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Sanger-Led Group Develops New Transposon-based Cancer Gene Screen in Mice

By Andrea Anderson

NEW YORK (GenomeWeb News) – In a paper appearing in the early, online version of Science yesterday afternoon, researchers from the Wellcome Trust Sanger Institute and elsewhere outlined their strategy for finding cancer genes in mice: an insertional mutagenesis method that relies on the PiggyBac transposon system.

After developing mouse lines genetically engineered to carry a PiggyBac transposon system, the team showed that these transposons can create mutations in the genome that lead to cancer. By looking at where insertions tended to occur in the genomes of dozens of hematopoietic tumors, the researchers have already started finding candidate genes for these cancers.

And, they say, the same approach — alone or in combination with other insertional mutagenesis approaches — can be used to look at the tissue-specific genes involved in cancer in mice as well as the timing of cancer development.

"These transposon tool kits in mice have the potential to increase the speed and efficiency with which cancer genes and oncogenic signaling networks are discovered," senior author senior author Allan Bradley, head of mouse genomics at the Wellcome Trust Sanger Institute, and co-authors wrote, "and should likewise facilitate functional analysis of these genes."

Inserting bits of DNA randomly across the genome disrupts specific sequences, creating mutations that may lead to cancer, the researchers explained. Determining where these insertions occur, in turn, offers clues about which genes are involved in cancer development and progression.

But prior to the latest study, researchers had relatively few tools available for doing genome-wide insertional mutagenesis-based screens in mammals, the team noted. To date, such screens have largely relied on either a transposon known as Sleeping Beauty, engineered from transposable elements in fish genomes, or certain retroviruses.

"Retroviruses have been used for cancer gene discovery in mice, but their application has been limited to the study of hematopoietic and mammary tumors due to viral tropism for these tissues," the team wrote. "DNA transposons, which are the key insertional mutagens in lower organisms, were inactivated in vertebrate genomes millions of years ago."

For the current study, the researchers decided to try a new transposon system for doing genome-wide insertional mutagenesis: a transposon called PiggyBac that originates in the cabbage looper moth, inserts itself at different sites in the genome than those favored by Sleeping Beauty, and moves larger bits of DNA than Sleeping Beauty.

Moreover, lead author and Wellcome Trust Sanger Institute researcher Roland Rad told GenomeWeb Daily News in an e-mail message that PiggyBac is less likely to do "local hopping" than Sleeping Beauty and does not cause the same sorts of undesired and undetectable damage as it moves around the genome.

After creating mouse lines containing PiggyBac transposase and transposons with various activating and inactivating regulatory sequences incorporated into them, the team tested nearly 400 mice, showing that mutations caused by PiggyBac-based transposition could cause cancer in the animals.

The types of tumors and the amount of time they took to develop depended on the specific regulatory elements incorporated into the transposons, they noted.

When the researchers focused in on 63 mouse blood cancers, they found 72 common integration sites affecting 67 loci the genome. Nearly half of these integration sites — about 42 percent — had not been found with mutational screens using retroviruses or Sleeping Beauty-based transposition.

Among the newly identified candidate genes: a transcription factor coding gene called Spic, a histone deacetylase gene known as Hdac7 , and Bcl9, a beta-catenin co-factor gene involved in the Wnt signaling pathway that was previously implicated in human leukemia.

The researchers plan to do additional studies using the PiggyBac system, including experiments combining PiggyBac screen data with insertional mutagenesis data generated using Sleeping Beauty and/or retroviruses.

"Some genes that are never hit with one system can be discovered with another system," Rad explained in his e-mail. "Thus, the three insertional mutagenesis tools are complementary. Their combined use will help to achieve saturation mutagenesis of the genome."

The team also touted the method as a promising way to look for cancer-related genes in a tissue-specific way. For instance, Rad said, he and his colleagues have already been using this approach to look for cancer genes in the mouse pancreas and intestine.

So far Rad said the team does not have plans to commercialize products based on the PiggyBac transposon screening system.

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