The mouse that roared

The ancient Greeks had their myth of the fire-breathing chimera with its head of a lion, body of a goat and serpent’s tail. Modern America has its own mythical chimeras — from the Teenage Mutant Ninja Turtles and Spiderman to monsters of grade B movie fame like The Creature from the Black Lagoon and The Fly. Pondering the differences among organisms has fired the imagination with the once-seemingly-outrageous notion that one species could come to share the same biological stuff with another. But what today’s real-world scientific researchers term a transgenic species — one species that contains one or more genes of another — has been a living reality since 1980. That is when Yale scientists became the first in history to implant a human gene successfully in another species — a mouse.

While a mouse with a human brain or other phenomenal powers may still be only for the cartoons, the tiny, programmed, transgenic rodent is having a powerful impact on medicine. Over the past decade, the transgenic laboratory mouse has emerged as an essential model for advanced biomedical research throughout the world and has begun to produce tangible benefits for healthcare. Small, easy-to-handle, relatively inexpensive, and quick to breed (gestating in about three weeks), the laboratory mouse has long been the most commonly used animal model for scientific research. Today, with one or more foreign genes implanted, the transgenic mouse provides an unparalleled research tool for virtually every field of biomedical research. The impending completion of the Human Genome Project to map out the entire DNA blueprint will make the transgenic mouse an invaluable model for the study of genetic functions. Many believe that the transgenic mouse and other transgenic species will help lead researchers to a biomedical revolution over the next decade or so that could dwarf all the achievements of the preceding century. Already, transgenic mice and other transgenic species are being used to produce human blood cells, hormones and other proteins needed to treat human disease.

In a sense, each new transgenic mouse line, containing a gene from another species, is an entirely new species of mouse, one that never existed before. While the potential benefits that result can be enormous, the process itself is by now technically routine. The main source at Yale is the Section of Comparative Medicine’s Transgenic Mouse Service, which has a full-time technologist whose sole job is to create new transgenics. Supported in part by the Yale Cancer Center, the service produces new strains at the rate of about 30 to 50 per year. More than a score of other laboratories around Yale have their own transgenic operations and probably add at least that many more new lines of mice each year. The mice are used to study a vast range of questions, including the workings of the immune system, the causes of cancer, ways to control drug addiction, how the brain and skeleton develop and the mechanisms of asthma. While separated by 200 million years of evolution from humans, the mouse is also proving a gold mine for researchers trying to explain how humans evolved.

CREATING NEW SPECIES

Although the procedure for producing a transgenic animal is by now straightforward and widely employed, it is by no means simple. A transgenic mouse is created by individually injecting a copy of a foreign gene into the nucleus of a fertilized mouse egg. The egg containing the foreign gene is then reimplanted into the uterus of the mother. The work is microscopic and requires very pure DNA and working conditions. Even under the best of circumstances, only around one in five procedures will be successful. Each new strain of transgenic mouse costs about $2,000 to produce. Three weeks after the transgenic embryo is reimplanted, the mother delivers an infant mouse with the foreign gene present in the nucleus of all of its cells.

A mouse is created for a specific purpose that cannot otherwise be accomplished by less expensive and less time-consuming means, such as work with cell cultures or computer modeling. “Human genes work pretty faithfully in the mouse,” says Richard A. Flavell, Ph.D., chair of the Section of Immunobiology and a Howard Hughes Medical Institute investigator. He uses transgenic mice to study the workings of the immune system. The transgenic mouse has become indispensable for deciphering human genetic functioning. “You can’t find out how genes work in whole organisms by other means.”

While research using transgenic mice is, for most purposes, still in its infancy, it is already clear that enormous advances are in the offing. “The genetically engineered mouse,” says Robert Jacoby, D.V.M., Ph.D., chair of the Section of Comparative Medicine and director of the Yale Animal Resources Center, “will be the E. coli of the 21st century,” referring to the bacterium that was used to derive much of our current knowledge about how genes work. “The diversity of animal models has become literally infinite. Theoretically all genes could be studied with a transgenic mouse model.” Adds Flavell, “The combination of genomics and the transgenic approach is going to lead to massive medical breakthroughs.”

