The Rodent Revolution
By Holly Ahern
A nude mouse
What do nudes, knockouts, and gnotobiotics have in common? Contrary to the images these terms might conjure up, they are not characters in an X-rated horror film set on an alien planet. These are all laboratory animals created expressly for use by scientists in several areas of biology and biomedical research.
The applications for laboratory animals are many and varied, including basic research in genetics, immunology, and cell biology as well as for determining the pathology of microbial infections. Animals carrying defective copies of genes are increasingly used in biomedical research laboratories as model systems for investigating the pathology of human disorders such as cancer, arthritis, and Alzheimer's disease. These same animal models aid researchers in their search for treatments and cures.
While all sorts of animals are used in basic and applied research, mice and rats are increasingly gaining ground as the laboratory animals of choice because of their suitability to newly emerging applications. "The mouse has become a valuable asset for advancing biomedical research," says Lawrence Cunnick, president of Rockville, Md.-based Biocon Inc., whose company provides services -- such as making monoclonal antibodies and housing transgenic animals -- for scientists who want to use animals in their research but lack the facilities or expertise.
"Mice are ideal animals for basic research applications because they are relatively easy to handle, they reproduce rapidly, and they can be genetically manipulated at the molecular level," notes Michael Hayre, director of the laboratory animal research center at Rockefeller University. "Mice have started to replace many of the other animals, such as dogs and cats, used in biomedical research because of their many advantages."
C57 black 6 inbred mouse
With some special types of mice and rats, the potential exists to "decrease the number of larger animals used in biomedical research," comments Tony Cruz, general manager of GenPharm International in Mountain View, Calif., a company specializing in genetically engineered mice. "For example, much of the current research on Alzheimer's disease has been performed with primates. Now that we have a good mouse model of the disease, it is likely that use of primates for these studies will decrease."
The reason for this shift from larger animals to rodents for research stems almost directly from the explosion in molecular biology research that began in the 1970s and spilled over into the 1980s. Just as foreign genes can be inserted into plasmid DNA, genes from other organisms can be inserted into the genomes of animals such as mice, creating so-called transgenic animals. Transgenic mice, for example, might carry DNA sequences from any one of several different organisms, from viruses to humans. With the state of the current technology, practically any gene that can be cloned can be added to the mouse genome.
Transgenic mice develop from eggs that have been microinjected with bits of foreign genetic material. The injected embryos are transferred to the oviducts of a female mouse to develop. A few of the embryos grow to term with the foreign DNA firmly inserted in their genomes. The transgene is often expressed just as are all of the other genes of the mouse, providing researchers with an opportunity to examine tissue-specific gene expression, for example, or to study the genetic controls that regulate development. If the transgenic animals are carefully bred, the transgene can be passed on to create future generations of the modified mice.
Particular strains of transgenic mice called knockouts are getting a good share of the attention that is being focused on these types of animals. "Genes are 'knocked out,' or disrupted, when transgenes are inserted into a particular genetic location," explains Cruz. Using transgenic technology, researchers can target specific mouse genes for inactivation. Because the genetic patterns of mice and humans are so similar, knockout mice provide valuable models of human diseases.
"This is a tremendously powerful tool because it makes data obtained from animal studies more applicable to human diseases," says Hayre. "Often with conventionally bred mice, the models represent the mouse equivalent of a human disease. Now we are able to manipulate these animals at a genetic level and actually reproduce the disease as it occurs in humans."
Knockouts and other types of custom-made mice are often created for use in specific research applications. But what do you do once you've created a mouse and want to grow more? "There are a number of stages involved in the development of a transgenic mouse," explains Sam Phelan, director of marketing at Taconic Farms Inc. in Germantown, N.Y. "Most of these animals are created by researchers at academic institutions. However, in order for the mouse to gain widespread acceptance within the scientific community, other scientists need the opportunity to work with it."
After the mouse has been created, animal laboratories, such as Taconic, step in. "We define the traditional genetics of the animal and bring it to pathogen-free status so that the mouse can be shipped to animal facilities around the country, and we serve as the animal's distributor," Phelan explains. Taconic maintains strains of rats and mice for its customers.
A major supplier of mice for genetic and biomedical research is the Jackson Laboratory in Bar Harbor, Maine. Jackson Lab is a nonprofit research institution that provides mice to universities, medical schools, and research laboratories worldwide. The lab maintains an extensive collection of transgenic and knockout mice to serve as models for several human diseases, including cancer, AIDS, cardiovascular disease, cystic fibrosis, and diabetes. Mice such as these are opening up whole new areas of research.
For example, consider a transgenic mouse carrying the human gene APOA1, which produces a protein component of the so-called good HDL cholesterol. Because the transgenic animals carrying this gene are highly resistant to heart disease, these mice are being used in studies to understand how HDL cholesterol protects people from heart attacks. Mouse models of cardiovascular diseases are studied in cardiac catheterization labs, just as their human counterparts are.
"Within the last month we have had to modify one of our catheters to a smaller size because so many of our long-term customers have changed to use mice in their research," says Connie Nordstrom, regional sales manager for Millar Instruments Inc. in Houston. Catheters for mice produced by Millar Instruments are miniaturized with a solid-state transducer in the tip. When the device is placed within an animal's heart, it provides an accurate measure of the mouse's blood pressure and blood velocity that originates right at the source.
Transgenic mouse from Taconic Farms in Germantown, N.Y.
