May 6, 2005 A powerful laboratory technique used by fruit fly geneticists for more than a decade is now available to scientists studying genes and diseases in mice.
Writing in the May 6 edition of the journal Cell, researchers from Stanford University describe a streamlined method for creating a "genetic mosaic mouse"--a rodent whose body is genetically engineered to produce small clusters of cells with mutated genes.
The new technique, called Mosaic Analysis with Double Markers (MADM), was developed in the laboratory of Liqun Luo, professor of biological sciences at Stanford who was recently named an investigator with the Howard Hughes Medical Institute.
"With MADM, you can look at a tiny subset of cells and study gene function at a very high resolution," says Luo, who also is affiliated with the Neuroscience Institute at the Stanford School of Medicine. "Our method can be used to study a variety of tissues, such as the skin, heart and nervous system."
Mosaics are designed to give researchers an opportunity to observe what happens when a specific gene is removed from a small cluster of cells in a living animal. With MADM, cells carrying an altered gene of interest actually turn green for easier observation.
"We use a green fluorescent protein," Luo says. "So now if you mutate a gene, you'll know in which cell the normal gene is lost. For example, if you delete a tumor suppressor gene, the green cells will proliferate, and you can actually study the tumor's progression. If you can image these cells in a live animal, you can potentially watch the tumor grow."
Luo points out that MADM is more precise than the widely used "knockout mouse" technique, in which a gene of interest is removed ("knocked out") of every cell in the animal's body. The knockout method can have unwanted, deleterious consequences for the mouse and the experiment, Luo adds, whereas MADM acts more like a scalpel, creating a handful of mutant cells in an otherwise normal animal. Mosaic mice
Geneticists have been using mosaic fruit flies for decades. In the early 1990s, scientists developed a more efficient technique that allows researchers to control when and where mutant cells are generated in the fly's body. However, scientists have had a much more difficult time designing mosaic vertebrates, such as mice. The mouse has long been considered an ideal laboratory model for studying human development and disease, primarily because mouse DNA and human DNA are remarkably alike.
The MADM technique, which Luo and his colleagues developed for mice, works on the same principal as the method currently used to create mosaic fruit flies. The researchers begin with two embryonic stem cells whose chromosomes have been engineered to carry two inactivated segments of a green fluorescent protein molecule. Mice derived from these embryonic stem cells are mated to each other. As their offspring grow, the cells in their body begin to divide--a normal process that results in the duplication of each chromosome. Before cell division is complete, a special enzyme causes the exchange, or recombination, of the two engineered chromosomes. If one of those chromosomes contains a bad copy (mutation) of a gene, the recombination event could cause an offspring to inherit two bad copies of the gene, which would result in a mutant cell. This process simultaneously activates the green fluorescent protein, which turns the mutant cell green.
"If there is no recombination, there are neither green nor mutant cells," Luo explains. "So even if only one cell turns green, we know it has to contain the mutated gene of interest."
In their study, the Stanford team focused on the cerebellum, the part of the brain whose main function is to coordinate motor activity and maintain balance. The researchers used MADM to study the development of cerebellum granule cells, which are the most abundant cells in the brains of mice and humans.
"Usually people think that all cerebellum granule cells are the same--they are born, and their final function is determined by their interaction with other neurons," Luo says. "But we found that there appears to be a certain degree of predisposition to these cells by their lineages. This comes back to an interesting problem in developmental neurobiology as to whether the brain is wired by genetics or by environment--nature or nurture. Our discovery makes us feel that cerebellum wiring is more genetically determined than previously thought."
In a companion article published in the same edition of Cell, scientists Todd E. Anthony and Nathaniel Heintz of Rockefeller University describe MADM as "an elegant method" that brings mouse geneticists "one step closer to the enviable experimental facility available to invertebrate geneticists."
There are "many potential applications of this powerful approach," Anthony and Heintz wrote, including the "opportunity for in-depth studies of molecular mechanisms that underlie the dynamic properties of specific neuronal populations." MADM also could prove important for analysis of complex developmental or degenerative diseases resulting from genetic mutations, they added.
In light of its potential commercial applications, Luo has begun the process of licensing MADM through Stanford University's Office of Technology Licensing. Meanwhile, he and his colleagues are returning to the lab to see if the technique can be applied to other aspects of developmental biology and disease in mice.
The study was co-written by the following affiliates of Stanford: postdoctoral fellow and lead author Hui Zong; graduate student J. Sebastian Espinosa; research associate Helen Hong Su; and medical student Mandar D. Muzumdar. Research was supported by a McKnight Technological Innovations in Neurosciences Award, the National Defense Science and Engineering Graduate Fellowship Program and the Stanford Medical Scholars Research Fellowship Program.
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