All mice are the same, until they're not

When most people think of the quintessential lab mouse, they think of a little white mouse with red eyes. Soft fur. A timid nature. But scientists think of something very different. This mouse is black, small and fast, with pink ears and a pinkish tail. It’s got black eyes to match. The fur may be soft, but the temper sure isn’t. This is the C57 Black 6 mouse.

Each Black 6 mouse should be almost identical to every other Black 6 mouse. They have been bred to their own siblings for hundreds of generations, so there should be very few genetic differences left. But even supposedly identical mouse strains have their differences. These take the form of mutations in single DNA base pairs that accumulate in different populations. Recently, researchers showed that one of these tiny changes in a single gene was enough to produce a huge difference in how two groups of Black 6 mice respond to drugs. And the authors identified a surprising number of other small DNA differences still waiting to be explored.

On one level, the new work offers scientists a novel tool for identifying genes that could relate to behaviors. But it also serves as a warning. “Identical” mouse populations aren’t as alike as many scientists had assumed.

The Black 6, the most common lab mouse in the United States, is used for everything from drug abuse studies to cancer research. The Black 6 is also the reference strain for the Mouse Genome Sequencing Consortium. Whenever scientists discover a new genetic change in a mouse strain, they compare it first against the Black 6. And it’s the mouse used by the International Knockout Mouse Consortium (now the International Mouse Phenotyping Consortium), which keep a library of mouse embryos with different deleted genes. The Allen Brain Atlas, a database of neuroanatomy and gene activity throughout the mouse brain, relies on the Black 6 as well.  

The Black 6 has grown so prominent partly because of its long history. The lab mouse, officially known as the C57BL/6 strain, was established in 1921 at the Jackson Laboratory in Bar Harbor, Maine (where its codename is the C57BL/6J, 6J for Jackson). Originally purchased from a farm and then carefully bred brother to sister to establish a mouse line of nearly identical mice (excepting the random mutations that happen to us all), the 6J became one of the original mouse lines for sale to laboratories, and they’ve kept a line up and running since 1948. In 1951, the National Institutes of Health took a subcolony of the 6J from Jackson and started up its own colony, soon called the C57BL/6N (6N for NIH — get the naming convention yet?). Charles River, another big mouse distribution hub, got some of the mice from NIH in 1974. And on and on. There may now be as many as 50 “substrains” of the C57BL/6. But no one particular substrain is the standard. The 6J is featured in the Allen Brain Atlas and the Mouse Genome Sequencing Consortium. The 6N is the mouse for the International Knockout Mouse Consortium.

Bred from the same stock, the 6J and the 6N should be the same mouse. To make sure, Joseph Takahashi of the University of Texas Southwestern Medical Center in Dallas and colleagues ran behavioral screens on the 6J and the 6N. All seemed normal until the team ran a test to look at how much animals run around in response to cocaine. The 6J mouse had twice the stimulated response to cocaine that the 6N did, a major difference in behavior. The result surprised Takahashi because the mice substrains are thought to be too closely related for such a difference. The populations diverged only 62 years ago. Obviously, something had changed.

To investigate any genetic differences that may underlie the behavioral difference, the researchers bred the 6J and the 6N together for two generations. The second generation had an intermediate response to cocaine halfway in between the 6J and the 6N. The difference came down to a single DNA base pair in a single gene called Cyfip2. A mutation in the gene meant that the 6N had an unstable form of the Cyfip2 protein, and thus a decreased stimulant response to cocaine. The group published the findings in the December 20 Science.

Cyfip2 is particularly important in the formation of dendritic spines, tiny appendages on the bodies of neurons that receive inputs from other cells. Fewer spines formed in the 6N mice with the mutation. The 6N mice also produced fewer electrical signals in the nucleus accumbens area of the brain, a region associated with drug reward.

This small difference has big implications. “What’s surprising,” Takahashi says, “is that it is a strain derived from another strain and only divergent by 62 years.” There are many inbred mouse strains out there — black, brown, white, naked and everything in between — and scientists are very used to seeing differences between them, from basic behaviors to drug responses. But the 6J and 6N are supposed to be the same strain. Takahashi noted that scientists are careful with different mouse strains, but “people who are not card-carrying mouse geneticists might think a Black 6 is the same no matter where it comes from. People would like to think they are the same, but they’re not.”

The study could make people look more closely at their lab mice. The 6N substrain appears to have acquired its mutation in Cyfip2 before the mice went from the NIH to Charles River labs, for example, so the Charles River Black 6 mice probably have it as well, as does every other substrain derived from the NIH group.

The research also raises some big questions about what other differences the 6N might have hiding beneath that furry coat. Takahashi’s group identified 100 other mutations in addition to the one in Cyfip2. Cyfip2 itself plays a fundamental role in how neurons join to each other. It could mediate functional changes in many other areas of the brain. Takahashi’s group has plans to look at the other mutations, as well as what additional effects might arise from the changes in Cyfip2.

On the bright side, comparisons of 6J and the 6N could also provide a new way to study mouse behaviors. Because the two substrains are so closely related, the number of gene differences is extremely small (yes, in the world of genetics, just 100 changes is a vanishingly small number). Many scientists compare mouse strains, but when they do, they have many, many more genetic changes to deal with. With just a few changes between substrains, comparing behaviors could help identify how specific changes in genes affect behavior. Scientists aren’t just limited to the 6J and the 6N. Many other substrains exist that could reveal genes associated with specific behaviors.

Still, going forward, scientists might need to consider not just what mouse strain they use, but what mouse substrain. It may no longer be so easy to classify studies together because they used a Black 6 mouse. Did they use a 6N or a 6J? Or another Black 6 all together? Going back to the original 6J might seem to offer a simple answer, but a lot of work already has been done on the 6N, Takahashi notes. “There’s no going back,” he says.

The take-away here? One tiny gene change can make for a “strained” relationship.