As discussed previously, rodents, i.e., mice and rats, account for the overwhelming majority of all laboratory animals. This is reflected in the present scientific development of the exponentially growing number of genetically engineered mouse strains produced. Therefore, after outlining the contributions of a wide range of laboratory animals to Physiology and Medicine Nobel Prizes, we discuss the present role of laboratory animals in medical science, with the focus on mice. However, a final section will introduce nontraditional species in the laboratory animal sciences.
Looking back at the Nobel Prizes for Physiology and Medicine between 1996 and 2001, five of the six selected Nobel awards were given to achievements that, at least in part, were based on experiments utilizing laboratory animals. The 2001 Nobel Prize in Physiology and Medicine was awarded to Leland Hartwell, Timothy Hunt, and Paul Nurse for their work on "key regulators of the cell cycle." Using sea urchins and yeast cells in independent experiments, their research led to the identification of key regulatory genes and their products, which are responsible for cell growth and division. Over 100 celldivision control proteins, later named cyclin-dependent kinases, or CDKs, were identified, as were molecules called cyclins that bind to CDKs to control their phosphorylation and activity. The cyclins are conserved in eukaryotic organisms, including yeast, plants, animals, and humans. This discovery may lead to the development of future therapeutic agents that target the cell cycle machinery and kill cancer cells.
The 2000 Physiology and Medicine Nobel Prize awarded to Arvid Carlsson, Paul Greenberg, and Eric Kandel recognized pioneering discoveries on slow synaptic transmissions between neurons. Eric Kandel received his prize for his contributions to the understanding of the neuronal pathways of learning and memory, which was deduced from research conducted in the sea slug Aplysia.
Nitric oxide formed the basis of the 1998 Nobel Prize in Physiology and Medicine, which was awarded to Robert Furchgott, Louis Ignarro, and Ferid Murad. In a serendipitous experiment conducted on rabbit aorta preparations, Robert Furchgott discovered an endothelium-derived factor that was later recognized as nitric oxide (NO). NO acts as a signaling molecule in the cardiovascular system, causing local vasodilation, thereby displaying protective properties for the vascular system against atherosclerosis via various effects on leukocyte vascular permeability. However, as a universal signaling molecule, NO is also involved in critical roles in inflammation, apoptosis, and neurotransmission.
Stanley Prusiner received the 1997 Physiology and Medicine Nobel Prize for his discovery of prions, tiny protein molecules that are etiologically linked to a variety of slow-acting, inevitably fatal diseases in humans and animals. "Prion" is an acronym for "proteinaceous infectious particles," and forms an entirely new genre of disease-causing agents. Prions cause neurodegenerative disorders in sheep and goats (scrapie), cattle (mad cow disease), elk and deer (chronic wasting disease), and also humans (Creutzfeldt-Jakob disease and kuru). Moreover, findings gained through the study of these diseases may contribute to the understanding of more common dementia disorders, such as Alzheimer's disease.
Discoveries elucidating interactions between viruses and the immune system resulted in the 1996 Nobel Prize awarded to Peter Doherty and Rolf Zinkernagel. Using both inbred and outbred strains of mice, Doherty, a veterinarian, and Zinkernagel pioneered work that explored cell-mediated lysis of virus-infected cells. Results from their experiments uncovered the central function of the major histocompat-ibility antigens in signaling self to the immune system. Discoveries associated with this work are significant for the understanding of autoimmune diseases and immunological surveillance mechanisms crucial in transplantation medicine.
These are just five recent examples in which biomedical research, utilizing a wide range of laboratory animals, contributed to significant progress in medical knowledge. Such research contributions leading to Nobel Prizes, the highest accolade in science, are testimony to the important role of laboratory animals in medical progress, as well as to the wide acceptance of animal-based research among scientists. This latter fact was further demonstrated in a petition to the then Surgeon General C. Everett Koop that was signed by 30 Nobel Prizewinners in 1989.8 The Nobelists strongly endorsed the use of animals in medical research, a recommendation prompted by the increasingly visible actions of animal-rights activists.
