One of the most remarkable technical advances in biomedical research
has been the development of transgenic mice. Transgenic mice can be made
by using a transgene constructed with recombinant DNA techniques to randomly
insert a new gene into the genome of the mouse or by mutating a targeted
gene. A randomly inserted transgene usually adds to the genetic repertoire
of the animal, whereas a targeted mutation usually produces a functional
"knock-out" of a gene. Transgenic mice are a unique tool for understanding
how interactions between individual genes and the environment affect human
health.
Randomly Inserted Transgenes
Most transgenic mice are made by injecting a transgene into fertilized
eggs. The eggs are removed from the oviduct, and an ultrafine glass pipet
is used to inject a solution containing a few hundred copies of the transgene
DNA into the nucleus. The injected eggs are put into the oviduct of a surrogate
mother, and pups are born 19 days later. Some of these mice will have the
transgene into their genome and their descendants for generations to follow
will also carry the transgene. A mouse receiving the transgene is referred
to as a transgenic founder; its descendants are members of a genetically
identical transgenic line.
Mighty Mouse.
Transgenic mice are powerful tools for solving human gene puzzles.
The methods to make transgenic mice were developed in the early 1980s
by John Gordon and by Ralph Brinster and Richard Palmiter and their co-workers.
The first use of transgenic mice was to study gene function in the whole
animal, in particular, how and why a specific gene is "turned on"
in some tissues and "turned off" in others. Although all of the
cells in the body contain an identical set of genes, some genes are active
in only one or a few tissues. It is this diversity of gene expression that
produces the distinct cell types and tissues of the body, making a muscle
cell different from a liver cell.
The two main parts of a gene are the regulatory region and the protein-coding
region. When the right combination of proteins binds to specific sites along
the DNA in the regulatory region, the gene is switched on, and the protein-coding
region becomes active. Transgenic mice have been used to identify the exact
parts of the regulatory region that turn a gene on in a particular tissue.
To do this, a reporter gene is made by fusing different parts of the regulatory
region of one gene onto the protein-coding region of another gene. The protein-coding
region is usually taken from a gene not found in the mouse, often the B-galactosidase
gene of bacteria. The B-galactosidase gene codes for an enzyme that produces
a blue color when exposed to a specific chromogenic substrate called X-Gal.
Only those mice that receive the part of the regulatory region needed to
activate the gene will develop the blue color in the appropriate tissue.
The knowledge gained about the regulation of expression of specific genes
using transgenic mice is likely to have important implications for future
environmental health research. For example, the level of activity of a gene
that codes for a detoxifying enzyme may determine a person's susceptibility
to a toxic environmental agent. This hypothesis could be tested in different
lines of transgenic mice designed to express the gene at high or low levels.
Such mice might also be used to detect when exposure occurs, define the
mechanisms leading to the toxic effects, and develop treatments for individuals
exposed to the agent.
Transgenic mice have also proven to be valuable for understanding the
roles of proteins. Using transgenic mice, is possible to increase or decrease
the amount of specific proteins, express proteins at a different time or
in a different tissue than normal, and test the function of a modified protein.
For example, one of the first transgenic mice lines had a transgene with
the regulatory region of the mouse metallothionein gene spliced to the coding
region of the rat growth-hormone gene. These mice grew to be twice as large
as normal mice. This study dramatically demonstrated that the protein coded
by the transgene was functional in the mouse and that rat growth hormone
produced an addictive effect on mouse growth hormone.
How to build a better mouse. Scientists are using two innovative techniques to create transgenic
animals.
Transgenic mice offer new ways to identify and characterize environmental,
occupational, genetic, dietary, and other factors that induce neoplastic
processes. Two classes of genes have been identified that influence tumor
formation: Tumor-suppressor genes act in a negative manner to control cell
growth, and oncogenes appear to function in a positive fashion. One gene
being studied is the p53 tumor-suppressor gene, whose product appears to
be involved in maintaining genomic stability. Mutations that inactivate
the p53 gene are the most common genetic alteration observed in a wide variety
of human cancers. Compared to normal animals, mice that carry a mutant p53
transgene are much more susceptible to lung tumor formation after treatment
with a chemical carcinogen. Tumors from these mice are being examined to
identify additional tumor-suppressor genes that interact with the transgene.
