No abstract

The lepidopteran transposable element piggyBac can mediate germline insertions in at least four insect orders. It therefore shows promise as a broad-spectrum transformation vector, but applications such as enhancer trapping and transposon-tag mutagenesis are still lacking. We created, cloned, sequenced and genetically mapped a set of piggyBac insertions in the red flour beetle, Tribolium castaneum. Transpositions were precise, and specifically targeted the canonical TTAA recognition sequence. We detected several novel reporter-expression domains, indicating that piggyBac could be used to identify enhancer regions. We also demonstrated that a primary insertion of a non-autonomous element can be efficiently remobilized to non-homologous chromosomes by injection of an immobile helper element into embryos harbouring the primary insertion. These developments suggest potential for more sophisticated methods of piggyBac-mediated genome manipulation.

Genetic transformation in insects holds great promise as a tool for genetic manipulation in species of particular scientific, economic, or medical interest. A number of transposable elements have been tested recently as potential vectors for transformation in a range of insects. Minos is one of the most promising elements because it appears to be active in diverse species and has the capacity to carry large inserts. We report here the use of the Minos element as a transformation vector in the red flour beetle Tribolium castaneum (Coleoptera), an important species for comparative developmental and pest management studies. Transgenic G 1 beetles were recovered from 32.4% of fertile G 0 's injected with a plasmid carrying a 3xP3-EGFP-marked transposon and in vitro synthesized mRNA encoding the Minos transposase. This transformation efficiency is 2.8-fold higher than that observed when using a plasmid helper. Molecular and genetic analyses show that several independent insertions can be recovered from a single injected parent, but that the majority of transformed individuals carry single Minos insertions. These results establish Minos as one of the most efficient vectors for genetic transformation in insects. In combination with piggyBac-based transgenesis, our work allows the introduction of sophisticated multicomponent genetic tools in Tribolium.

Due to its small size, short life cycle, and easy maintenance, the flour beetle Tribolium castaneum is well suited for the genetic analysis of development. One drawback of Tribolium as a genetic system is, however, the difficulty of keeping embryonic lethal lines. Presently, only few lethal mutations can be kept as balanced stocks. Therefore, heterozygous carriers must be identified anew in every generation in order to maintain a recessive embryonic mutation. To alleviate this problem we have devised a block system that allows the simultaneous processing of many mutant lines or test crosses for visual inspection of larval cuticle phenotypes. Using this technique, one person can maintain about 100 embryonic lethal stocks, which makes feasible the thorough genetic analysis of embryogenesis in this species.

INTRODUCTIONThe procedure for introducing transgenes into the Tribolium genome is similar to that for Drosophila and is of comparable efficiency. Purified transposon vector and helper plasmids are injected through the posterior pole of precellular embryos. The injected eggs are incubated under humidified conditions until they hatch. Adults resulting from injected eggs are mated inter se, and transformants are identified among their offspring with the help of visible genetic markers. The helper plasmids express the respective transposase under control of the Drosophila hsp70 promoter, but no heat shock is required. Transgenic animals are identified by fluorescent proteins that are expressed in the eye (and several other tissues including the central nervous system [CNS]) under the control of the artificial 3xP3 promoter. Among the fluorescent proteins enhanced green fluorescent protein (EGFP), enhanced yellow fluorescent protein (EYFP), enhanced cyan fluorescent protein (ECFP), and dsRed, it is EGFP and EYFP that provide the strongest signals. An alternative marker system based on rescue of the white-eye mutation in the eye pigmentation gene vermilion makes scoring transgenic animals even easier. Using these dominant markers, transgenic lines tagged with different markers can be crossbred, and offspring carrying both constructs can be easily identified, which is useful for remobilization experiments and misexpression experiments using the Gal4/UAS system. This protocol provides a detailed method for the generation of transgenic lines, which also can be applied toward the generation of improved mutator and helper strains.