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The Use of Animal Models in Studying Genetic Disease: Transgenesis and Induced Mutation

By: Danielle Simmons, Ph.D. (Write Science Right) © 2008 Nature Education 
Citation: Simmons, D. (2008) The use of animal models in studying genetic disease: transgenesis and induced mutation. Nature Education 1(1):70
You are more like a mouse than you might think! Today, scientists are creating models of human genetic disease using mice, flies, worms, and other animals. But what do these models reveal about us?
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Except in the case of highly controlled and regulated clinical trials, geneticists and scientists do not use humans for their experimental investigations because of the obvious risk to life. Instead, they use various animal, fungal, bacterial, and plant species as model organisms for their studies. Some such species are described in Table 1.

Model Organism Common Name Research Applications
Saccharomyces cerevisiae Yeast Used for biological studies of cell processes (e.g., mitosis) and diseases (e.g., cancer)
Pisum sativum Pea plant Used by Gregor Mendel to describe patterns of inheritance
Drosophila melanogaster Fruit fly Employed in a wide variety of studies ranging from early gene mapping, via linkage and recombination studies, to large scale mutant screens to identify genes related to specific biological functions
Caenorhabditis elegans Roundworm (nematode) Valuable for studying the development of simple nervous systems and the aging process
Danio rerio Zebra fish Used for mapping and identifying genes involved in organ development
Mus musculus House mouse Commonly used to study genetic principles and human disease
Rattus norvegicus Brown rat Commonly used to study genetic principles and human disease
Table 1: Models used to study genetic principles and human diseases

When animal models are employed in the study of human disease, they are frequently selected because of their similarity to humans in terms of genetics, anatomy, and physiology. Also, animal models are often preferable for experimental disease research because of their unlimited supply and ease of manipulation. For example, to obtain scientifically valid research, the conditions associated with an experiment must be closely controlled. This often means manipulating only one variable while keeping others constant, and then observing the consequences of that change. In addition, to test hypotheses about how a disease develops, an adequate number of subjects must be used to statistically test the results of the experiment. Therefore, scientists cannot conduct research on just one animal or human, and it is easier for scientists to use sufficiently large numbers of animals (rather than people) to attain significant results.

Rodents are the most common type of mammal employed in experimental studies, and extensive research has been conducted using rats, mice, gerbils, guinea pigs, and hamsters. Among these rodents, the majority of genetic studies, especially those involving disease, have employed mice, not only because their genomes are so similar to that of humans, but also because of their availability, ease of handling, high reproductive rates, and relatively low cost of use. Other common experimental organisms include fruit flies, zebra fish, and baker's yeast.

Methods of Inducing Human Disease in Other Organisms

Despite their genomic similarities to humans, most model organisms typically do not contract the same genetic diseases as people, so scientists must alter their genomes to induce human disease states. In attempting to engineer a genetic mouse model for a human disorder, for example, it is important to know what kind of mutation causes the disease (for example, is the disease null, hypomorphic, or dominant negative?), so that the same kind of mutation can be introduced into the corresponding mouse gene.

Scientists approach this task in two main ways: one that is directed and disease driven, and the other that is nondirected and mutation driven (Hardouin & Nagy, 2000). The nondirected, mutation-driven method uses radiation and chemicals to cause mutations. One common technique associated with this method is the large-scale mutation screen. On the other hand, the directed, disease-driven approach can employ any one of a number of techniques, depending on the exact type of mutation involved in the disease under study. Common directed techniques include transgenesis, single-gene knock-outs and knock-ins, conditional gene modifications, and chromosomal rearrangements.

Large-Scale Mutation Screens

As the name might imply, indirect approaches attempt to randomly make mutations in animal models' genomes. Then, the animals are screened in an attempt to determine which ones show phenotypes that are similar to human diseases. Thus, instead of being driven by the disease mutation, these methods are based on screening the phenotypes.

