A moth’s gene helps discern gene functions

A piece of DNA from a moth could signal a major leap in the understanding of what specific genes do.

A new tool for genome research, developed in the Yale laboratory of Tian Xu, Ph.D. ’90, professor and vice chair of genetics, professor of molecular oncology and development and a Howard Hughes Medical Institute investigator, promises to greatly accelerate the work of assigning purpose to thousands of unexplored human genes.

The tool is a jumping gene, a small piece of DNA called a transposon that moves around the genome, usually settling in other genes and allowing scientists to suppress the activity of existing genes or insert new ones.

Transposons are active in many plant and insect genomes and have helped to make the fruit fly Drosophila the darling of geneticists, as these mobile DNA fragments were used to decipher the role of nearly every gene in that model organism. But for decades scientists could not find an equivalent transposon for mammals.

As reported in the August 12 issue of the journal Cell, Xu and his colleagues manipulated a transposon called piggyBac, found in the cabbage looper moth, so that it can be easily cut and pasted into the genomes of higher organisms, including mice and humans. “With this transposon, we now have the ability to systematically inactivate each and every gene in a model organism like the mouse,” Xu said.

In mouse studies, scientists have traditionally used chemicals to modify genes, but this approach is painstakingly slow, and it can be difficult to locate the genes that have been mutated. The piggyBac transposon, when injected into fertilized mouse eggs along with an enzyme called transposase, is remarkably efficient at inserting itself into important coding regions of the genome, and as its name implies, it carries genetic tags that allow researchers to locate mutations quickly.

Moreover, piggyBac has the added feature of total reversibility, which should allow scientists to verify that particular mutations have particular effects. In the presence of transposase, piggyBac easily hops into genes, and it remains in place in any offspring in subsequent generations that do not inherit the enzyme. But when these mice are mated with others who carry the transposase gene, piggyBac hops back out of genes without leaving a trace.

These traits make piggyBac a “dream tool” for geneticists, Xu said. “This new technology will completely change the game of using mutagenesis to understand the function of mouse genes and, by extension, their human counterparts.” PiggyBac could also be a promising new vehicle for human gene therapy, according to Xu, who said that, in addition to carrying tags, piggyBac can be engineered to carry new genes into the genome.

To demonstrate the potential of this genetic piggybacking, Xu and his colleagues used piggyBac to insert a gene for a protein that glows red under ultraviolet light into a mouse. However, many more experiments will be required to determine whether the transposon, or some variation of it, could reliably and safely transfer therapeutic genes to humans.

Xu’s immediate goal is to use piggyBac to inactivate every gene in the mouse, one by one, a project that would be unthinkable with traditional mutagenesis methods. “For the past two decades, it has routinely taken about a year to mutate one gene in a mouse, and altogether about 3,000 genes have been knocked out in mice, out of a total of about 25,000 that are in the genome,” Xu explained. With the help of piggyBac, he said, “in three months, with two students, we have done 75 genes.”

—Pat McCaffrey

Using lasers to determine cell function

Through genetic tweaking, researchers at the School of Medicine have made fruit flies walk, jump and fly on command—by flashing a light at them. The scientists inserted rat ion channels into nerve cells that control flies’ escape movements, then injected the flies with a chemical that would activate the ion channels when exposed to light. Gero A. Miesenböck, M.D., associate professor of cell biology at the medical school, who led the study that appeared in the journal Cell in April, said that the research offers a new way to learn how nerve cells govern behavior.

A livestock virus may offer a new approach to treating glioblastoma

Viruses are hijackers, wreaking infectious havoc by taking over a cell’s machinery and using it to replicate. But their wily ways may not be all bad. Yale professor of neurosurgery Anthony van den Pol, Ph.D., is harnessing their destructive power to develop a novel treatment for glioblastoma, the most common and aggressive form of brain cancer.

Glioblastoma strikes about 7,000 Americans each year, and most patients live just a year after diagnosis. Although it can be treated with surgery, radiation and chemotherapy, said van den Pol, the cancer usually comes back.

The idea of unleashing viruses to destroy tumor cells is beginning to gain validity, not just for brain cancers but also for ovarian, prostate and other kinds of tumors. When this line of research began two decades ago, scientists feared that the viruses would spread to healthy cells, so they genetically altered them to prevent them from replicating. But those inactivated viruses kill relatively few brain tumor cells. The Yale team hypothesized that a replicating virus would be much more effective.

Van den Pol and his colleagues bred several generations of vesicular stomatitis virus on glioblastoma cells, selecting for strains with the highest tumor-killing capacity. Then they tested the virus and saved those strains that did not infect normal cells. In a study published in the Journal of Virology in May, the researchers reported that the strain they developed selectively killed glioblastoma cells in vitro, and was able to infect and kill whole tumors in mice.

The results are promising, but van den Pol stressed that they are still preliminary. The team plans to expose the virus to different types of cells found in the brain to make sure that it will not infect them. “This is a high-risk strategy,” he said, “but we’re dealing with a disease for which at present there is no cure.”

—Alla Katsnelson

et cetera

New target for melanoma

Using a technology devised at Yale five years ago, researchers have found what may be a new target for treatment of melanoma. The Yale team used AQUA (automated quantitative analysis) to measure protein expression in melanoma tissue microarrays. In a study in Nature in July they reported that microphthalmia-associated transcription factor (MITF), a protein involved in cell survival, abnormally copies itself many times over. This over-expression was prevalent in metastatic disease and correlated with decreased rates of patient survival.

This suggests, said David L. Rimm, M.D., Ph.D., HS ’93, an author of the study and associate professor in the Department of Pathology, that MITF may represent a distinct class of oncogene that is necessary for tumor progression. Reduction of MITF activity sensitizes melanoma cells to chemo-therapeutic agents, and targeting MITF in combination with other drugs may offer a new approach to treating melanoma.

—John Curtis

A toll on infections

In 1997 the late Charles A. Janeway Jr., M.D., and Ruslan M. Medzhitov, Ph.D., professor of immunobiology, discovered toll-like receptors, or TLRs, molecules that alert the body’s acquired immune system to the presence of microbial or viral invaders.

Scientists have since identified over a dozen types of TLRs, which detect proteins in bacteria and viruses but not those in the eukaryotic cells that make up our bodies. Some pathogens, however, are also eukaryotes, and a team at Yale and the National Institutes of Health wondered whether TLRs could recognize them.

In the June 10 issue of the journal Science, the group reported that TLR11, discovered in mice at Yale last year, triggers an immune response after it detects a protein in the virus that causes toxoplasmosis.

Team member Sankar Ghosh, Ph.D., professor of immunobiology, said that while it is not yet clear whether humans have a functional version of TLR11, these studies should lead to development of novel strategies to combat these infections.

—Peter Farley


			Originally published in Yale Medicine, Autumn 2005. Copyright © 2005 Yale University School of Medicine. All rights reserved.