Rodent Embryonic Stem Cells and the Advances of Molecular Biology
One of the key advances in animal-based research in the last two decades was the development of technologies that allowed researchers to grow embryonic stem (ES) cells in tissue culture. In mammals, the fertilized oocytes and the 4- or 8-cell-stage blastomere are totipotent, i.e., when transferred by injection into early embryos, they can develop into the entire complex animal. Bradley9 first demonstrated in the 1980s that ES cells could give rise to all somatic cell types of the embryo. This finding formed the basis for targeted gene technology, which resulted in the creation of "knockout" and other transgenic animals.10 Such transgenic animals can be made by randomly inserting a new gene, called a transgene, into the genome of the recipient animal embryo. Alternative technologies include direct mutagenesis of the transgenic animal's genome using targeted approaches, which results in a gene knockout animal, or the use of spontaneous mutations in animals that results in disease models for specific conditions. Mice occupy the key position in this expansion of transgenic animal development, but efforts continue to employ these and other technologies in rats and other species.
The development of genetically engineered mice is one of the most significant achievements that is moving biomedical research forward into a new area. This accomplishment, combined with the publication of the first drafts of the human and murine genome maps (see "Murine Genome Map Completed"), represents a pivotal step in understanding the genetic components of human diseases. Such understanding of the contributions of genes and their products to the pathogenesis of disease will further help identifying illnesses earlier, leading to advances in prevention, prognostication, and therapeutic development. The potential of altering the genetic makeup of laboratory mice allows researchers to create animal models that are not just "workable" approximations, but are, in fact, close replicas of the human disease under study. Genetic alterations in animal models allow the study of single or multiple gene effects of human diseases. However, since many complex diseases are multifactorial, not every single pathologic condition or genetic defect may be replicated in a genetically altered laboratory animal model.
A surprisingly large number of the estimated 30,000-40,000 human genes have murine equivalents. Scientists who study how these genes function in animal models will discover the roles that homologue animal genes play. Such new knowledge deduced from experiments using laboratory mice and their genetically engineered siblings, as well as other species, will allow scientists to study a wide variety of illnesses. It is expected that thousands of new mouse strains with altered genetic backgrounds will be generated and used within the next decade.
Among the many examples of genetically engineered mouse strains that have resulted in advances in biomedical sciences are the following models:
The Cystic fibrosis knockout mouse provides an outstanding animal model for cystic fibrosis (CF). Human CF is caused by mutations in the gene encoding the cystic fibrosis transmembrane regulator (CFTR). The CF knockout mouse, CFTR-/-, was created by expressing CFTR under an intestinal-specific promoter in a mouse that carries the mutated CF gene, thereby becoming a double transgenic animal. This site-specific expression of CFTR corrected the single-knockout animal's early death because they now show functional ileal goblet and crypt cells. The double CFTR-/- mouse maintains the pulmonary CF phenotype, thus providing an animal model for CF.11 Recent investigations demonstrated reversion of the lethal CF phenotype in this double CFTR-/- mouse model by in utero gene therapy using an adenovirus containing the CFTR gene.12
The atherosclerotic lesion mouse contributes to progress in the fight against atherosclerosis and related illnesses. Apo E3 transgenic mice express the human dysfunctional apo E variant of the apoli-poprotein. The Apo E3 transgenic mouse develops hyperlipidemia and atherosclerosis on a high fat/high cholesterol diet. Plasma cholesterol levels in the apo E3 mice increase in 4 months, and they develop atherosclerotic lesions in their aortas. Because these lesions resemble those in human patients with atherosclerosis, the apo E3 mouse is a valuable animal model for studies on the genetics, development, and therapy of atherosclerosis.13
The Amyotrophic Lateral Sclerosis (ALS) mouse serves as an animal model for Lou Gehrig's disease, which is caused by a mutation in the enzyme superoxide dismutase-1, or SOD-1. Transgenic mice expressing mutant forms of the SOD-1 gene, detectable in familial ALS human patients, display clinico-