Two of the most effective ways to generate mutations are by exposing organisms to X-rays or to the chemical N-ethyl-N-nitrosourea (ENU). X-rays often cause large deletion and translocation mutations that involve multiple genes (Bedell et al., 1997a), whereas ENU treatment is linked to mutations within single genes, such as point mutations (Hardouin & Nagy, 2000). ENU can produce mutations with many different types of effects, such as loss and gain of function (Rosenthal & Brown, 2007), and it is frequently employed in screens in model organisms such as zebra fish. These types of models are particularly useful for identifying new genes and pathways that contribute to disease.


As opposed to the use of X-rays and ENU, transgenesis is a directed approach. Transgenic animals are generated by adding foreign genetic information to the nucleus of embryonic cells, thereby inhibiting gene expression. This can be achieved by either injecting the foreign DNA directly into the embryo or by using a retroviral vector to insert the transgene into an organism's DNA. The first mouse gene transfers were performed in 1980 (Hardouin & Nagy, 2000); however, at that time, the methods for transgenesis were not optimal. For instance, the foreign DNA was incorporated into only a small percentage of embryos and was inconsistently passed to the next generation. Also, small transgenes were inserted into random sites in the genome, and depending on their location, they weren't always expressed. More recently, scientists have developed a way to increase the size of the DNA fragments used in transgenesis by cloning them in yeast or bacterial artificial chromosomes (YACs or BACs, respectively). These larger transgenes are more likely to contain regulatory sequences necessary for normal gene expression and are usually more comparable to the endogenous gene (Bedell et al., 1997a). As a result, the use of transgenic mice has dramatically increased in the past two decades, and this type of animal model has contributed greatly to our knowledge of disease development.

Single-Gene Knock-Outs and Knock-Ins

Both knock-out and knock-in models are ways to target a mutation to a specific gene locus. These methods are particularly useful if a single gene is shown to be the primary cause of a disease. Knock-out mice carry a gene that has been inactivated, which creates less expression and loss of function; knock-in mice are produced by inserting a transgene into an exact location where it is overexpressed. Over the years, more than 3,000 genes have been knocked out of mice, and most of these genes have been related to disease (Hardouin & Nagy, 2000).

Both knock-out and knock-in animals are created in the same way: a specific mutation is inserted into the endogenous gene, and then it is conveyed to the next generation through breeding. The use of embryonic stem (ES) cells is required for this technology. This is because ES cells can contribute to all cell lineages when injected into blastocysts, and they can be genetically modified and selected for the desired gene changes. Homologous recombination creates the mutations; this is a process that physically rearranges two strands of DNA for the exchange of genetic material. Many types of mutations can be introduced into a model gene in this way, including null or point mutations and complex chromosomal rearrangements such as large deletions, translocations, or inversions (Bedell et al., 1997a). Many knock-out and knock-in mice have similar, if not identical, phenotypes to human patients and are therefore good models for human disease.

Conditional Gene Modifications

One drawback to using transgenic, knock-in, and knock-out mice to study human diseases is that many disorders occur late in life, and when genes are altered to model such diseases, the mutations can profoundly affect development and cause early death. These effects would preclude using animal models to study adult diseases. Thankfully, new technology has made it possible to generate mutations in specific tissues and at different stages of development, including adulthood. To do this, mice with two different types of genetic alterations are needed: one that contains a conditional vector, which is like an "on switch" for the mutation, and one that contains specific sites (called loxP) inserted on either side of a whole gene, or part of a gene, that encodes a certain component of a protein that will be deleted (Bedell et al., 1997a). A conditional vector for the gene is made by inserting recognition sequences for the bacterial Cre recombinase (loxP sites) using homologous recombination in ES cells. The vector contains a drug-resistant marker gene that allows only the targeted ES cells to survive when exposed to the drug. Thus, the mutant ES cells can be selected and injected into the host mouse embryo, which is implanted into a foster mother. The resulting offspring are chimeras and have multiple populations of genetically distinct cells. Chimeric offspring are then crossed, and the resulting generation of offspring has the recombinase effector gene. The mice containing the Cre recombinase under the control of tissue-specific or inducible regulatory elements are crossed to the mice with the desired loxP sites. When Cre is expressed, recombination occurs at the loxP sites, which delete the intervening sequences, and the resulting mutation is induced in specific regions and times. These conditional mutant models are becoming increasingly popular, and international initiatives have been created to accommodate their demand (Rosenthal & Brown, 2007).

