[From CASE Reports, Vol. 11, No. 3, 1996]

Transgenic Animals Key to Human Disease Research

Neither science nor society may be ready for the direct manipulation of the human genome, but a technique for directing the genetic make-up of animals is being used in some of the most exciting biomedical research today. This technique involves inserting a foreign gene into the chromosome of an animal. The animal, called transgenic, then expresses that gene along with its normal genes.

Transgenic animals are being used in basic science research to elucidate events that range from the development of the embryo to events that occur decades later in people who have autoimmune diseases. These studies will help scientists understand the cause of many diseases and may point the way to their treatment. Transgenic animals are also proving to be valuable tools in the drug development process itself, providing a more accurate way to test the effectiveness of drugs. In another exciting development, transgenic animals are providing organs for transplantation that are less likely to be rejected than the organs currently used.

To add a gene, called a transgene, to the normal chromosome set of an animal, researchers remove a one-cell embryo (the fertilized oocyte) from the oviduct of a pregnant animal and inject a cloned segment of DNA, including a gene of interest, into the male pronucleus. The researcher then puts the embryo into the oviduct of a surrogate pregnant female who will carry the transgenic animal to term. The transgenic animal will be born with all of its normal genes plus the transgene that was inserted into the one-cell embryo. Transgenic animals can be bred and their offspring will carry the transgene.

The transgene inserts randomly into the cell's chromosome, not necessarily at the location where its normal counterpart exists. Because it carries all of its own regulatory elements, the transgene functions normally even in the new location. This technique has proven to be widely applicable in biomedical research, and is being used by research-ers in several Connecticut laboratories.

Transgenic Animals and Embryological Development

CASE member Frank Ruddle, Sterling Professor of Biology at Yale University, led the Yale research team that first developed transgenic animals (a term that they coined) in the early 1980s. Today, Professor Ruddle is using transgenic mice to study the control systems of a family of developmental genes, called homeobox genes, that specify the morphological structures of animals during development.

The homeobox genes are important in the normal development of animals-mutations in these genes can lead to abnormal development or to cancer. These genes are highly conserved between species: similar genes have been found in humans, mice, birds, and fish. Slight differences in the genes between species contribute to the vastly different appearances of these animals. Professor Ruddle is working with the homeobox gene HoxC8, whose expression is important in the morphogenesis of the neck, forelimbs and trunk. HoxC8 is turned on during development of the embryo when organ systems are being formed. Professor Ruddle is interested in certain regulatory elements that occur in the hox genes and how they regulate gene expression; these elements are called enhancers. "Homeobox genes have an important role in evolution," according to Professor Ruddle, "Small changes in the enhancers elements of these genes can make a big difference in how animals develop." For example, a modification in the HoxC8 enhancer in whales may account for modification of their anatomy adaptive to a marine lifestyle (whales are believed to have evolved from a common ancestor related to cows and camels).

Professor Ruddle's group uses an iterative process to dissect out the function of the enhancer genes. First they create a transgenic mouse embryo with the entire HoxC8 gene and observe the development of the embryo. Then they chemically remove some of the regulatory elements of the gene, and create a transgenic mouse embryo with the smaller gene segment. They repeat this process with smaller and smaller gene segments until they reach a point at which expression of HoxC8 is lost, which indicates that they have removed the enhancer genes. Since they have kept track of the gene segments that they removed, the researchers can identify the segment that contains the enhancer genes. Professor Ruddle's group has narrowed down the location of the HoxC8 enhancer to a 300-base-pair segment of DNA-in a gene that is approximately 30,000 base pairs in length.

Gene Targeting

Researchers at the Institute for Research Technologies at Bayer Corporation in West Haven are using a relatively new technique to produce transgenic animals. This technique, called "gene targeting," inserts the transgene into a specific site in the chromosome, replacing the targeted gene. In this way, the product of the transgene is produced, but not the corresponding product of the original targeted gene.