The use of the mouse as a study subject is of course nothing new. Inbred laboratory mice have been a staple of 20th-century medical research, from basic biological and behavioral studies to trials for new medications. Even Gregor Johann Mendel, the monk who first derived the laws of genetics in the mid-1800s through his studies of pea plants, is believed to have bred mice to observe inheritance of coat colors. Geneticists discovered early in this century that inbreeding among sibling mice over a span of about 50 generations could create a particular strain with identical inheritance characteristics. With no genetic variation except chromosomal differences between male and female, the effects of environmental manipulations and chemical reactions can be studied under controlled conditions, even in separate laboratories.

Everything about the inbred laboratory mouse could be easily controlled and studied — everything except for the most fundamental aspects of life: genetic structure, function and variation.

Prior to the creation of the transgenic mouse procedure, novel strains of mice emerged only by naturally occurring mutations, or a mutation could be induced by means of exposure to chemicals or radiation and then inbred over 50 generations as a novel strain. This was how most knowledge about the effects of chemotherapy, radiation therapy and most modern medications came about. Prior to the development of molecular biology techniques in the 1970s, identification of genes and their functions could come about only through this route. Progress was slow and often only happened serendipitously. “The process took a fair amount of time because of the number of generations needed to breed genetic uniformity,” says Jacoby. The chair of neurobiology, Pasko Rakic, M.D., Ph.D., worked on spontaneous neurological mutations in mice in the 1970s. “Although some of the random mutations have proved to be very useful and tantalizingly promising,” he recalls, “the chances of obtaining the next, functionally relevant mutation was unacceptably low.” A TRANSGENIC LANDMARK

In the late 1970s, revolutionary new methods for cloning and handling DNA were still being perfected. The laboratory of Frank Ruddle, Ph.D., now Sterling Professor of Molecular, Cellular and Developmental Biology and professor of genetics, had been using the new methods to pioneer techniques for mapping and modifying genes. The way to test the success of the techniques was to implant the modified gene in a cell in tissue culture and see how it functioned. “We knew we could transfer DNA into a cell in tissue culture,” recalls Ruddle. “We were curious about how it would function in a whole organism.”

Ruddle had already shown that nuclear injection of DNA into cultured cells was the most effective method of gene transfer, so he focused on that method for transforming mouse embryos. In 1980, he set up a cross-disciplinary team to tackle the challenge. He persuaded Jon Gordon, M.D. ’80, Ph.D. ’78, who was finishing his medical degree at the time and had prior graduate training in mouse reproductive biology, to join the laboratory group. Ruddle constructed what is called a transfer gene, made from the thymidine kinase gene of the human herpes virus and an enhancer region of the simian virus 40. Gordon suggested a novel approach of injecting the male pronucleus containing the transfer gene into the one-cell mouse embryo. All previous attempts to transfer genes into multi-cellular embryos had failed, and many in the scientific community simply doubted that gene transfer into a living embryo could be done. Says Ruddle: “The most important ingredient for a technological breakthrough is a sustained effort in the face of probable failure. I convinced the team that we should analyze no fewer than 100 mice before giving up. We hit pay dirt on mouse 48.” When the mother delivered a living infant mouse with the foreign gene in all of its cells, Ruddle’s team had created the first transgenic animal in history. (Experiments elsewhere to create transgenic plant species were under way around the same time.)

Ruddle coined the term “transgenic” for the resulting species. The team replicated its results and published them that year in the Proceedings of the National Academy of Sciences. That was followed by a paper in Science that showed that the gene was preserved through generational breeding and would continue to act like one of the mouse’s own genes. The transgenic revolution had begun.

The technique of pronuclear injection that Gordon developed for the purpose remains largely unchanged. (Gordon joined the faculty at the Mount Sinai School of Medicine in New York in 1982, where he now works on human reproduction and transgenic animal models for human cancer.) When compared to the hoopla surrounding the one-time cloning of Dolly the sheep three years ago, this far-more-revolutionary achievement received relatively little attention. Ruddle, however, recognized its importance. “We understood very thoroughly,” he says, “what the implications were for studying how development occurs and the potential for spin-offs of understanding of disease and how to treat disease in different and more powerful ways. It’s gratifying how the applications keep multiplying.”

