Researchers at the University of Utah have created a less-expensive, less-time-consuming way to mutate large, non-gene stretches of DNA. And that could speed understanding of different disease processes and help development of treatments.
Their technique is reported this week online in the journal Nature Genetics.
Diseases may develop from gene mutations. But they can also result from mutations in the non-gene DNA. And it is those portions of DNA that the research targets, according to Mario Capecchi, distinguished professor and co-chairman of human genetics at the U. and an investigator for the Howard Hughes Medical Institute.
Capecchi likens the human genome to a large book — about 5 billion letters or 1,000 volumes of 1,000 pages each. The new technique is a "way to cut out as much text as we want" from the DNA sequence, which forms the genome.
DNA, or deoxyribonucleic acid, is a molecule made from numerous "base pairs" of four nucleic acids designated A, C, G and T. Genes are about 2.5 percent of that DNA "text." The rest is noncoding sequence. To remove it using existing methods has been "enormously expensive."
The new method "is significant because it makes it practical to do this for a vast amount of the total genome," he said. "We can look at a lot of DNA that's been neglected completely."
Mice are used to model human disease. In a release regarding the study, one of the researchers, Sen Wu, a postdoctoral fellow in human genetics at the U., said a key to understanding the function of the genetic blueprint is to take out part of the DNA sequence and see what happens. This discovery makes it simple and practical.
Other researchers on the project were human geneticists Guoxin Ying, a postdoctoral fellow, and Qiang Wu, an assistant professor (no relation to Sen Wu).
Besides figuring out how to delete long pieces of DNA in a simpler, cheaper fashion, they also devised an "efficient" method for mixing and recombining pieces of two chromosomes, making it easier to breed mice with human cancers.
The non-gene DNA sequences are important because they turn genes on or off, up or down. Still other sequences fold and pack DNA into the nucleus of each cell. Much of it is considered "useless junk," but Capecchi and the other researchers believe much of it will turn out to be important. They believe that mutations in the non-gene sequences can cause genes to malfunction. And making their removal faster and cheaper has the benefit of targeting the genes they regulate, as well.
A number of diseases are known to result from "chromosome translocation." Chromosomes break and their branches, which look like the letter X, recombine incorrectly. Leukemia and more than 30 different sarcomas are among diseases that start with chromosome translocation. But when researchers try to duplicate the translocations in mice, it may take many, many mice to get the desired effect. The researchers have found a much more efficient way of generating the translocation they want, making it, too, more efficient and inexpensive, Capecchi said.
"That's an important advance," he said, with significant cost savings to breed a mouse genetically engineered the way they want it.
The researchers used short pieces of DNA called loxP, which act as "signposts" that say to cut the DNA there. In the new method, loxP is inserted in a chromosome on one mouse and somewhere else in the same chromosome in a second mouse, which has a gene named Cre in its cells. Those two mice are bred to produce a mouse with loxP DNA on two sites in the same chromosome and which has the Cre gene. The protein Cre makes is the knife, cutting the DNA where the loxP is found.
That offspring mouse is then bred with a normal mouse and in 10 percent of the offspring, the desired segment of DNA is either deleted or duplicated and scientists can see what happens, thus learning what role that piece of DNA has.
They use the same method to make two chromosomes break and recombine to form a desired translocation, making it easier to breed mice with certain human cancers.
They enhanced their new technique with a "jumping gene" from a moth that inserts loxP DNA randomly in various spots on the mouse genome. That way, they can easily breed mice with loxP surrounding various large stretches of DNA. Then they go back to their new method and breed the mouse with one that has the Cre gene so it will mutate the stretches between those loxP DNA signposts.
Using the map of the mouse genome, they can study mice that have the loxP where they want it and make the desired mutation for their research. It means besides deleting stretches of non-gene DNA, they can also knock out genes more easily.
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