Because this technique eliminates the targeted gene, it is particularly useful in three areas of research. First, one can test whether it is reasonable to pursue a certain therapeutic approach. For example, if the researchers want to know if a disease could be treated by reducing the amount of a certain protein or the activity of a particular enzyme, they can produce transgenic animals missing the corresponding gene ("gene-knockout animals"). If symptoms of the disease are ameliorated without serious side effects, then it might be worth the time and expense of producing a drug that would have the same effect. (In many cases, another system in the body might take over the function of the gene product that was eliminated, and it would not be worth pursuing that line of research.) "Proof-of-principle" experiments with transgenic animals can save researchers time and money, and accelerate research along more fruitful lines.

Another use for the gene-targeted animals is to analyze early in the development of a drug whether it is really effective against human disease without experimenting on humans. It is important to test drugs in animals before trying them in humans, especially when the consequences of drug failure are serious, but because the biochemistry is slightly different, the animal responses often do not reflect exactly what happens in humans. Use of gene targeted animals has been particularly useful in research on compounds that bind to hormone receptors. Researchers in Bayer's Metabolic Diseases Institute, headed by Peter M. Rae, are using them in research on compounds to treat obesity and diabetes. The Bayer researchers have created transgenic mice that have a receptor gene replaced with the corresponding human gene, so that all of the particular receptors on the cells are human. Drugs that are tested in these animals will reflect the anticipated results in humans more accurately than drugs tested in normal rodents.

A third area in which transgenic animals are particularly useful is in testing the efficacy of drugs to treat diseases for which there are no good existing animal models, for example, Alzheimer's disease. Primates are essentially the only animals that develop an Alzheimer's-like disease, and both ethical considerations and expense preclude testing potential new drugs on primates. The Bayer group is working to develop colonies of transgenic mice that develop the plaques and tangles in their brain tissue that are characteristic of Alzheimer's disease. The plaques are an accumulation of a certain protein that is present in all people but is misprocessed

in patients with Alzheimer's. The researchers hope to learn from the transgenic mice how to interfere with the misprocessing so that the plaques do not form. Genetic mutations have been identified in patients with early onset Alzheimer's disease. The Bayer group is creating mice that have these genetic mutations, and the preliminary results suggest that the mice exhibit the expected molecular defects. They are now waiting to see if the mice develop the plaques that signify Alzheimer's disease.

According to Dr. Rae, "Transgenic technology is a powerful research tool that is finding increasing uses in the pharmaceutical industry. It's still a relatively young technology, and its success in these early applications will dictate how widely it is incorporated into preclinical research programs in the industry. At Bayer, its utility has been established for a couple of applications, and we're using it in an increasing number of drug discovery projects."

Regulation of Bone Development

Alexander Lichtler, Associate Professor of Pediatrics at the University of Connecticut Medical School, is using transgenic mice to try to understand the regulation of an important bone protein, type I collagen, during development.

"Bone is constantly being remodeled by cells that chew up existing bone and cells that lay down new bone-this process is important not only during development, but also in aging," says Professor Lichtler.

As people age, the cells that chew up the bone continue to work efficiently, but those that produce new collagen do not, so bones get thinner and are more likely to break. "By understanding this process, we may be able to develop therapeutic agents to intervene and stimulate the synthesis phase," explains Professor Lichtler. "This could be important for people with osteoporosis, and for elderly people who are prone to hip fractures."

Type I collagen is the most important protein made in bone. It is responsible for the strength of the bone; without it, the minerals of the bone would not hold together and the bone would not form. Collagen is also made by cells in other tissues, including skin and tendons, but in different amounts in each of these tissues-if skin cells made the same amount of collagen as bone cells do, the skin would be too thick, and if bone cells made the same amount that skin cells do, the bone would be too weak. Professor Lichtler's group wants to find out how the cells know to make the right amount.

As bone precursor cells develop, they suddenly start to produce more collagen. In cooperation with the University of Connecticut/VA transgenic animal facility in Newington, headed by Stephen Clark, Associate Professor of Medicine, Professor Lichtler's group has made transgenic mice that have the promoter section of the rat collagen gene attached to a marker gene, to learn what part of the promoter is responsible for the increased collagen production. They started with mice with the entire promoter, and measured production of the marker. They then cut away increasing lengths of the promoter, making new transgenic mice and measuring the marker each time. Using this technique, they have identified a thirteen-base-pair-long section of the promoter that contains the activity.

