A method for interfering with disease genes in experimental animals will enhance drug discovery for cancer and other disorders, said a Baylor College of Medicine researcher.

A new system called lentiviral pINDUCER enables researchers to turn a gene on or off whenever they want, said Dr. Thomas F. Westbrook, assistant professor of biochemistry and molecular biology, molecular and human genetics and pediatrics at BCM.

"This allows us to test any gene in the genome for its role in disease in live animals," said Westbrook, also a member of the NCI-designated Dan L Duncan Cancer Center at BCM.

Westbrook and collaborators from the National Institute of Environmental Health and Sciences in Research Triangle Park, N.C., and Harvard Medical School in Boston describe their work in an online article in the Proceedings of the National Academy of Sciences.

RNA interference

A lentivirus slowly but surely infects cells and integrates its own genetic material into the cell's genetic machinery, ensuring that the lentivirus and whatever it carries will be passed on to subsequent generations of cells. Westbrook and his colleagues use the lentivirus to take the critical piece of RNA into a cell. With special techniques, they can then turn on the RNA interference and watch as it stops a particular gene's activity.

Over the past decade, RNA interference (RNAi) has made it possible for scientists to study what happens in an animal or a tissue culture when a gene is turned off. Their research is based on that fact that a specific bit of RNA actually acts as a trigger to shut off a particular gene. This is called RNA interference.

The RNA that "interferes" with a gene is a single strand with a specific genetic code. It looks for a strand of messenger RNA that "complements" it. The alphabet of RNA is adenine (A,) uracil (U,) cytosine (C) and guanine. The A seeks out the U and the C seeks the G to make a strand. When the RNAi finds the messenger RNA that has a matching strand, they bind together. This prevents the messenger RNA from taking its genetic "message" to the protein-making machinery of the cell. In effect, it shuts the gene off, preventing the protein from being made.

Westbrook and his mentor, Dr. Stephen Elledge of Harvard Medical School, have developed many large RNAi "libraries" for use in mammalian studies. However, despite this important tool, there was still a problem in applying RNAi in studies of mice that grow human tumors.

Provides closer look

The inducible system enables scientists to study the tumor in a situation closer to that found in humans. The tumor grows in the animal, and then the researcher turns on the RNA interference, which shuts off the gene of interest. Then researchers can watch what happens to the tumor in the absence of that gene or protein.

"This most closely reproduces what happens in a patient," said Westbrook. "You give the patient a drug after the tumor is established."

They can use their toolkit to infect almost any kind of tissue and turn off the particular gene of interest, said Westbrook.

The lentivirus is particularly valuable because it becomes part of the cell's genome. If you put it in a single tumor cell and put that cell into a mouse, every other tumor cell has the same lentivirus.

"When we turn off our gene of interest, we can do that in every cell," said Westbrook.

Provides visible marker

Specific tools in the kit that can cause different parts of the cell to fluoresce green or red give them a visible marker of how they are inducing the RNA interference within the cell.

"This is a direct step toward doing genetic screens for cancer genes in living animals," said Westbrook.

Others who took part in this work include Kristen Meerbrey, Dr. Jessica Kessler, Kevin Roarty, Justin Fang, Jason Herschkowitz, Tingting Sun, Earlene Schmitt, Dr. Ronald Bernardi, Dr. Christopher Bland, Dr. Tom Cooper and Dr. Jeffrey Rosen, all of BCM; Mingwei Li, Anna Burrows and Alberto Ciccia, both of Harvard; and Guang Hu of the NIEHS.

Funding for this work came from The V Foundation for Cancer Research, the Mary Kay Ash Charitable Foundation, the Susan B. Komen for the Cure Foundation, the Specialist Program of Research Excellence from the National Cancer Institute, the National Institutes of Health, the U.S. Army and the Howard Hughes Medical Institute.