Little mouse, big science

Sitting in a standard-issue clear plastic cage among hundreds of other white laboratory mice, the piggyBac mouse looks absolutely ordinary, not a bit like an animal poised to turn human genetics research on its head. But when Tian Xu, Ph.D., professor of genetics, switches on an ultraviolet lamp, the mouse emits a faint pink glow—an aura that holds the secret of its transformative power. If Xu, professor and vice chair of genetics and the mouse’s inventor, has his way, he will breed up to a million more pink animals. Those animals, he says, will reveal the causes—and in some cases the cures—for myriad human diseases.

What is piggyBac’s secret? Xu knows, because he engineered the mouse to carry a small piece of moth DNA, a “jumping gene” called the piggyBac transposon, which in turn carries a genetic marker to turn the mouse pink for easy identification. The jumping gene makes the mouse a mutant factory: when the animal breeds, the transposon causes random genetic mutations in the mouse’s offspring—one gene per mouse is disabled. Compared to current methods for making experimental mice, known as knockouts, using piggyBac is 100 times quicker and cheaper.

Xu eventually plans to produce 100,000 new strains of mice with missing genes. Among them, he expects to find a knockout for the majority of the estimated 25,000 to 30,000 genes in the mouse genome. More than 99 percent of mouse genes have direct equivalents in humans, and so these mice will provide the first glimpse of the functions of many of our genes, most of which remain mysterious half a decade after the human genome project first cataloged their existence.

From China to Yale

Tian Xu came to New York City from China in 1983, a 21-year-old refugee from the Cultural Revolution pursuing graduate work in genetics at City College. After six months of living on a partial teaching assistant’s stipend in an abandoned building in Harlem, he received an offer he couldn’t refuse: a full scholarship to Yale. He was soon in New Haven, studying the development of the fruit fly Drosophila melanogaster with cell biologist Spyros Artavanis-Tsakonas, Ph.D.

His good fortune did not impress his mother in China.

“It was the first time she’d heard from me in six months, because I couldn’t afford to call home. But this was big news.” When Xu’s mom asked what he intended to study, Xu replied, “I’m gonna work on flies, Mom.” After a very long silence, his mother spoke: “Son, we have a lot of flies right here in our hometown.”

For a geneticist, fruit flies are a model organism for figuring out what genes, and the proteins they encode, actually do. The ability to produce hundreds, even thousands, of mutant flies quickly by using chemicals, radiation or even transposons lets researchers look for traits they are interested in, such as slow growth or crippled wings.

This kind of large-scale mutagenic analysis, called a forward genetic screen, has been a staple of fly research for decades. And at his lab in Yale’s genetics department Xu has used the technique to unravel the biochemical pathways involved in cancer cell growth and metastasis in Drosophila.

Forward genetic screens have played a pivotal role in our understanding of modern biology in lower organisms, including bacteria, yeast, flies, worms, plants and zebrafish. But the lack of comparable genetic screens in mammals has impaired our ability to understand many aspects of human biology and disease. Xu felt that his fly work could only begin to approximate human disease because of the wide evolutionary distance between the two organisms. Moving his research closer to humans meant moving to the mouse.

Are you a mouse or a man?

Like humans, mice are mammals, with similar anatomy, physiology and developmental stages. They breed rapidly and can be inbred to produce large numbers of identical animals. Their care and feeding are more complicated and costly than that required for fruit flies, but as higher animals go, they are small and inexpensive.

For years, the obstacle to genetic studies in mice was a lack of tools for mutating genes en masse for forward genetic screening. Starting in 1981 with Frank H. Ruddle, Ph.D., Sterling Professor of Molecular, Cellular and Developmental Biology at Yale, researchers developed genetic engineering methods that allowed them to add and subtract genes from mice, and these methods revolutionized the use of mice for targeted genetic research.

Today, researchers can selectively mutate mouse genes at will. Typically, they use genetically altered embryonic stem cells to create embryos, which mature and pass the altered genes on to their offspring. The process is investigator-driven—scientists decide which genes to add or eliminate. From choosing a gene and designing a piece of DNA to disrupt it, to creating embryonic stem cells and injecting the cells into embryos to produce mice, to breeding out the final mutants, each new knockout is a custom product that takes a year and approximately $100,000 to bring to life. But the goal of large-scale systematic mutations in the mouse genome has remained elusive. Efforts to use chemical mutagens or viruses to disrupt large numbers of genes have been abandoned as too expensive and unpredictable.

Xu had a different idea. He wanted to create mutant mice as easily as he had made mutant fruit flies. He didn’t want one or two or even 10 mouse mutants—he wanted a complete collection, one mouse strain for each gene, that would allow him and scientists around the world to discover the roles of genes in human disease. “I thought if we could do the kind of work in mammals that we do in flies, that would be tremendous.”

For that, he needed a new tool.

