Mouse tool offers new disease insights
than a decade is now available to scientists studying genes and
diseases in mice. This technique offers a powerful new tool to
study genetics, particularly in disease.
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.
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 method called Mosaic Analysis with Double Markers (MADM), a tiny subset of cells and study gene function at a very high resolution and can be used to study a variety of tissues, such as skin, heart and nervous system.
Liqun Luo, professor of biological sciences at Stanford explained: "We use a green fluorescent protein, so if you mutate a gene, you'll know in which cell the normal gene is lost."
"For example, if you delete a tumour suppressor gene, the green cells will proliferate, and you can actually study the tumour's progression. If you can image these cells in a live animal, you can potentially watch the tumour grow," he added.
There are many potential applications of this powerful approach 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.
MADM claims to be 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 consequences for the mouse and the experiment whereas MADM acts more like a scalpel, creating a handful of mutant cells in an otherwise normal animal.
Geneticists have been using mosaic fruit flies for decades. In the early 1990's, 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 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.
The theory is that if no recombination occurs, there are neither green nor mutant cells. Even if one cell turns green, researchers can almost be certain it will 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 said.
"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," he added.
