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