Chromosomal Rearrangement

The aforementioned advances in ES cells and Cre/loxP conditional mutations have helped pave the way for the creation of models for complex human diseases involving chromosomal rearrangements. Mouse models of these disorders can be created using indirect approaches, such as radiation, but their usefulness is restricted because pathological endpoints are unpredictable and undefined (Yu & Bradley, 2001). Using the Cre/loxP recombination system overcomes these setbacks by allowing site-specific mutations necessary to produce accurate models of defects caused by human chromosomal rearrangements. These mutations can include chromosome deletions, duplications, inversions, and translocations, as well as nested chromosome deletions.

Mouse Models Exist for Many Human Genetic Diseases, but Are They Effective?

Human genetic diseases affect a wide range of tissues throughout the body and are caused by numerous types of mutations. Creating mouse models for all these disorders is understandably a daunting task for scientists. Nevertheless, over 1,000 mutant strains exist, and most of these mutants are models for inherited genetic diseases (Hardouin & Nagy, 2000). Models for human disease have been made by mutating the same gene in mice that is responsible for the human condition for about 100 genes (Bedell et al., 1997b), and in most cases, these models replicate many of the corresponding human disease phenotypes. These diseases include several types of cancer, heart disease, hypertension, metabolic and hormonal disorders, diabetes, obesity, osteoporosis, glaucoma, skin pigmentation diseases, blindness, deafness, neurodegenerative disorders (such as Huntington's or Alzheimer's disease), psychiatric disturbances (including anxiety and depression), and birth defects (such as cleft palate and anencephaly) (Rosenthal & Brown, 2007).

Animal models have greatly improved our understanding of the cause and progression of human genetic diseases and have proven to be a useful tool for discovering targets for therapeutic drugs. Nonetheless, despite promising results with certain preclinical treatments in animal models, the same treatments do not always translate to human clinical trials. As a result, many diseases are still incurable. Most available animal models are made in mice, and they recreate some aspects of the particular disease. However, few, if any, replicate all the symptoms. This statement is particularly true for neurodegenerative diseases, most of which involve cognitive deficits. One reason that mouse models might not completely mimic human disorders is that mice simply might not be capable of expressing some cognitive human disease symptoms that are apparent to the observer. For example, Huntington's disease patients show dyskinesia (involuntary movements), whereas mice do not. Perhaps using nonhuman primates might alleviate some of these discrepancies because their physiology is closer to that of humans. In fact, some researchers have pursued this possibility despite the technical difficulties and additional costs to perform transgenesis in primates (Wolfgang & Golos, 2002). For example, a transgenic model of Huntington's disease was recently developed using rhesus macaques that replicated some of the characteristic pathologies of the disorder as it occurs in humans (Yang et al., 2008). Indeed, because of the tremendous genetic resources that are currently available, use of nonhuman primate models might become more accessible and might lead us into a new era of disease research and drug discovery.

References and Recommended Reading

Bedell, M. A., et al. Mouse models of human disease. Part I: Techniques and resources for genetic analysis in mice. Genes and Development 11, 1-10 (1997a)

———. Mouse models of human disease. Part II: Recent progress and future directions. Genes and Development 11, 11-43 (1997b)

Hardouin, S. N., & Nagy, A. Mouse models for human disease. Clinical Genetics 57, 237-244 (2000)

Rosenthal, N., & Brown, S. The mouse ascending: Perspectives for human-disease models. Nature Cell Biology 9, 993-999 (2007) doi:10.1038/ncb437 (link to article)

Wolfgang, M. J., & Golos, T. G. Nonhuman primate transgenesis: Progress and prospects. Trends in Biotechnology 20, 479-484 (2002) doi:10.1016/S0167-7799(02)02052-8

Yang, S. H. et al. Towards a transgenic model of Huntington's disease in a non-human primate. Nature 453, 921-924 (2008) doi:10.1038/nature06975 (link to article)

Yu, Y., & Bradley, A. Mouse genomic technologies: Engineering chromosomal rearrangements in mice. Nature Reviews Genetics 2, 780-790 (2001) doi:10.1038/35093564 (link to article)


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