for average litter size
Total number of pups required to be born
Average litter size
Total number of litters required
If 97.5 pups are required to be born in total and the average litter size for the strain is 7.2 pups, then 97.5 ÷ 7.2 = 13.6 litters are required in total.
4. Account for breeding productivity
(Total number of litters required
% productive matings)
Total number of matings
If 13.6 litters are required in total and on average for the strain 90% of matings result in pregnancy then (13.6 ÷ 90) x 100 = 15.1 matings are required in total**.
*According to Mendelian ratios (e.g. 1 out of 4 pups from a heterozygous intercross should be homozygous, so the number of pups required to produce a homozygote is 4).
**The final number of matings is rounded up. Therefore, in this example 16 females would be set up in 8 trio matings or 16 paired matings. Bear in mind that about half the time this number of matings will not give the desired number of homozygotes, and will not necessarily give the same number of males and females. There should be a contingency plan to use slightly fewer animals or to extend the breeding slightly. This is more relevant when smaller are numbers being bred.
For an alternative method to calculate the number of animals required to breed a colony see the Breeding Colony Size Planning Worksheet from The Jackson Laboratory.
We recommend that matings should not be left running for a continuous supply of animals. It is best practice to minimise animal numbers, and often to optimise experimental design, to know the number of mice that are needed and calculate numbers for breeding at each stage. This is true even for colonies that are not currently being used for experiments.
There is not a generic answer to this question as it is very dependent on the inbred strain and the genetic alteration. Monitoring breeding performance should indicate when younger mice should be used.
Maintaining strains of mice can result in a quick turnover of generations and a build-up of spontaneous mutations in a strain (genetic drift). Spontaneous mutations can arise at a surprising rate and by chance some will become homozygous (The Jackson Laboratory estimates that there is one mutation with potential impact on research findings in every 10 generations [based on C57BL/6J mice separated by 69 inbred generations over 19 years of continuous breeding] see Strategies to minimise genetic drift and maximise experimental reproducibility in mouse research). These mutations can affect the phenotype and the rate of drift is higher within small research-focused colonies (because a smaller colony increases the likelihood of mutations becoming homozygous). Whilst genetic drift cannot be avoided completely it can be minimised through careful forward planning. It is recommended to keep colonies as backcrosses where possible, with wild type mice supplied from a genetically controlled colony either in house or at a commercial supplier. If this is not possible, due to having multiple alleles or complex genetics, it is recommended to refresh the strain by backcrossing it with the inbred background strain approximately every five generations. This will help keep the colony as genetically similar to the control as possible and ensure that results are reproducible. Allied to this is the necessity to keep accurate records of pedigree information and the number of inbred generations that have occurred.
I am interested in using a mouse strain that is not available from a commercial supplier. Where can I look to see if the strain has already been created?
We maintain a list of individually searchable mouse repositories. The International Mouse Strain Resource also has a useful function which searches repositories from around the world. Please note that mouse strains may not have been generated on a suitable background for your specific scientific question and/or for your planned breeding strategy, so it is important to read the full details of how the strains are created and maintained.
If repositories do not hold the strain you are looking for, try local strain sharing networks such as the Mouse Locator Network in the UK. If requesting a strain through a sharing service, it is important to ensure you have the correct permissions (e.g. a Materials Transfer Agreement) and that you have information on how the strain was created and maintained (background strain, frequency of backcrossing, etc.). We recommend you confirm your strain upon arrival including genotyping/SNP assays – for more information, see our guidance on colony management best practice.
I am ordering a new mouse strain and will receive it as frozen sperm. Where can I find information on how to deal with the frozen sperm when it arrives?
If you do not have experience receiving frozen sperm and/or embryos, consider contracting a third party to recover the strain for you. The Infrafrontier website has information on receiving and unpacking cryopreserved sperm that arrives on dry ice, which can be found in the shipment protocols section. The website also includes video tutorials, information on receiving frozen material and cryopreservation protocols, including how to freeze sperm yourself should you choose to in the future.
We would advise that you proceed with caution if it is the first time you are using these protocols. If you need assistance or guidance, please contact our advice service.
I need to breed my animals in several different litters. How do I design my experiment so I can analyse results from different litters together?
If the objective of the study is not specifically to look at litter differences, litter can be used as a blocking factor. This means setting up each litter as a mini-experiment – with some animals from each litter being used as control animals, and some receiving the intervention(s). Including litter as a blocking factor in the randomisation ensures that any variability introduced by the different litters is evenly distributed across all groups and hence ensures that any comparisons between the interventions will be unbiased and a true reflection of the effect of the intervention. Including litter as a blocking factor in the statistical analysis, reduces the variability caused by the different litters within the treatment groups, and thus increases the ability to detect a real effect of the intervention. If you are testing the effect of a single treatment, you can analyse your results using a one-way ANOVA with blocking factor.
I want to include both sexes in my study but am not specifically looking for sex differences. How can I design my experiment to use both sexes without doubling my animal numbers?
How you design an experiment to include both sexes depends on your scientific question, and what comparisons you will be making when analysing the results. It is important to make sure that the same proportion of male to female animals receive the control treatment and each intervention (ideally half and half).
One option to take account of the variability induced by sex is by including sex as a blocking factor in the randomisation and statistical analysis. Including sex as a blocking factor in the randomisation ensures that any variability introduced by the different sexes is evenly distributed across all groups, implying any treatment comparisons are not biased by any sex effects (which may be the case if the allocation is not even across sexes). Additionally, including sex as a blocking factor in the statistical analysis reduces the variability caused by the different sexes within the treatment group, and thus increases the ability to detect a real effect of the treatment. You can analyse your results using an ANOVA with blocking factor. Be aware though that this will not allow you to draw any conclusions about the effect of sex on your results (either the overall difference between males and females or whether the effect of treatment varies between sexes). Instead, you can plot the data from the sexes separately using boxplots to help you decide if you want to study sex differences in more detail in the future.
Another option is to use sex as a factor of interest and design the experiment using a factorial design. If you are not planning comparisons to test the effect of the intervention in males and in females separately (i.e. you are not planning on using within sex post-hoc comparisons), the sample size should be determined using a power calculation for a two-way ANOVA. This experiment will be able to test whether the effect of the intervention depends on sex (i.e. is there an interaction between sex and the intervention?), but will not necessarily have enough animals per group (i.e. be sensitive enough) to reliably test the effect of the intervention in females and males separately using post-hoc pairwise tests.
More information to help you decide what is most appropriate for your experiment can be found on the Experimental Design Assistant website, and if you are still unsure seek advice from a statistician.
For answers to more specific questions about colony management or breeding difficult strains, email firstname.lastname@example.org. Please include 'Breeding and colony management' in the subject line, to ensure your email reaches the correct team. Staff from the NC3Rs will anonymise your query, and forward it to a relevant expert from the breeding and colony management working group. Any advice given is the opinion of the specific expert contacted and is not intended to be exhaustive.
Your query will be treated as confidential and will be processed in accordance with current UK data protection legislation.
The information you provide will be processed by the NC3Rs solely for the purpose of answering your query. Your information will be retained in accordance with the UKRI’s retention policy.
Information on how we use personal data can be found in the UK Research and Innovation Privacy Notice.