Since then, Ruddle has been using the transgenic mouse technology to study the evolution of the so-called master control genes. His laboratory is focused on the Hoxc8 gene. This gene regulates various aspects of early development, such as the formation of the upper body and limbs in humans, and is seen across a very broad spectrum of species, from the primitive fishes to humans. Ruddle is interested in the evolutionary variations in the expression of genes that have taken place over the course of hundreds of millions of years. “The mutations in the master control gene allow transition in evolution from one form of species to another,” he says. To study how the gene is expressed in different organisms, he has created transgenic mouse species with the Hoxc8 gene from whales, birds and fishes.

There are no five-ton white mice with flukes swimming around New Haven Harbor. In most instances, Ruddle inactivates the gene, using it to report on how it would be expressed in different organisms. In current experiments, he has transferred live whale genes into the mouse to see how the whale Hoxc8 gene affects mouse development. “We hope to see modifications in the vertebral column that correspond to the structural modifications in whale skeletal design as a result of adaptation to the marine habitat over the past 30 million years.” Early results have provided useful clues about the evolutionary pathways that lead from one species to another.

Ruddle has also developed new, patented tools for targeting and isolating variations in coding and control regions of genes. Among the implications for this work is looking at ways that different populations respond to medications in order to develop new drugs that are more effective in treating individuals. This is part of an entirely new field called pharmacogenetics, which aims to create personalized medicines. Ruddle serves as an advisor to a former student of his, Gualberto Ruano, M.D., Ph.D., founder and chief executive officer of Genaissance Pharmaceuticals, a New Haven-based biotechnology firm pursuing applications for developing new disease treatments and better utilizing existing therapies using genomics and information technology.

A MOUSE GUIDE TO HUMAN GENETICS

Transgenics has proven of great use in the study of a wide range of disease mechanisms and biological processes. Among recent firsts at Yale, Prabir Ray, Ph.D., an associate professor of medicine, and Jack Elias, M.D., section chief of pulmonary and critical care medicine, created the first transgenic mouse that expresses human interleukin 11 in the lung, which is known to be a factor in the development of asthma. Thus, this mouse provides an important animal model for understanding the development of asthma.

Immunobiologist Flavell has collaborated with Ray in his transgenic work. In his own laboratory he works with more than 50 different strains of transgenic mice to study how immune responses are regulated at the molecular level. Transgenic mice were valuable in his and colleagues’ work in creating LYMERix, which is the first vaccine against Lyme disease and is being marketed by pharmaceutical company SmithKline Beecham. Flavell is now focusing on understanding autoimmune disease. In juvenile-onset [or insulin-dependent] diabetes, a potentially lethal disease that most often begins in childhood and affects about 750,000 Americans, the pancreas destroys its own insulin-making cells. Flavell’s laboratory has created transgenic models for this process, giving them some of the first clues as to why it takes place. He says, “We now think that you get an infection, which causes the pancreas to fill up with immune cells that pick up the dead cells and turn on the immune system. It, in turn, sends lymphocytes to clear the dead cells. You’ve started an immune process that leads the lymphocytes to attack other pancreatic cells, eventually destroying their insulin-producing function and causing juvenile diabetes.” While the system of genetic coding, expression and regulation of expression is overwhelmingly complex, involving thousands of genes, the possibility exists that a genetic therapy could be created to halt the immune response and reverse the damage to the pancreas.

The paths that result from basic research are never entirely predictable. Ensign Professor of Medicine Arthur Broadus, M.D., section chief of endocrinology, began in the 1970s by studying the calcium build-up, or hypercalcemia, that is often seen in late stages of certain cancers. In 1988 he identified the parathyroid hormone-related peptide (PTHrP) as the tumor product responsible for hypercalcemia in cancer patients. Acting like a hormone, PTHrP breaks down bone, resulting in an often-lethal increase in blood calcium.

