Red Flour Beetle (Tribolium) Germline Transformation and Insertional Mutagenesis

Abstract

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.