Living mice glow when
genes turn on
BY TIM STEPHENS
Borrowing glow-in-the-dark
chemistry from fireflies, scientists have developed a
technique enabling them for the first time to observe
gene activation as it occurs in a living mammal. In a
series of experiments performed at the School of Medicine
in mice and rats, the researchers used ultrasensitive
cameras to detect light emitted when certain genes were
turned on, even deep inside the animals' bodies.
The technique may find use
as a way of monitoring gene therapies, tracking
infectious processes in various diseases, possibly
including AIDS, and studying gene expression in animals
as they grow and develop, said Christopher Contag, acting
assistant professor of pediatrics and director of
bioluminescence research at Stanford.
"This is a powerful
approach for looking at any number of biological
phenomena, because you can study gene regulation in a
living animal over time, in superficial or deep
tissues," Contag said.
The investigators describe
the technique in the October issue of the scientific
journal Photochemistry and Photobiology. Contag's
co-authors include six researchers associated with the
Department of Pediatrics: research assistant Stanley D.
Spilman; visiting scholar Dr. Pamela Reilly Contag, CEO
of Xenogen Corp.; research assistant Brian F. Eames (now
at UCSF); assistant professor Dr. Phyllis Dennery; Dr.
David Stevenson, the Harold K. Faber Professor; and
assistant professor Dr. David A. Benaron.
Natural bioluminescence,
such as the glow of fireflies, happens when the enzyme
luciferase reacts with an energy-rich chemical called
luciferin to generate light. Contag and his co-workers
used the firefly luciferase gene and a genetic promoter
to generate a molecular "light switch" that
revealed when gene activation occurred in their
experimental systems.
One set of experiments
tested the use of luciferase as an indicator light for
monitoring gene therapy. The idea behind gene therapy is
to transfer healthful genes into patients with diseases
caused by genetic defects, such as cystic fibrosis.
Contag's group transferred
the luciferase gene into the lungs of rats. After
injecting luciferin into the rats, the researchers were
able to detect, from outside of the animal, light coming
from the lungs. The glow indicated that the luciferase
gene was active.
The new technique will
help researchers know when they have succeeded in
transferring a potentially therapeutic gene into lab
animals. In the past, this has often required analyses of
tissue biopsies or postmortem analysis to look for the
products of the transferred gene. "But then you no
longer have that animal to study to find out if the
therapy was effective, so you need many more groups of
animals in order to evaluate efficacy," Contag said.
By linking the luciferase
gene to a therapeutic gene, researchers can now use a
sensitive imaging device as a noninvasive way to monitor
the success of gene transfers in living animals.
"You can reduce the
number of animals used for experiments tenfold while
getting more information more quickly," Contag said.
"This will help streamline development of many types
of therapies, including DNA-based gene therapies and gene
vaccines."
His group performed
another set of experiments in mice genetically altered to
carry the luciferase gene in their chromosomes. In these
transgenic mice, created by Dr. John Morrey at Utah State
University, expression of the luciferase gene is
controlled by the regulatory region of the genome of HIV
(the virus associated with AIDS).
The Stanford researchers
used a chemical signal to switch on the HIV regulatory
region, then detected the glow of luciferase activity in
the cells of various tissues. They were able to detect
luciferase activity both in the skin and in deeper
tissues such as the colon after chemical induction or
after activation by bacteria in the gut. Weak signals
originating from especially dense tissues, such as the
liver, might not be detectable, Contag noted.
The HIV regulatory region
in this experimental model is more than just a convenient
tool for developing the technology, he added. Scientists
around the world are hoping to develop a mouse model for
studying replication of the AIDS virus. The ability to
study HIV in a small animal, rather than using
chimpanzees or other primates, would be a major
breakthrough for AIDS researchers. "But to have an
indicator light in these animals that tells you where and
when the virus is replicating in the living animal, in
real time would be amazing," Contag said.
"Such a tool would
have tremendous impact on antiviral drug
development," he said.
In their studies, Contag's
group also used luciferase as an indicator of HIV
replication in cell cultures. This technique could be
used to develop a rapid assay for screening large numbers
of potential antiviral drugs, Contag said.
Other areas in which he
plans to use the molecular light-switch technique include
studies of tumor progression, gene expression in cancer
cells, cancer therapies, and host responses to bacterial
and viral infections.
The work was supported in
part by the California Universitywide AIDS Research
Program, the Stanford Office of Technology Licensing, the
Office of Naval Research, the Baxter Foundation, Mary L.
Johnson, and the Packard Fund at Lucile Packard
Children's Hospital. SR
|
