“P(acman) overcomes a key limitation of currently available methods because it allows you to study large chunks of DNA in vivo,” said Dr. Hugo Bellen, professor of molecular and human genetics at Baylor College of Medicine and director of the program in developmental biology. He is also a Howard Hughes Medical Institute investigator. The new technique allows researchers to study large genes and even gene complexes in the fruit fly, which was not possible before.
P/phiC31 artificial chromosome for manipulation, or P(acman), combines three recently developed technologies: a specially designed bacterial artificial chromosome (BAC) that allows maintenance of large pieces of DNA in bacteria, recombineering that allows the manipulation of large pieces of DNA that can then be inserted into the genome of the fly at a specific site using phiC31-mediated transgenesis.
It is a new technique with far-reaching promise, said Bellen.
P(acman) overcomes certain obstacles that have hampered research. It allows the cloning of large pieces of DNA to be used to transform the genome, and it permits that DNA to be inserted into specific places in the genome. Bellen credits the report’s first author, Koen J.T. Venken, a graduate student in the BCM Program in Developmental Biology, with putting the technologies together to come up with a new methodology in the field.
Current technology has certain problems for researchers seeking to understand the structure and function of genes, said Bellen. Often, when scientists breed flies that lack a particular gene and then try to put that gene back into the fly, it inserts itself randomly into the genetic blueprint.
In some cases, it makes too much protein, and in others, too little. In other instances, it may disrupt the message from another gene.
“You are really comparing apples and oranges when you do this,” said Bellen. The technique is also limited to small DNA chunks.
“Koen set out to develop a new transgenesis system using the three techniques,” said Bellen.
The bacterial artificial chromosome, or BAC, he used allows the scientist to maintain large chunks of DNA in the bacteria, but it is present in only one or few copies. However, the bacteria can be induced to produce many copies of the DNA when needed.
Koen then integrated a technique called “recombineering” into the strategy, which facilitates the scientist to clone large chunks of DNA and subsequently allows them to make specific mutations anywhere he or she wants in the gene.
The third technique allows the researcher to pinpoint where he or she wants to the mutant gene to go in the genetic blueprint of the fly, eliminating the apples-and-oranges problem. This third technique – phiC31 – works also in mouse and human cells, implying that this new technique could be used in those cells as well.
Ross Tomlin | EurekAlert!
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