These studies may identify novel genes involved in cancer development and
help determine the relationship between genetic diversity and variable susceptibility
to cancer.
The protein-coding region of oncogenes has been introduced into mice
with regulatory sequences that target certain tissues and allow activation
of the transgene by a specific chemical or genetic mutation. For example,
transgenic mice carrying an activated oncogene that is sensitive to carcinogenic
and promoter effects of environmental chemicals applied to the skin are
being studied. These mice respond to certain chemicals with a different
pattern of papillomas or carcinomas than animals lacking the transgene.
Such transgenic mice provide a sensitive way to study the rate and pattern
of tumor formation, the range of tissues affected, and the interaction of
different factors that influence tumor formation. Researchers hope to make
some transgenic mice more sensitive to the carcinogenic effects of certain
chemicals than other animals to more rapidly detect carcinogens.
Big Blue transgenic mice mice were developed in a collaborative effort
between NIEHS and Stratagene. All of the cells in these mice contain an
inactive B-galactosidase reporter transgene. Exposure of the mice to mutagenic
chemicals causes mutations that activate the transgene. These mutations
are identified by isolating DNA from various transgenic mouse tissues, transferring
it to bacterial hosts, and scoring the activity of B-galactosidase in
vitro This approach is a relatively rapid and efficient way to screen
in vivo for chemical mutagens. In addition, mouse DNA can be extracted
from the bacterial colonies, and the specific nature of the mutation can
be determined from the nucleotide sequence.
increased - genetic - repertoire
Transgenic mice have also been used to identify previously unknown genes.
Transgenes are incorporated randomly into the genome of the fertilized egg
and occasionally disrupt a gene by being inserted into its regulatory or
protein-coding region. The result is mice with genetic mutations such as
limb deformities, infertility, reversal of left-right asymmetry, or lethal
developmental anomalies. However, the transgene serves as a marker for the
site of mutation and can be used to clone the disrupted gene. This allows
identification of a candidate gene for comparable birth defects in humans.
Such findings have led to unique approaches for identifying genes involved
in development. In "gene trapping," transgenes that lack regulatory
sequences but that contain the bacterial B-galactosidase coding region are
injected. These transgenes are expressed when they are incorporated into
a location in the genome that allows them to be activated by the regulatory
portion of a gene. The X-Gal labeling approach can then be used to define
the temporal and spatial expression of the trapped gene, and the transgene
can be used as a marker for cloning that gene.
The NIEHS, National Academy of Sciences Institute of Laboratory Animal
Resources (ILAR), and Oak Ridge National Laboratory Human Genome Group have
combined efforts to establish a database that catalogs information on transgenic
animals. Researchers use standardized nomenclature to submit information
about their transgenic animals lines. The computerized database includes
details of the transgenic constructs, expression patterns of transgenes,
the morphological and functional effects of the transgenes, and other information
about the genetics and availability of the animals. [For additional information
about the transgenic animal database contact Karin Schneider at Oak Ridge
National Laboratories, (615) 574-6529.]
Targeted Gene Mutations
It became possible to mutate specific genes in the late 1980s with the
convergence of advances in developmental, reproductive, and molecular biology.
Targeted mutations have allowed the development of mouse models of human
genetic diseases and tests of the roles of specific genes by causing loss
of function ("gene knockout"). One key technical advance was the
development of methods for growing embryonic stem cells from mouse embryos
in tissue culture. When embryonic stem cells are injected into mouse embryos
at an early stage of development, they affect all the tissues of the mouse.
Embryos are removed on the third day of development, injected with embryotic
stem cells, and transferred to surrogate mothers. Some of the mice born
are chimeras, composed of a mixture of cells from the original embryo and
from embryonic stem cells. The sperm or eggs in the chimeric mouse give
rise to offspring carrying the embryonic stem cell genes.
The other key advance in producing mice with genes knocked out was techniques
to cause mutations that block the function of specific genes in embryonic
stem cells. This first requires cloning the gene and modifying it using
genetic engineering methods. The modifications inactivate the normal function
of the gene, but add the ability to inactivate a toxic drug. The normal
gene in an embryotic stem cell is replaced with the modified gene, by a
procedure known as homologous recombination. Embryonic stem cells that contain
the modified gene are selected by their resistance to the drug and subsequently
injected into mouse embryos to make chimeric mice.
