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Title: PIGGYBAC-MEDIATED GERMLINE TRANSFORMATION IN THE BEETLE TRIBOLIUM CASTANEUM

Author
item LORENZEN, MARCE - KANSAS STATE UNIV
item BERGHAMMER, ANDREAS - UNIV MUNCHEN, GE
item BROWN, SUSAN - KANSAS STATE UNIV
item DENELL, ROBIN - KANSAS STATE UNIV
item KLINGLER, MARTIN - UNIV MUNCHEN, GE
item Beeman, Richard

Submitted to: Insect Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/9/2003
Publication Date: 11/30/2003
Citation: LORENZEN, M.D., BERGHAMMER, A.J., BROWN, S.J., DENELL, R.E., KLINGLER, M., BEEMAN, R.W. PIGGYBAC-MEDIATED GERMLINE TRANSFORMATION IN THE BEETLE TRIBOLIUM CASTANEUM. INSECT MOLECULAR BIOLOGY 12:433-440. 2003.

Interpretive Summary: Recent advances in genomics and bioinformatics have opened the door to rapid discovery and analysis of genes that regulate important biological phenomena. Gene transfer technology is needed to fully understand and exploit potentially useful genes discovered through genomics. In this research, we showed that a jumping gene called "piggyBac", isolated from a moth, can insert itself randomly into many different sites on flour beetle chromosomes, and that it can be "loaded" with other genes, which also insert along with the piggyBac carrier. Furthermore, a piggyBac gene that has inserted into beetle chromosomes can be either stabilized or remobilized (made to insert into new locations on other chromosomes). These new techniques can be used to "label" target genes with a molecular tag to help identify and clone those genes. The new techniques can also be used to cause mutations in genes whose functions are unclear, or to identify sets of genes that have particular expression patterns (e.g., genes affecting the nervous system, or genes affecting the molting process). Such techniques will be used to discover, characterize and exploit insect control genes in pest insects.

Technical Abstract: The lepidopteran transposable element piggyBac shows promise as a broad-spectrum transformation vector for insects, but applications such as enhancer trapping and transposon-tag mutagenesis are still lacking for this element. We created a set of piggyBac insertions in the red flour beetle, Tribolium castaneum, and cloned, sequenced and genetically mapped the resulting insertion junctions. We found that this transposon inserts via a precise, transposase-mediated mechanism, specifically targeting the canonical TTAA recognition sequence. We detected several novel reporter-expression domains among a random set of transformed lines, indicating that piggyBac could be used in beetles to identify enhancer regions. We also demonstrated that a primary insertion of a nonautonomous element can be efficiently remobilized to nonhomologous chromosomes by injection of an immobile helper element into embryos harboring the primary insertion. These developments suggest potential for more sophisticated methods for piggyBac-mediated genome analysis and manipulation.