Princeton Weekly Bulletin, February 16, 1998
Of experimental mice and men
By JoAnn Gutin
Shortly
after neuroscientist Joe Tsien came to Princeton last August to
take up his new job as assistant professor of molecular biology,
he got a DHL delivery from Boston. The package was an ordinary
cardboard animal shipping crate -- air holes, containers of
jelly-like liquid food -- but if the shippers had taken the
value of the contents into consideration, they would have used a
blue Tiffany box.
Inside were about 40 brown mice from Tsien's old lab at MIT. The product of thousands of hours of complicated and painstaking genetic tinkering, these mice have a tiny mental defect: a flaw in their memories. At MIT, where Tsien and colleagues developed new genetic technologies to engineer them, the mice helped clarify the molecular basis of memory; at Princeton, Tsien is determined that they and their descendants will help scientists understand not only human cognition but also neurological ills such as Alzheimer's, Parkinson's and drug addiction. In fact, he predicts that researchers in nearly every field of biomedical research will engineer mice with his techniques and use them to study every sort of problem. "They'll be spreading like wildfire," he says.
Global knockout
About 10 years ago the hottest research tool in biology was the so-called "knockout" mouse: animals in which a single gene was disabled, or knocked out, in every cell of the body. This engineering technique, nicknamed the "global knockout," allowed researchers to observe what a particular gene contributed to functioning, just as omitting yeast from bread would show what it was good for.
When the mice first came on the scene, neuroscientists were as excited as other researchers at the potential of this tool. But when they tried knocking out genes suspected of being implicated in behaviors, their experimental animals would sometimes die or develop serious physical defects. And even if a behavioral effect did occur, there was no way to be sure its origin was in the brain and not the muscles, say, or the eyes. In other words, global knockout mice were a great technological leap, but for brain research they were a sledgehammer when a scalpel was needed.
At about the time the first knockout mice were being developed, Tsien was an undergraduate at East China Normal University in Shanghai with a burgeoning interest in the brain. Neuroscience was in its equivalent of the Stone Age, and researchers had almost no understanding of how the brain worked. "We knew nothing, really, about the molecular nature of proteins involved in brain function, about the neurological machinery sending signals," Tsien recalls. Determined to tackle these problems, he says, "I decided I needed a PhD in biochemistry."
Blue skies, clear water
This was easier said than done. There was no place in China to earn such a PhD, and no one to ask for advice. "In the library we had a huge book of all the graduate programs in the United States, and I just kept flipping through it," he recalls. "I didn't know anything -- I saw `Indiana,' and I thought `Oh -- maybe there are Indians there. That sounds interesting.' When I saw `Minnesota' -- the Chinese character means `blue skies and clear water' -- I thought, `Okay, that sounds good.'"
In 1986, armed with a lot of determination and very little English, Tsien got on a plane and flew to Minneapolis. The shift from tropical Shanghai to frigid Minnesota required a certain attitude adjustment. "It was very ... cold," says Tsien, now a naturalized American citizen. Then he adds, brightening, "But it proved to me that I could live anywhere."
After a few anxious months sitting in lectures that he could barely understand, Tsien got the hang of both the language and American-style academics. In four years he had his PhD, and he spent the ensuing decade honing his skills with postdoctoral positions at Columbia University with Eric Kandel, a leading memory researcher, and then at MIT, where he worked with Nobel laureate immunologist Susumu Tonegawa.
New methodology
The question Tsien had been preparing himself to tackle was one that had intrigued scientists for a century: What exactly goes on in the brain when we learn and remember?
"How is it," he says," that we can register new information and later on retrieve it? Clearly, changes are taking place; are they chemical or structural or both?" Researchers knew that a structure in the brain called the hippocampus was important in memory, and they reasoned that learning there must involve a strengthening of communication in the synapses, or gap between neurons, but they had no way of testing this idea. So, says Tsien, "When I got to MIT I decided to develop a new methodology."
Again, easier said than done. But within a few years, Tsien made a technological breakthrough: he devised a highly precise, brain-region specific knockout technology -- the neuro-scientific equivalent of the smart bomb. Armed with this advance, he was ready to test the century-old idea that changes in the brain are the basis for learning and memory.
Enter the mice
Tsien and his team developed a number of mutant mouse strains. In one strain, they introduced bits of DNA that would bracket the gene controlling the production of receptors for a particular neurotransmitter. (Decreases in the number of these receptors, many neuroscientists believed, would impair learning and memory.) Informally, Tsien calls these mice his "pencil marked" mice.
In another strain, the group introduced a gene that would cause dissolution of everything between the introduced brackets, but only in cells in restricted areas of the brain; these mice Tsien calls his "smart scissors."
A final strain of mouse is called the "clever reporters." Their DNA contains a gene that causes cells to turn blue in the presence of the dissolving enzyme secreted by the "smart scissors."
When researchers crossed the "smart scissors" with the "clever reporters" and recrossed their offspring with "pencil marked" mice, they got an animal that lacked the receptor gene in a localized region, and whose affected nerve cells were stained blue. The upshot?
"Without these receptor proteins, these mice couldn't turn up the volume in the synapses of the spatial memory area of their brains," Tsien says. The way this showed up behaviorally was that they couldn't learn mazes that depended on spatial clues for solution, though they were normal in every other respect. "Clearly, learning-induced changes in this region are crucial for memory formation," Tsien observes. "With these experiments we went from the molecules to the cells to the neural circuit to the behavior." Linking the four levels in this way "has always been a dream of neurobiology," he adds.
Inducible knockout
In genetic engineering, as in any other type, the trick is to keep improving the product. In several Cell papers last winter, Tsien reported his work, including the recipe for his forgetful mice, so researchers in labs worldwide are using this new technology and breeding similar animals to answer questions of their own. "The real challenge for me is how we at Princeton are going to maintain a lead," says Tsien. "This is something that I've been spending a lot of time thinking about."
Among the ideas he's come up with is to figure out how to knock out the receptor genes and then turn them back on, in the same animal -- the so-called "inducible knockout mouse."
"That way we can let the animal learn and then switch off the receptor to see if synaptic changes during the consolidation period affect how long memories can last. Maybe eventually we can dissect apart learning, consolidation and retrieval. There are so many experiments I can think of doing with this new technology -- this is a really exciting time to be doing brain research."
With enough time, and enough mice, the sky appears to be the limit.