Since then he and his colleagues have been using transgenic mice to explore the normal function of the PTHrP gene. The surprising results point to the importance of the gene in basic bone and skin formation, as well as in diseases from skeletal and cartilage malformations to Alzheimer’s disease. The gene appears to have important activity that even affects aging. “It’s been a gold mine,” he says.

A competing group succeeded in knocking out or removing the functioning of the gene and learned that the absence of the gene led to lethal bone formation problems during gestation because of excessive calcification. In other words, the gene has an essential role in the regulation of bone formation during development. Broadus went a step further by combining the knock-out with a created transgenic replacement gene targeted specifically at cartilage that allowed the mice to live to maturity. He could then observe what deficits, if any, resulted in the developing mice. “It was like a gene therapy experiment,” he says. His research shows that the gene regulates a large number of physical characteristics, including tooth, skin and mammary-gland development. “It’s a tightly regulated gene,” he says. “Over- or under-expression causes major problems in development.” To his great shock, he says, “We’ve become developmental biologists.”

More recently, he and his colleagues have discovered that the gene also has a vital, ongoing function of protecting neurons by regulating calcium in the brain. Calcium is necessary for brain functioning, but too much calcium can contribute to a wide range of very serious and common disorders, including Alzheimer’s, Parkinson’s and Huntington’s diseases. “They each have different structural targets,” he says, “but in all cases there is calcium-driven toxicity.” Moreover, increased expression of calcium is at the root of a widely credited theory of aging. The transgenic mouse could potentially serve as an animal model for different disorders, creating new platforms for developing treatments, and possibly explain some of the biological mysteries of aging. “The implications,” he says, “are big.”

Transgenic mice have opened new vistas on the brain. Several laboratories at Yale are studying complex brain development processes using transgenic mice. Essential cell death in the brain has long been known to take place after birth in order to allow the “sculpting” of neurons and their connections that must take place for learning to occur. It was long believed that this cell death process only began with birth. Neurobiology chair Pasko Rakic, M.D., and his colleagues have shown through their work with knock-out and transgenic mice, however, that specific types of cell death also must occur for proper development of the brain during gestation. By preventing cell death during embryonic brain development, they created a mouse with a larger-than-normal cerebral cortex. In another study, Rakic and his colleagues examined the effect of deleting the genes in the so-called JNK family that are involved in stress-induced cell death in the adult organism. Mice lacking both copies of some of these genes are resistant to noxious agents that in normal mice induce neuronal loss. This finding suggests strategies for preventing cell death in neurodegenerative diseases, as well as after a stroke.

The complexities of the brain are very difficult to study in humans. It is ethically impossible to induce disease states in normal brains to study what changes occur. The transgenic mouse, however, can provide models for many diseases. Jameson Professor of Psychiatry Eric Nestler, M.D. ’80, Ph.D. ’82, and colleagues have looked at the genetic processes behind complex behaviors such as drug addiction and anxiety disorders. They made a double transgenic mouse — one with two new genes. By raising the mice on a diet that includes a low dose of the antibiotic tetracycline, they have inactivated a gene, delta FosB, that regulates other genes in specific neural regions known to be involved in drug addiction, from nicotine to cocaine. By then removing the tetracycline, the gene of interest in the brain becomes active. This process is called an induced transgenic mutation. Once that gene is induced, the animals become hypersensitive to drugs of abuse. In a very interesting finding, reported in the Sept. 16 edition of the journal Nature, the researchers have shown that delta FosB persists in the mouse brain far longer than similar genes. This may help explain why even years after addicts cease taking drugs they are still so susceptible to relapse.

Although developing a therapy for drug addiction based on this discovery is years off, the Yale group is beginning to look for ways to control the gene’s activity. “It’ll be a fantastic advance,” says Nestler, director of the Division of Molecular Psychiatry at Yale’s Abraham Ribicoff Research Facilities, “when we have the genetic basis not just for abnormal states, but normal functioning as well. Eventually every disease and normal function will be attributed to specific genes. As these genes are found in humans, the best way to develop new therapies — and even to optimize normal functioning — is to put the human gene in a mouse and make novel animal models. For the first time, we have ways to design therapies that will target very specific genetic functions. We’re still just at the beginning of that process. We’re at the tip of the iceberg.”