Professor Lichtler's group further analyzed this section of the promoter and found that it contains a binding site for a certain protein. They mutated the binding site and made transgenic mice for the mutant. This mutation did not have much effect on collagen production in skin, tendons, or by the precursor cells in bone, but did eliminate the sudden increase in collagen production by the more mature bone cells. "We think that during development a protein binds to this site in the promoter and causes an increase in collagen production," says Professor Lichtler. "We have found a candidate protein but have not yet identified it. That's the next step."

Transgenic Organ Donors

The demand for human organs for transplantation has increased in recent years, but availability has not. In many cases, a person who needed a heart, kidney, or lung transplant died before a human organ was available. So researchers have tried using animal organs in their place. Whereas human organs for transplantation are carefully matched with the recipient, and the recipient receives immunosuppressive drugs to prevent rejection of the transplant, these strategies do not work with transplantation of organs across species (xenotransplantation).

The surface of a cell is a jungle of proteins, carbohydrates, and other mole-cules filling all the available surface area. One specific carbohydrate resi-due, galactose, is present on the surface of cells from all species except humans and other old-world primates. Humans have antibodies in their blood that bind to these galactose residues and activate complement, a protein that then binds to the cells and destroys them. This phenomenon, called hyperacute rejection, can destroy a transplanted organ in a matter of hours and is not affected by any existing immunosuppressive drugs.

Researchers at Alexion, Inc. in New Haven, collaborating with US Surgical Corp. in North Haven, are using transgenic strategies to provide non-human organs for transplantation into humans. They have chosen swine as organ donors for several reasons. Swine organs function similarly to human organs, so it is hoped that a person who receives a swine heart, lung, or kidney will be able to lead an active, productive life. Swine also produce large litters and have relatively rapid breeding cycles, so organs could be made available at a rate that would meet the clinical need. In addition, swine are very disease-resistant and US herds have been kept disease free.

The safety of xenotransplantation is of great concern to the researchers, patients, and the general public. The US Food and Drug Administration (FDA) will oversee xenotransplantation when it reaches the stage of large-scale human clinical trials. According to Louis Matis, Vice President of Research and Immunobiology at Alexion, "Regulations for the use of swine tissues in humans are in the process of being evaluated at the FDA. The regulatory issues are quite likely to be surmountable and we do not foresee many problems with using swine organs in humans."

Although swine organs are physiologically compatible with humans, swine cells do bear the carbohydrate residues that activate complement and result in hyperacute rejection in humans. This is where the transgenic research comes into play. Swine cells, like cells from other species, including humans, also bear proteins that inhibit complement. These regulatory proteins are species-specific, however, which means that the proteins on swine cells will not inhibit the complement in the human transplant recipient.

Using transgenic techniques, the researchers at Alexion have created swine that bear the human complement inhibitory proteins on their cell surfaces, which should be able to inhibit complement activation and, therefore, hyperacute rejection. Organs from these animals have been transplanted into non-human primates; preliminary results indicate that hyperacute rejection is avoided by this technique-the transplanted organs survive for significantly longer periods of time.

A second transgenic strategy is also being followed at Alexion. The galactose residue that initiates hyperacute rejection is placed on the cell surface by a specific enzyme called galactose transferase. Researchers at Alexion are creating transgenic swine that have, in addition to the normally occurring galactose transferase, a fucosyl transferase, which puts a fucosyl residue, rather than galactose, on the cell surface. The fucosyl residue is what the human antibody system expects to encounter, and so this residue does not initiate hyperacute rejection. Since galactose transferase is still functioning in the transgenic animals, some galactose may be present on the cell surface, but only in small amounts, so complement activation is greatly reduced and hyperacute rejection does not occur. Studies in transgenic swine are in the early stages, but studies in transgenic mice have demonstrated the feasibility of this technique.

These experiments using transgenic animals constitute the beginning of a revolution in biomedical research and applied science-a revolution that scientists believe will inevitably lead to major advances in the practice of medicine.--Lisa Christenson, science writer.

Back to CASE Reports TOC