Mice have been freeloading on humans for millennia. Now, in laboratories around the world, scientists are returning the favor. Model systems such as the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans are actually easier to work with than mice, but mice are more closely related to humans, making them better models of human physiology. "For modeling genetic diseases like breast cancer, you definitely need mammals," says Kay-Uwe Wagner, a molecular biologist at the Eppley Institute for Research in Cancer and Allied Diseases, Omaha, Neb.
Early in the 20th century, geneticists used inbred mouse strains for much of their work.1 Long before the true nature of a gene was understood, geneticists were using inbred mice-genetically identical animals, akin to identical twins-to map murine genes. These early mice had other uses, too: they served as xenograft hosts and helped researchers tease apart the nuances of the immune system.
In the 1970s and 1980s, as molecular biology ascended, transgenic technology gave researchers the power to insert exogenous, human genes into mice. Mus musculus became the preferred model organism for investigators keen to understand the functions of human genes. But transgenic technology, while elegant, was crude by comparison with the gene "knockout" strategies that emerged later. This gene-targeting technology-also known as targeted mutagenesis and gene replacement technology-gave investigators a powerful new tool to study the genetic basis of mammalian development and disease. Most recently, researchers have refined these tools and begun exercising ever more precise control over the changes they make as they seek to discern a specific gene's function.
Transgenic or gene transfer technology originated in the early 1980s with the develop- ment of a technique known as pronuclear microinjection: the transfer of genetic material into rodent embryos.2 Re-searchers needed only an inverted microscope and a microinjector (see table), and by the end of the decade, hundreds of transgenic mouse lines were being produced in laboratories worldwide. The problem was, researchers could neither predict nor control where in the genome the foreign genetic material would insert. As a result, mouse lines carrying the same transgene could display wildly varying phenotypes. Then, Mario Capecchi, a human geneticist at the University of Utah, changed all that.3
"There was a breakthrough in altering the gene in the late '80s, early '90s, when the gene targeting technique was pioneered by Capecchi," explains Wagner. Using homologous recombination-the exchange of equivalent DNA sequences between the host genome and the introduced genetic material-Capecchi proved it was possible to aim the transgenic insertion, or transgene, at a precise location in the mouse genome.4,5 This gave scientists the ability to replace, or knock out, a specific gene with an inactive or mutated allele. By comparing the phenotypes of these knockout mice to those of wild-type mice that still have all their genes intact, researchers can readily deduce the function of the targeted gene. Conversely, scientists can also "knock in" a wild-type gene to test whether a particular mutation causes a given phenotype. Last year, Capecchi, Oliver Smithies, and Martin Evans shared the Lasker Award for their seminal work in the development of knockout technology in mice.3
One of the most exciting applications of murine knockout technology is in biomedical research. Scientists are using the models to study the molecular pathologies of a variety of genetically based diseases, from colon cancer to mental retardation. The hope is that by better understanding how a certain gene contributes to a particular disease, researchers can then take the knowledge a step further and look for drugs that act on that gene.
Still, knockout technology has its limitations. Because of developmental defects, many knockout mice die as embryos before the researcher has a chance to use the model for experimentation. Even if a mouse survives, several mouse models have "somewhat different phenotypes" from their human counterparts, says Wagner. The p53 knockout is a good example. p53 has been implicated in as many as half of all human cancers, but p53 knockout mice develop a different spectrum of tumors than do humans. In particular, mice develop lymphomas and sarcomas, whereas humans tend to develop epithelial cell-derived cancers. This phenotypic difference limits the utility of knockout mice as models of human disease.6 Now, a new technology known as conditional knockout or tissue-specific gene targeting is providing a way around these limitations.
The Next Generation
The goal of conventional knockout technology, explains Wagner, is to knock out both alleles so that the gene is entirely absent from all cells. The purpose of conditional knockouts, in contrast, is to delete a gene in a particular organ, cell type, or stage of development. Researchers can use the technique to knock out certain portions of specific genes. These conditional knockout mice offer at least two benefits. First, they typically survive longer than traditional knockout mice. In addition, if knockout mouse technology is more precise than transgenic technology, conditional knockout methods are even more so.
For more than a decade, for example, researchers have known of the link between BRCA1 mutation and breast cancer. But BRCA1 knockout mice, in which the entire gene was deleted, did not survive, hindering efforts to understand this gene's function. It was not until Chu-Xia Deng and his colleagues at the National Institutes of Health engineered BRCA1 knockout mice with a disabled exon 11, that scientists began to understand the gene's precise function and role in tumorigenesis.7 Indeed, Deng says that much of the attention that his work has received is from scientists who are just as, if not more, interested in his animal model than the results of his research.
There are several different ways to make conditional knockout models, but the most widely used method is the Cre-loxP recombinase system. Scientists use Cre recombinase like scissors, Wagner explains, to excise a gene that has been flanked by two loxP target sequences. Because Cre recombinase is expressed only in certain cell types, the targeted gene will be knocked out of only certain cells.
Like Deng, Wagner and his colleagues have been using the Cre-loxP recombination system to generate transgenic mice with mammary tissue-specific gene deletions. "You can narrow the window down even further to a particular time frame you want to study," says Wagner. "You can really refine the whole technology to do exactly what you want to do."
