Maybe it’s not you, maybe it’s your mice!
Have you found yourself unable to reproduce previous experiments with your mice? Have you spent hours scrutinizing the tiniest details of your protocol trying to determine what went wrong? Have you looked into the health and genetic integrity of your mice? It’s very easy to underestimate the impact that the health status and genetic quality can have on the phenotype(s) of your mice.
Health Status is not just about “sick” mice
Pathogens are infectious agents that can cause overt disease (sick mice) or alter phenotypes and biologic responses during experimentation. If your mice are infected with a pathogen, then you might notice problems with the animals’ phenotypes. Some of the common symptoms of infected mice include hunched posture, ungroomed and “ruffled” fur, squinted eyes, reduced movement, and loss of body weight. If your mice look sick, be sure to inform and consult with your veterinary staff and to take appropriate measures.
When monitoring the health status of your mouse colonies, you need to be aware of not just the presence of harmful mouse pathogens, but also of opportunistic organisms. Opportunists are infectious agents that are not normally pathogenic, but may cause disease or alter biologic responses under certain circumstances. Immune phenotypes can be particularly sensitive. For example, health status has a strong influence on the susceptibility to and onset of Type 1 Diabetes in the NOD/ShiLtJ (001976) inbred mice. When the immune system of NOD/ShiLtJ mice is activated, even by an opportunistic organism, the onset of diabetes can be significantly delayed. In contrast, Il10 and Il2 knockout mice, which are commonly used mouse models for Inflammatory Bowel Disease (IBD) and ulcerative colitis, require conventional housing conditions with the presence of a Helicobacter-positive enteric flora to exhibit the disease phenotypes. When housed in germ-free or specific pathogen-free conditions, the mice develop a less severe or even absent IBD phenotype, depending on their genetic background.
Therefore, be sure that you read the literature to familiarize yourself with any known effects of health status on strains’ phenotypes. You also need to monitor the health reports for your mouse facilities, to pay attention to any changes as soon as they are reported, and, if necessary, take appropriate action before your colonies are significantly affected.
Genetic Differences Influence Phenotype
There are many, many examples of how genetic changes can lead to phenotypic changes for mutant and transgenic mouse models. Mice that are homozygous for the Leprdb mutation (a model for obesity and diabetes) on the C57BLKS/J genetic background are obese, have an uncontrolled rise in blood sugar, show progressive loss of the insulin-producing beta-cells, and die early. In contrast homozygotes for the same mutation on the C57BL/6J genetic background are obese, but are only transiently hyperglycemic, and live a normal life span.
Another great example is the considerable variation observed in mammary tumor latency for the MMTV-PyVT transgenic mice on 28 different F1 hybrid backgrounds. Depending on the background, the average tumor latency ranges from as short as 38 days to as long as 85 days. These are just two examples; there are countless more.
The two biggest threats to the genetic integrity of your mice can be compromised by either genetic contamination or genetic drift. Genetic contamination is the accidental introduction of alleles from one mouse strain into the genome of a second strain via a breeding error. It typically occurs through human error when setting up breeding cages, and occurs very quickly – just a single cross can mess up a strain’s background. 129X1/SvJ (000691) and C57BLKS/J (000662) are two classic examples of accidental genetic contamination in inbred colonies giving rise to new inbred strains. (Note: the contaminations occurred prior to arriving at JAX).
The Jackson Laboratory imports more than 500 new mutant and transgenic mouse strains every year. We perform Genetic Quality Control testing on these new strains to confirm their genetic backgrounds, and have found that approximately 1/8 (12.5%) of these strains have genetic backgrounds that don’t agreed with the background information provided by the donating investigators. Clearly, this is a much more common problem than you might think. Just imagine a situation where you get a mouse strain from another laboratory, and are told that they are on a C57BL/6 genetic background. You set up your experiments and use C57BL/6 as your controls. But what if the mice are not on C57BL/6? Then your controls are not appropriate, and you are in danger of misinterpreting your data!
Fortunately, genetic contamination is easy to control. The following steps can assist you in reducing, if not completely eliminating, genetic contamination risks.
- Maintain clear and comprehensive colony records
- Prevent unintended inter-strain breeding
- Watch for changes in phenotype
- Verify periodically the genetic background of all strains by Genome Scanning
Genetic drift is “the constant tendency of genes to evolve even in the absence of selective forces. Genetic drift is fueled by spontaneous mutations that disappear or become fixed in a population at random.” (Lee Silver, “Mouse Genetics” Oxford University Press, 1995). In simpler terms, spontaneous mutations constantly arise in every mouse colony. Some of these mutations may spread throughout your colony until all the mice are homozygous for them. Although many, maybe even most, spontaneous mutations are benign, some may affect the phenotypes of your mice.
Unfortunately, genetic drift is a reality that anyone working with mouse models – or other animal models, for that matter - must accept. Fortunately, if you take the proper steps, you can greatly reduce genetic drift’s impact:
- As mentioned above, maintain clear and comprehensive colony records and watch for any phenotypic deviations (including changes in breeding performance).
- Refresh your colony every 5-10 generations. If your strain was originally obtained from a vendor, order new breeders from that same vendor. The Jackson Laboratory uses a unique, patented Genetic Stability Program (GSP) for our most popular strains, like C57BL/6J, that effectively limits cumulative genetic drift. If you are using one of these GSP strains, then by ordering new breeders, you can take advantage of our efforts to limit genetic drift. The way to refresh a mutant or transgenic mouse strain that has been backcrossed onto an inbred background, is to backcross your mice to that same inbred strain every 5-10 generations. This ensures that your mice stay as genetically similar to your controls as possible.
- Cryopreserve any mouse strain that is critical to your research program that either you created yourself, or that may be difficult to reacquire. Cryopreservation protects your mouse strains and your research programs from disasters such as genetic contamination and genetic drift. If a breeding error, genetic drift, loss of phenotype, or a pathogen outbreak hits your colony, AND you have the strain cryopreserved, you can recover the original strain and continue your studies with minimal lost time.