Journal of the National Cancer Institute Advance Access originally published online on December 25, 2007
JNCI Journal of the National Cancer Institute 2008 100(1):6-10; doi:10.1093/jnci/djm301
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© Oxford University Press 2007.
NEWS |
MEDAL FOR MOUSE MEDDLING
Knockout Mouse Creation Wins Nobel Prize
The ability to delete or mutate any gene of interest in mice has transformed the landscape of mammalian biology research. In early October, the Nobel Prize committee acknowledged the work of three scientists who pioneered gene targeting in mice: Mario R. Capecchi, Ph.D., Martin J. Evans, D.Sc., and Oliver Smithies, D.Phil.
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Cancer biologists were among the first to begin using this method, which has dramatically increased their knowledge about how cancers form and grow. Since the early 1990s, researchers have created hundreds of strains of mice with precise mutations, which they are using to gradually piece together the puzzle of the deadly disease.
"There have been so many mouse models that have played an invaluable and illuminating role in cancer," says Ron DePinho, M.D., of the Dana-Farber Cancer Institute in Boston. Cancer biologist Larry Donehower, Ph.D., at Baylor College of Medicine in Houston explains that the models span a wide range of malignancies. "There are a lot of tough cancers to model, and almost all have been covered."
Gene targeting was a substantial improvement over other genetic engineering techniques in use by the early 1990s. Researchers had created the first transgenic mice a decade before by injecting DNA into fertilized eggs and looking for adult animals that produce their protein of interest. Because this method inserted DNA into the genome randomly, researchers couldn’t delete normal copies of genes. That meant the transgenic mice were limited to gain-of-function mutations, instances where the two copies of normal genes were joined by additional copies, which would overproduce the normal protein or a mutant copy , says cancer biologist Tyler Jacks, Ph.D., at the Massachusetts Institute of Technology in Cambridge. Scientists had to infer a gene's function by observing what happens when there was too much of the protein or when a mutant copy interfered with the normal one.
The Nobel Prize–winning technology allowed researchers to take out specific genes. To do so, they would deliver a snippet of DNA into embryonic mouse cells in culture, which would then recombine with its sister DNA in the genome. The recombination cuts out the resident DNA and inserts the mutant DNA, which can then form a damaged protein or no protein at all. The embryonic stem cells then grow into a whole mouse with a specific mutation exactly where it is supposed to be in the genome. The first human disease model created with this technology was cystic fibrosis, from the labs of both Smithies and Evans.
Although working separately, Smithies, Evans, and Capecchi piggybacked on the work of the others. Evans had originally shown that embryos could carry foreign genes and be carried by mouse mothers. Capecchi and Smithies managed to inject DNA directly into a cultured cell's nucleus and have that DNA recombine with its sister DNA. Then Smithies used Evans’ embryonic stem cells, which were deficient in a particular enzyme, and selected for his embryos of choice by reintroducing the enzyme's DNA and growing the embryos in special culture media. Capecchi developed yet another way to select for embryos carrying desired DNA by tagging the DNA with an antibiotic resistance gene and growing the embryos in the antibiotic. Using these advancements, any gene of interest can be inserted or removed from mice.
DePinho says the advancements that enable the mouse models of today ranged beyond the Nobel Prize–winning work. For example, Ralph Brinster, V.M.D., Ph.D., at the University of Pennsylvania in Philadelphia had previously figured out how to culture embryos. "The ability to put something in culture and back into the reproductive tract in the mouse was a huge, huge advance," DePinho says.
The first animals came down with cancer through gene targeting in the early 1990s. The first two models were ones that lacked the well-known cancer stoppers p53 and retinoblastoma (Rb). Donehower and colleagues in Allen Bradley's laboratory at Baylor College of Medicine in Houston cut out p53, which was known to be deleted or mutated in half of human cancers. The team expected the developing mice to have problems, but the mice didn’t. Older mice, however, did develop a variety of cancers, including many lymphomas. "We found proof that p53 is a tumor suppressor," Donehower says. "It was a nice validation of the p53 model."
Around that time, Jacks was a new postdoc at the Whitehead Institute, where a team led by Bob Weinberg, Ph.D., had just cloned the Rb gene, whose absence caused sporadic tumors in children. And Nobel Prize winner Capecchi had just created the antibiotic-resistant mouse embryos. "I thought, wouldn’t it be great to make a mouse model with mutant retinoblastoma and do experiments that can’t be done in humans?" Jacks says. "So I asked [Capecchi] if it would be okay if I came out to Salt Lake to learn the technique. He spent a couple of days with me showing me how to do it and how to work with embryonic stem cells. He was incredibly helpful and gracious," Jacks says.
Mice engineered with a bad Rb gene indeed developed tumors, but not the ones researchers were expecting. Instead of eye tumors, the animals suffered from pituitary and thyroid gland cancers. "In 2004, we finally succeeded in making a mouse that got retinoblastoma," he says. Along with two other research teams, they found that a second gene protected the eye cells from cancer, and both mutations were required for tumors to form.
"The technique allows for a whole range of very accessible models of cancer. It allows us to put the right mutation into the right cell at the right time," Jacks says. "We can approach this problem in a meaningful, thoughtful way." One important result the models revealed is that cells need more than one bad gene to turn cancerous. "Even when you knock out p53, it takes many months for cancer to arise. We know the cells need more genetic alterations than just that one," Donehower says.
Those were just the first of many cancer models. For example, DePinho later developed an important knockout mouse in which telomerase, the protein responsible for keeping chromosome tips long and healthy, is deleted. "The most important factor that drives cancer is age," he says. Work with mice that lack telomerase suggests that cancers arise when chromosome tips shorten over the course of a lifetime of cell reproduction. The failing tips cause chromosomes to break and recombine incorrectly, until finally "cells reach the large number of mutations needed for transition to malignancy," he says.
H. Shelton Earp III, M.D., director of the University of North Carolina's cancer center in Chapel Hill, says that mouse models have come a long way from the simple deletion of one gene. "They allow us to learn things about organismal cancer that we can’t learn any other way," he says. Many models are developing sporadic cancers, similar to what happens in most human cancers. Also, Jacks and colleagues now have a mouse in which they can turn p53 on or off at will. UNC's Terry Van Dyke has a model of brain cancer in which three genes are altered. "The resulting tumors actually look like glioblastomas," Earp says.
However, researchers have yet to determine how accurate the array of mouse models are at predicting human response to therapy, Earp says. "The closer we get to making a mouse cancer pop up in an organ and have it be invaded by stroma and immunocytes, the more likely they will be similar to human cancers, we believe." For example, when researchers put human glioma cells into mice, they grow into a ball. "That's not what a human tumor looks like. Human tumors have these fingerlike pathologies and invasion of other cells," he says. "We’ll know in the next 18–24 months how accurate these models are."
The mouse models designed to study cancer have also been helpful in studying mammalian development, Jacks says. "One of the things [deleting genes] lets you do is understand the function of genes in development. Very often cancer genes are found to be critical in normal development."
The importance of gene targeting is evident not only by the awarding of the Nobel Prize but also by the effort put into engineering mice. Jacks says that large consortia in a variety of countries are developing libraries of mouse embryonic stem cells that carry mutations in all genes, one mutation per stem cell. Researchers will be able to check out new genes that they become interested in as easily as checking out a book.
"The field is just blossoming." Earp says, which makes many people wonder what took the Nobel committee so long to acknowledge gene targeting.
"The award was overdue," Donehower says.
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