A Mightier Mouse

One of molecular biology’s mightiest tools has been the knockout mouse. Although its name conjures up images of a cartoon animal that boasts, “Here I come to save the day” before duking it out with evil characters, this mouse is no fantasy. The knockout mouse has served as a model for human diseases and shed light on the workings of the brain. Now a new generation of knockout mice developed at MIT packs an even more powerful punch.

Knockout mice are laboratory strains engineered to lack a specific gene. By studying what happens when that gene is missing, researchers gain clues to its function. Until now the clues were fuzzy, says Susumu Tonegawa, director of MIT’s Center for Learning and Memory and a Nobel laureate. Researchers could make a mouse lacking a key gene, but it would be missing throughout the mouse’s body and its lifetime-from the earliest stages of embryonic development to death. Because a gene may have different functions in different parts of the body or at different times, investigators were hard pressed to draw conclusions from such “first-generation” knockout mice about exactly where and when the missing gene would have otherwise played a role.

To zero in on a gene’s function, researchers need to knock it out only in certain locations or during a narrow window of time. That is what Tonegawa, Matthew Wilson-an assistant professor in the departments of Biology and Brain and Cognitive Sciences-and investigators at California Institute of Technology and Columbia University have done. They have created a new kind of knockout mouse in which a specific gene is deleted only in certain cells in the brain and only after neural circuitry has developed. The researchers have worked with a gene thought to affect the development of spatial memory, the mechanism by which we navigate familiar environments.

To create the new strain, the scientists attached an “on-off switch”-a stretch of DNA called a site-specific promoter because it controls activity in particular types of cells-to a molecular device that chops out genes. German researchers had used a similar approach to create a mouse strain lacking a gene in immune-system cells, but applying the technique to the brain was especially tricky, says Tonegawa. His group knew the gene chopper would work in actively dividing cells such as immune cells, but wasn’t sure about brain cells, which stop dividing around the time of birth. The researchers also weren’t sure that the on-off switch would restrict gene deletion as precisely as they wanted. Although subregions of the brain have quite different functions, the anatomy and organization of brain cells are virtually identical from one subregion to another. The team feared the promoter wouldn’t be able to distinguish cells in a specific subregion.

Still, they tried the technique-and fortunately, says Tonegawa, “nature was kind.” The system worked in the brain, and moreover, because the promoter happens to be inactive during development, it worked only after the neurons had fully formed. In a series of papers published late last year in the journal Cell, the scientists revealed that their “regional gene knockout technology” allowed them to determine which molecule is responsible for strengthening the neural connections that lead to spatial memory, and where that molecule exerts its effect. Stopping the gene from producing the particular molecule after neural development resulted in mice that, unlike normal mice, could not learn how to get out of a maze after being placed in it day after day. To confirm that the brains of the knockout mice were not undergoing the synaptic strengthening necessary to form mental maps of the maze, the researchers implanted tiny electrodes in the animals’ brains and recorded activity from the neurons where the gene had been knocked out. By contrast, “first-generation” knockout mice previously developed to study spatial memory had died within a day or two, before researchers could study them. Apparently the gene was critical to survival early on.

The Cell papers created a stir among neuroscientists, who immediately saw ways to use regional and time-specific gene knockouts to explore other problems, including neurological disorders. Parkinson’s disease, for example, results from the death of cells in a part of the brain called the substantia nigra. These cells normally release the neurotransmitter dopamine, so researchers have assumed that the disease results from the lack of dopamine, explains Zach Hall, director of the National Institute for Neurological Disorders and Stroke. Giving Parkinson’s patients L-dopa, a dopamine precursor, lessens their symptoms, but only partly and temporarily. Maybe, says Hall, that’s because substantia nigra cells supply not just dopamine but some other essential but unidentified product. Comparing the effect of knocking out just the genes that make dopamine at a particular time with removing the substantia nigra cells altogether could help pinpoint whether one or more other products play a role, he explains.

Applying the Technique Elsewhere

One neuroscientist eager to apply the new technique is Michael Stryker of the University of California, San Francisco. Stryker wants to understand the molecular mechanisms that underlie development of the visual cortex-the part of the brain that receives and processes input from the eyes. Work in his lab and others has shown that connections among neurons in the visual cortex form rather imprecisely at first. During normal development, the connections reorganize into the specific pattern necessary for normal vision-a process referred to as visual cortical plasticity. If that doesn’t happen, the result can be diminished vision, often to the point of blindness.

Stryker, Alcino Silva, a senior staff investigator at Cold Spring Harbor Laboratory in New York, and their colleagues have previously experimented with first-generation knockout mice in an attempt to pinpoint what happens at the molecular level when connections in the visual cortex reorganize. But the results have been inconclusive. Stryker hopes that refining his experiments using regional and temporal gene knockout mice could better test his group’s theory that connections in the visual cortex do not reorganize properly unless a particular molecule is present at a specific time in development.

Applying the new knockout technology to other parts of the brain such as the visual cortex will depend on the discovery of additional DNA sequences that turn genes on or off in particular regions. Toward this end, investigators can create many strains of transgenic mice with the same on-off switch. By chance, in each strain the switch tends to incorporate into a different DNA site. The intrinsic property of the switch and the DNA site where it integrates, in turn, dictate the part of the brain where the switch is active. The researchers are then picking out for further research those strains in which the switches are working in very specific regions of the brain.

The Tonegawa team is also trying to more precisely control the timing of knocking out genes. One way of doing this is by injecting a compound that triggers gene deletion. The experimenter can control the timing of the deletion by injecting the compound at a specific stage of development.

According to Stryker, this kind of work may eventually help researchers resolve a question of fundamental interest to neuroscientists: discovering “whether the mechanisms responsible for learning and memory are really just the same ones that are used in development.” Scientists know much more about the molecular mechanisms that underlie general development than they do about the mechanisms responsible for learning and memory. If similar processes are involved, many of the mysteries of the brain will be made clear rapidly. Moreover, says Wilson, research that identifies particular molecules at work in memory could eventually lead to techniques for reducing memory loss or even for improving normal memory.