While the research benefits of transgenic animals have exponentially increased, the commercial value has lagged somewhat behind. "Transgenic mice are a growing area of the animal market, but acceptance of these mice as research tools is occurring more slowly," says Cruz of GenPharm. "To increase the market for these animals, it is necessary to look for specific niches where a particular type of transgenic mouse can be used." GenPharm has moved into the applied research market with the development of transgenic mice that can serve as model systems for studies in pharmacology and toxicology.
A particular transgenic mouse with a knockout mutation in the tumor suppressor gene p53 (the Stratagene Big Blue mouse, created by Stratagene Inc. of La Jolla, Calif., and offered by GenPharm) is proving useful as an animal model to test chemical compounds for their potential to cause cancer. A knockout in the p53 gene results in mice that are prone to tumors. When these mice are exposed to carcinogenic compounds, cancers are detected earlier than in normal mice. "Performing bioassays with the p53 knockout mouse may decrease the amount of time it takes to perform the assay and also the cost," continues Cruz. The p53 knockout is also in demand by cancer researchers because of the link between mutations in the p53 gene and several forms of human cancer.
Not all of the animals used in research are as exotic as knockout mice. Many breeds of mice are the result of carefully controlled inbreeding or outbreeding of mouse strains over several years. Inbred and outbred mice are available from several different suppliers of laboratory animals, including Charles River Laboratories in Wilmington, Mass.; Hilltop Lab Animals in Scottsdale, N.J.; and Indianapolis-based Harlan Sprague Dawley Inc.
Balb/c mice and nude mice, two strains popular with immunologists making monoclonal antibodies, are mice that have been inbred to generate desired qualities. When mated, brother and sister mice carrying one copy of a defective gene may produce offspring with two copies, leading to unique phenotypes that may be genetically useful. Nude mice are an example. "Due to inbreeding, nude mice have no thymus gland and therefore lack T lymphocytes," explains Cunnick of Biocon. Why nude? "The genes for hair are associated with the genes for the thymus gland," says Cunnick. Therefore, mice with no thymus also have no hair.
Because the presence of pathogens, particularly viruses, in research animals may have profound effects on the outcome of an experiment, mice are maintained in as sterile an environment as possible. "To limit the number of variables a researcher is working with, every effort is made to eliminate opportunistic organisms from the animal's environment," Taconic Farms' Phelan points out. Therefore, animals are kept in sanitized environments -- where even the water and air are sterilized -- and are shipped to facilities pathogen-free.
To ensure the pristine status of its mice, Taconic ships its animals in special plastic containers with fixed polypropylene filters to decrease the possibility that the mice will come in contact with airborne contaminants.
Mice called gnotobiotics live their entire life in sterile conditions, usually in a plastic bubble where their only contact with humans is through gloves built right into the side of the container. Gnotobiotic mice are born through cesarean delivery to prevent even the natural contamination that occurs during the delivery process. The babies are removed from the mother in germ- free condition and transferred immediately to a sterile environment.
Researchers use gnotobiotic mice to study the effects of pathogenic organisms in isolation. A gnotobiotic mouse can be infected with a single microbe, such as Chlamydia, which causes a sexually transmitted disease. The pathological effects of that single organism can then be investigated. "It's the perfectly controlled study for infectious disease," claims Taconic Farms' Phelan.
Regardless of whether laboratory mice are genetically designed, inbred, or outbred, their life in the laboratory is generally not as unpleasant as the general public may perceive. "Animal research is heavily regulated by the federal agencies that provide grant money to researchers," notes Hayre, who is a veterinarian. "One thing in particular that we've seen as mice become the preeminent research model in many laboratories is that the scientists themselves require that the animals be maintained in an absolutely exquisite state of health."
Gone are the days of mice and rats living in crowded cages with minimal care. Today, animals used for research are kept in state- of-the-art facilities where the air and water are purified, the cages are cleaned daily, and the food is outstanding, at least by mouse standards.
Mobile Rodent: GenParm uses special containers to ship animals like its p53 knockout agouti mouse.
Because all laboratory animals, particularly genetically engineered ones, are extremely valuable to researchers, every effort is made to keep them in facilities that are free of disease. "Research animals live in very controlled environments where a great deal of attention is paid to parameters such as air flow, ammonia levels, humidity, and temperature," maintains Hayre.
Animal cages, once simple boxes of plastic and wire mesh, are now high-tech pieces of equipment. Cages manufactured by Allentown Caging Equipment Inc. of Allentown, N.J., for instance, are designed with full ventilation systems and HEPA (high-efficiency particular air) filters to keep the air that the animals breathe fresh and free of contaminants.
"The ventilated systems maintain animals for long periods and are especially useful for specialized animals, such as transgenics and nude mice," says Steve Benijni, a technical sales representative for Allentown Caging Equipment. "Because such animals are valuable and expensive, they are kept in isolation conditions with a minimum of handling. The better the environment that the animal lives in, the healthier the animal."
To keep the animals germ-free, cages often contain special watering systems that minimize their exposure to people and unfiltered outside air. Even their food is sterilized, either by autoclaving or by irradiation.
The current trend in many areas of research does seem to be moving away from using animals, both small and large. Mouse models are replacing primates in many aspects of biomedical research. However, "higher organisms have not been abandoned as test systems," notes Cunnick of Biocon.
"Where it can be done, there is clearly a trend away from using animals in research," says Jonathan Mansbridge, senior scientist at San Diego-based Advanced Tissue Sciences Inc., a company that has developed in vitro test kits based on human fibroblasts grown in tissue culture to replace animals in product testing.
"Researchers don't use animals unless there are no other options, and smaller animals are preferred if it is possible to use them. However, there are things that you just can't do otherwise."
Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.