Finding piggyBac

Based on his experience with Drosophila, Xu believed a transposon could do the trick for wide-scale mutagenesis in mice. Transposons, also known as jumping genes, were first described in corn in the 1950s by Barbara McClintock, Ph.D., who won the 1983 Nobel Prize in physiology or medicine for her discovery that the varied colors of Indian corn kernels arise from the disruption of pigmentation genes by transposons.

Mere snippets of genetic material, transposons insert themselves at random in the middle of the long DNA molecules that make up chromosomes. If they happen to land in the middle of a gene, the sequence becomes hopelessly garbled and the gene becomes nonfunctional. Over the course of evolution, transposons have remained active in plants and insects, presumably because they generate genetic diversity. For reasons that aren’t entirely understood, the abundant transposons in mammalian genomes (they make up as much as 40 percent of human DNA) have been “disabled,” perhaps to protect organisms from unwanted mutations. Despite decades of efforts by many researchers, no one had succeeded in discovering or engineering an efficient transposon in mammals.

Xu imagined using transposons like buckshot to pepper the mouse genome, where they would randomly insert themselves into genes and generate large numbers of mice with uniquely altered outward characteristics, or phenotypes. “People said I was crazy,” Xu recalls. “They said, you’ve never trained in mouse genetics. You’ve never even touched a mouse.”

Xu didn’t give up his day job; he kept his lab pushing ahead with its regular Drosophila work. On the side, though, he continued to search for the elusive transposon. In 1996 he applied for funding from the Howard Hughes Medical Institute, a foundation known for supporting risky but promising ventures. With its support, over the last eight years he has tested a succession of transposons and viruses from plants, insects and wherever he could find them.

Finally, he tried a strange-looking transposon known as piggyBac, found in the cabbage looper moth. It was different from anything else he had seen, and it was so exotic Xu figured it might work. PiggyBac had been discovered several years earlier by Malcolm Fraser, Ph.D., at the University of Notre Dame in Indiana, and it had already been shown to suppress gene activity in insects.

After engineering the piggyBac transposon to adapt it for mammalian cells, Xu found that it worked, and Xu’s mutant mice were featured on the cover of the journal Cell on August 12, 2005. “We don’t know why this one works while others don’t,” Xu said at the time, “It just works. We have a magical tool now.” Xu and colleagues further engineered a red fluorescent protein gene from jellyfish, which they added to the transposon, allowing them to identify visually which mouse carries a mutation and which is a mutant.

Mutants-R-Us

With quick and easy generation and recognition of mutants, the mammalian piggyBac transposon system is ideal for the large-scale project Xu envisioned. Besting the standard time requirement of one year per mutant mouse, within months the researchers had created 460 unique mutant mice.

Among the first batch of 95 mutants they examined in depth were mice with tusks, mice with neurodegeneration, mice that could not sense pain, mice that turn only leftward, mice that don’t grow and mice with bad manners. Then there were the sterile mice, and mice never even born because their defects were lethal early in development. In each case, these afflicted mice will lead the researchers to single genes critical for growth and development, autoimmune disease, social behavior, spinal cord defects and neurodegeneration.

“This is only looking at the first 95—imagine if we mutate every gene and look through them all, what we will find,” Xu says. The hit-or-miss nature of piggyBac mutations gives researchers a decided advantage over knockouts produced using embryonic stem (ES) cells, he says. PiggyBac allows scientists to study genes they didn’t even know existed. “We don’t pick which genes to get rid of. We just make the mutants randomly and let the animals tell us which ones are important. This is a critical difference, because scientists are not that smart that they always pick the right gene to knock out.”

Richard P. Lifton, M.D., Ph.D., chair and Sterling Professor of Genetics, an expert on hypertension genes and a Howard Hughes Medical Institute investigator, agrees. Lifton says, “That’s the power of forward genetic screens. They allow us to find genes that affect these phenotypes that up to now we’ve had no idea about at all.”

So unlike the ES cell method, which focuses exclusively on known genes, the unbiased approach of transposon-based mutagenesis can open up exploration of the entire genome, including areas of the genome previously dismissed as “junk” DNA.

Time for a new mouse

Xu’s development of the piggyBac mouse came as the drawbacks of the knockout technique were holding back mouse genetics, despite international support for a centralized program to knock out every gene in the mouse. First, the process of producing knockout mice via ES cell engineering is slow and expensive. According to the National Institutes of Health (NIH), about 11,000 mouse knockouts have been generated since the late 1980s, but that number is less impressive than it seems. Many genes have been knocked out more than once by different labs—one gene was knocked out independently at least 28 times. Of the estimated 25,000 mouse genes, only about 4,000 have been published, and studies of those genes have been limited to specific areas.

And not all mouse mutants are available to all researchers. Given the time and resources invested in making knockouts, some researchers want to keep their own mice close to home, impeding the sharing of reagents and increasing the chances for redundancy and waste.