Wagner and his colleagues are using the Cre system to target and knock out transcriptional stop sequences as a way to permanently activate genes, enabling their use as cellular labels. By staining for the gene activity of these constitutive genes, says Wagner, researchers can follow the cells at any physiological stage.
Wagner's team is using this genetic labeling system in its search for cellular clues to why women who have been pregnant are at lower risk for breast cancer.8 "We have identified a certain population of cells in the mammary gland that genetically are different but morphologically the same. Maybe these are the cells that protect." Wagner says that by labeling cells and making them traceable, researchers can learn much more about the kinds of cells that give rise to cancer. "It is very exciting," he concludes.
Mice for Sale
Conventional knockout technology has become so well established that its methods are described in cookbook-style manuals. Conditional knockouts, on the other hand, are "a bit more complicated to make," says Wagner. Technical challenges aside however, there are two good reasons to obtain knockout mice from a third party, commercial or otherwise: time and money. Donna Gulezian, product manager at New York-based Taconic, estimates that it takes at least one to two years to make a new knockout mouse, and then the animals must be carefully scrutinized. Commercially available mice, on the other hand, tend to be fairly well characterized, she says. "If you're building on work that someone else has done," says Gulezian, "you can use the same mice without a loss of integrity."
Custom knockout mice are also big-ticket items. "Making a knockout is very, very expensive," explains Wagner. A core facility might charge from $3,000 to $10,000 to make a knockout mouse, while a contractor might charge as much as $30,000. Commercially available mice, in contrast, generally sell for several hundred dollars.
One of the primary suppliers is the not-for-profit The Jackson Laboratory (Jax) in Bar Harbor, Maine. With more than two million mice from over 2,700 different strains shipped to thousands of labs worldwide every year, Jax (http://www.jax.org/) is the world's largest supplier of laboratory mice. And with more than 300 knockout mice already available or currently under development, they are also the largest supplier of knockouts.
Taconic (http://www.taconic.com/) is the largest commercial supplier of transgenic mice and rats. According to Gulezian, the company sells about 40 different knockout lines to hundreds of institutions worldwide. About a third of their clients are academic institutions, one-third are government, and the remainder are for-profit institutions.
Except for about 30 of the in-house knockout strains that come from Jax, neither Jax nor Taconic actually creates the mouse models themselves. Rather, they work in collaboration with outside investigators who are engineering the models, says Gulezian. Most, but not all, of these investigators are in academia. Taconic then imports the line and does additional work to stabilize the genome and ensure the health of the line. Despite some overlap with lines from Jax, Taconic's aim is to produce unique lines. "Our p53 knockout is definitely our top selling model," says Gulezian, finding wide use in short-term carcinogenicity studies. The MDR [multidrug resistance] knockouts, which are used for drug transport and availability studies, are also popular.
Although Jax and Taconic are the primary suppliers of already-engineered knockout mice, the UK-based B & K Universal (http://www.bku.com/) also offers knockout mice commercially; they sell about a dozen different strains. And the MMHCC (Mouse Models of Human Cancers Consortium) Repository at the National Cancer Institute in Frederick, Md., which was founded just last year, has about 10 knockout strains currently available web.ncifcrf.gov/researchresources/mmhcc/information/about.asp).
Mouse Houses on Campus
Many universities have on-campus transgenic core facilities, where staff can assist researchers with everything from engineering the knockouts to housing them. Washington State University's (WSU) Center for Reproductive Biology, for example, has a transgenic core facility for which space was set aside in one of the campus vivariums, according to Eric Nilsson, a researcher at WSU and one of the staff who oversees the facility. The core has existed for about two years, according to Nilsson, with usually three or four researchers using it at any one time. Most of the knockouts that the core scientists use come from Jax and Taconic, but the core also subsidizes researchers who develop their own knockouts.
One of the primary advantages to having this kind of for-hire resource on campus is that scientists need not know anything about knockout technology to use the facility. If the prospect of incorporating knockout technology into your research program is too daunting to set out on your own, you just ask for help. "That's part of my job," says Nilsson. "To help lead you through."
But before committing the resources to make a knockout mouse, researchers should be certain that nobody has beaten them to the punch. Contact the primary mouse vendors, as well as prominent researchers in the field. A small bit of legwork could save months or even years of bench work. "I would bet lots of animal models are shipped between researchers for collaboration," observes Wagner. Indeed, he says, many researchers don't make their models commercially available at all.
1. B.A. Maher, "Test tubes with tails," The Scientist, 16:22-4, Feb. 4, 2002.
2. Taconic Transgenic Models and Services Division, Access to Transgenic Technologies, Taconic, NY, 1997.
3. B.A. Maher, "Lasker ceremony: Homage amidst angst," The Scientist, 15:10, Oct. 15, 2001.
4. M.R. Capecchi, "The new mouse genetics: altering the genome by gene targeting," Trends in Genetics, 5:70-6, 1989.
5. M.R. Capecchi, "Altering the genome by homologous recombination," Science, 244:1288-92, 1989.
6. J.M. Perkel, "Telomeres as the key to cancer," The Scientist, 16:38-40, May 27, 2002.
7. L. Pray, "The role of BRCA1 in breast cancer," The Scientist, 15:23, June 11, 2001.
8. K.U. Wagner et al., "An adjunct mammary epithelial cell population in parous females: Its role in functional adaptation and tissue renewal," Development, 129:1377-86, March 2002.
© Copyright 2002, The Scientist, Inc. All rights reserved.
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