pathological features of the disease. The SOD-1 mouse model has helped to elucidate the pathogenesis of ALS and establish new therapeutic agents.14 Similar studies to those that resulted in the creation of the SOD-1 mouse led to a model for another neurodegenerative disorder, Huntington's disease.15
Murine models with Germ-line p53 mutations contribute to a better understanding of tumorigenesis. A variety of different lines of mice carrying the p53 alteration on different background strains is available. Specifically, one mouse strain carrying a mutant p53 transgene (135Valp53) develops pulmonary tumors more frequently after exposure to carcinogens as compared to controls. Therefore, the mutant p53 transgene may have a negative effect, and the mouse model offers a potentially useful tool for studies on chemoprevention and chemotherapy.16
In April 2001, the private enterprise Celera Genomics announced that they completed sequencing the genome of three different mouse strains, covering 15.9 billion base pairs for approximately 99% coverage of the full murine genome.17 Among the three sequenced mouse strains, Celera identified about 2.5 million single-nucleotide polymorphisms, or SNPs. It is believed that this genetic diversity will help in the characterization of mouse models of human disease. Comparative genomics will enable researchers to align the assembled and annotated human genome18 and the assembled mouse genome19 to identify pathologic mechanisms of diseases and their therapeutic treatment via drug development.
The public-private Mouse Sequencing Consortium (MSC), an international effort composed of six NIH institutes, three private companies, and the Wellcome Trust, announced in May 2001 that it also has successfully completed the genetic map of the mouse. The data, which were generated using the same sequencing techniques — called shotgun approach — that produces random bits of sequences, are now being assembled into the finished sequence of the mouse genome. The annotated data in this murine genetic map will be made freely available for the unrestricted use of researchers worldwide.20,21
Nontraditional Species — Zebrafish, Xenopus, Drosophila
In recent years, increased attention has been devoted to studies using fish. Fish are used for several reasons, including the fact that many species are egg layers and produce large numbers of eggs on a frequent basis. These eggs are transparent and thus, early developmental events can be closely monitored. The effects of a variety of chemical and other stimuli can be detected early, and the pathogenesis of the resulting abnormalities can be dissected in controlled environments. Furthermore, a single pair of fish can produce an extraordinary number of offspring. The major types of fish used in such studies have been derived from species formerly based in the aquarium trade and include zebrafish (Danio rerio), swordtails and platys (Xiphophorus sp.), and the Japanese medaka. Large numbers of inbred stocks of animals have been developed and are used in genetic studies, including determination of those changes that correspond to genetic abnormalities in human and other mammalian populations. They are also used in large studies of chemical mutagenesis and carcinogenesis. Advances in the genomics of zebrafish are contributing significantly to these efforts.
Fish-based studies pose unique problems. Water quality is of paramount importance, and intercurrent diseases that affect the various fish species are often not well defined. However, advances continue to be made in these areas, especially in the large, centralized laboratories that serve as centralized repositories.
African clawed toads (Xenopus sp.) are other aquatic animals that are of considerable importance in studies of embryogenesis, early development, and of factors that can affect these events. Embryos of these species are accessible for surgical and chemical manipulations, and the effects of manipulating or removing specific cells in the early embryo can be determined. In Xenopus, early developmental events occur more slowly than in other species, and thus, they can be more easily dissected out. As frequently pointed out by the scientific community using Xenopus, much of the understanding of early embryonic development has come from studies done using these animals.
Work with the fruit fly, Drosophila melanogaster, has been carried out over many decades and has resulted in major advances in animal genetics. More recent efforts have focused on delineation of the Drosophila genome, which will then be followed by studies on expression of products of specific genes. As with many other nonmammalian species, the costs of maintaining individual animals are greatly reduced and large numbers of individuals can be used in studies.
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