To solve some of these shortcomings, the NIH has set aside $50 million to establish a central repository for 10,000 mutant ES cell lines. The lines, which will be produced over the next five years, will be freely available to all researchers, who can request the ES cells and then use them to produce their own knockout mice in their labs. A parallel effort, using different strains of mice and creating a different type of knockout, is being started by an international consortium that includes Canada and several European countries.

Xu thinks that the key is to produce mutant animals that will reveal defects and diseases. Producing mutant animals from ES cells is a long process. The piggyBac mouse system can reach the goal faster and cheaper because it produces mutant animals in a highly efficient and cost-effective fashion, and allows rapid identification of mutants for analysis. His plan is to produce up to 100,000 mutant strains, each of which carries a single transposon mapped to a known site. A bank of frozen piggyBac mutant embryos would then be generated for distribution to researchers around the world. The resource would enable researchers in all fields of medicine to study the genes regulating the disorders they encounter, Xu says.

Going with piggyBac for genome-wide mutagenesis has other advantages, too. Transposons may be the only way to generate mutants in dozens of strains of mice for which ES cell cultures are not available. There are more than 200 breeds of mice used by researchers, each with its own personality. Some are preferred by immunologists, while others are better for neurological studies. To move a mutation between strains takes two or three years, which might be skipped altogether by utilizing transposon technology. Transposons could theoretically be used in other species, too, like the rat.

From Yale back to China

To produce the million mice it will take to find 100,000 mutants, Xu and the School of Medicine have embarked on a joint research project with his alma mater, Fudan University in Shanghai. The mutagenesis of the mice will be done at Fudan, where Xu and his colleagues have set up a state-of the-art mouse facility and production lab supported by Chinese government funds. Researchers in China have produced the first 500 mutants already, including the 460 Xu’s team produced. But finishing the job will require much more funding, and Xu is on the stump for that now.

He has applied for NIH funding for the research at Fudan, to produce 500 more mouse mutants in a pilot project. Xu wants to demonstrate that piggyBac can work in a different strain of mouse, and specifically in the strain that was selected for the NIH ES cell-based knockout project. One way or another, the mutants will all be made within five years, according to Xu. “We hope that our project will be supported by the NIH so that the mutants will be available to the scientific community throughout the world as soon as possible.”

Making the mutants is just the first step of Xu’s plan, and not even the most ambitious part. Once the mutants are created, the real work begins. Every one of the million mutant mice will need a thorough physical exam. The project, which Xu envisions carrying out at Yale, calls for a full workup for each mouse, and could include a CT scan, blood work and measures of immune, kidney, lung and cardiovascular function, as well as of behavior. Not all the mice will appear sick—many will seem perfectly normal until researchers take a closer look.

“Now, researchers generate their own mutant mice and study only the processes they are interested in,” Xu explains. “For example, they make one or a few mutations and look for hypertension. If they see hypertension, that’s great. But if they do not see hypertension, that’s the end of the story, and they will most likely abandon the mice. But those mice could easily have diabetes, a very significant piece of information that would be totally missed. Furthermore, researchers working on different diseases and biological processes are now repeatedly mutating the same genes in ES cells and/or taking the same ES cell line to repeatedly produce the same mutant mice. There is a significant waste of resources.”

The centralized and comprehensive phenotyping will allow researchers to choose only the genes of interest for in-depth mechanistic studies and ensures that the mice rapidly make their way into the labs of experts who can best utilize them to discover new treatments for disease. Right now, Yale scientists are defining a scientifically solid and practical panel of phenotyping procedures that will cover the widest possible range of diseases.

They believe their integrated approach will provide the biggest return on the piggyBac investment. They plan to make their results—and their mice—freely available to researchers all over the world. Xu wants to see all data posted on the Web, where interested scientists can troll for new genes for their favorite disease, then order off-the-shelf mice for their experiments.

Besides having an impact on the most common major diseases, the mouse studies will advance research into orphan diseases—neglected conditions affecting so few people that they do not attract interest from for-profit drug companies. “There are about 6,000 orphan diseases. While each one affects only a small part of the population, all together they affect many people. We have a solution to the problem of lack of interest, because by the process of systematically mutating every gene and screening through our mice, we will identify many of the genes that are responsible for these diseases,” says Xu.

Ultimately, Xu hopes to create more than just mice.

“I want to make Yale the premier international center for human disease studies. The aim is to set up a center, based on these mice, which will attract researchers from all over the world. Each one can focus on a disease, and identify the causes of that disease right here. Then, they will move on to develop a career studying the mechanism of each disease and finding a cure,” Xu says. “When I came to the United States 23 years ago, I had $50 to my name. Yale gave me a research fellowship and changed my life. Now, we have a chance with these mice to cultivate a new generation of physician-scientists, who will mushroom out to solve disease and help millions of patients. That would really be my dream come true.” YM