Coat-color markers are used to identify the mice carrying the genes from
embryonic stem cells. Most embryonic stem cells were originally derived
from mice with white coats. When the embryonic stem cells are injected into
embryos from mice with a black coat, the chimeric mice that result have
a mixture of black and white or light brown hair. When these chimeric mice
are mated to mice with a black coat, the offspring receiving a copy of the
genes from embryonic stem cells often have tan coats. However, other coat
colors are possible for offspring of the original chimeric mouse due to
the multiple genes involved in determining mouse coat colors. Since an offspring
of a chimeric mouse inherits only one copy of each embryonic stem cell chromosome,
additional matings are necessary to produce offspring with two copies of
the mutated gene in the absence of the normal gene.
The gene responsible for cystic fibrosis was one of the first to be targeted
for knock-out to produce a mouse model of a human disease. Cystic fibrosis
is caused primarily by a defective chloride-channel gene. Beverly Koller
at the University of North Carolina School of Medicine successfully targeted
a mutation to this gene. The mice produced have defects in chloride secretion
and sodium absorption that affect the fluid environment of the lining of
the lungs and airways and other epithelial tissues such as those in the
digestive and reproductive tracts. These mice have been a major advantage
in studying cystic fibrosis. The mice are being used to better understand
how mutations in the gene lead to the disease, to test different drug treatments,
and to develop gene therapy strategies.
Inherited mutations in the p53 tumor-suppressor gene give rise to the
Li-Fraumeni syndrome in humans, characterized by a profound susceptibility
to several forms of cancer. When the p53 gene was mutated in mice by Lawrence
Donehower and Allan Bradley at the Baylor College of Medicine, these animals
developed normally but were prone to spontaneous development of a variety
of neoplasms by 6 months of age. About 75% of mice with mutations that inactivate
both copies of the p53 gene develop multiple types of tumors by this age.
One of the surprising results of many gene knock-out studies is that
the tissues in which the proteins are usually expressed are not disrupted,
and there are no other obvious effects of the mutation. An explanation frequently
proposed has been that some proteins are redundant, and closely related
proteins can replace the ones eliminated. Other explanations are that the
animal can function normally without the protein, that loss of a protein
might up regulate compensatory mechanisms, or that proteins may be expressed
in tissues where they have no function at all. Harold Erickson of Duke University
argues that protein synthesis is cheap from the perspective of cellular
metabolism, but that gene control mechanisms are expensive. He suggests
that unless the protein is deleterious to the cell, there is no compelling
reason for the cell not to make a superfluous or junk protein. However,
the number of examples where gene knock-outs have no obvious effects are
small, and it remains to be seen if there are subtle but important effects
in these animals.
Gene knock-out technology is still at an early stage of development and
is difficult and inefficient. To make it more usable, the availability and
stability of embryonic stem cell lines, the efficiency of targeting mutations
to specific genes in embryonic stem cells, and the frequency by which chimeras
produce offspring that carry the gene knock-out must be improved. The recent
development of embryonic germ cell lines from primordial germ cells may
reduce some of these technical problems. In addition, gene knock-outs are
often made by removing part of a gene and inserting a piece of foreign DNA
in its place, using a drug-resistance gene isolated from bacteria. Continuing
improvements in technology are allowing more subtle changes to be introduced
into genes, such as producing specific deletions or substitutions in the
regulatory or coding regions that mimic human disease mutations without
permanently inserting foreign DNA. As more genes are identified from experimental
studies and the human genome project, transgenic mice will provide more
powerful ways to test how genes interact with the environment to affect
human health.
Mitch Eddy
Suggested Reading
First N, Haseltine FP. Transgenic animals. Stoneham, UK:Butterworth-Heinmann,
1991.
Grosveld F, Kollias G. Transgenic animals. San Diego, California: Academic
Press, 1992.
Hogan B, Costantini F, Lacy E. Manipulating the mouse embryo. Cold Spring
Harbor, New York:
Cold Spring Harbor Laboratory, 1986.
Robertson EJ. Teratocarcinomas and embryonic stem cells: a practical
approach.
Oxford:IRL Press, 1987. |
Mitch Eddy is head of the Gamete Biology Group in the Laboratory of Reproductive
and Developmental Toxicology at NIEHS.
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Last Update: August 26, 1998