Regulated gene expression in pest insects

A major challenge for modern agriculture is to control pest species while minimising the adverse consequences to the environment. An attractive option is the Sterile Insect Technique (SIT) in which sterile insects are released to mate with wild pest insects1. Successful SIT programs have been conducted against the New World screwworm across the Americas and against Medfly worldwide. We have demonstrated that major improvements to this technique are possible through the use of genetically engineered insects2. Oxitec is developing this 'RIDL' technology to provide improved methods for the control of insects of agricultural and medical importance. This is clearly dependent on the ability to construct suitable genetically modified (GM) insects. Transposable element technology is the only method for stably integrating DNA into the germline of insects3-4. The DNA of interest is cloned between the ends of the transposon, deleting the transposase gene. The engineered transposon can no longer transpose without added transposase. The construct is micro-injected into embryos, along with a source of the transposase. This is normally another plasmid encoding transposase, but can be purified protein or mRNA. At a low frequency, the transposase will cause the engineered transposon to 'jump' from the injected DNA to the insect's genome. These insertions are stable unless re-exposed to transposase. The generation of a field-usable strain will require the production of multiple insertion lines, followed by elimination of unsuitable ones to leave one (or more) suitable for field use. This is a well-trod path in the development of plant varieties. However, the number of strains required initially for GM crop breeding, typically hundreds or a thousand, is simply impractical using current insect transformation methods. Therefore it is imperative to develop a method for rapidly generating many lines. Since the micro-injection method is very labour-intensive, this project focuses on a way to avoid this. The approach of choice is the use of jumpstarter lines. These have transposase genes inserted into their genome and so transposase can be supplied and insertions remobilised by simple breeding experiments, rather than micro-injection. By this method very large number of insertion lines can be generated with relatively little effort The student will develop jumpstarter lines for several pest species. We know this is possible because we have done it for the Mediterranean fruit fly6. However, the method used was rather crude and we know (because we have tried) that it will not work in at least several other pests and needs considerable refinement. Specifically, we need to move away from the ubiquitous expression of transposase that we used in the Medfly version. Expression of piggyBac transposase seems quite toxic in some species, so that expression is either poor initially or lost over time. Rather we need to go to germ-line specific expression and, preferably regulated germline expression. The student will therefore develop germline-specific expression systems for pest insects, testing and demonstrating their function by expressing fluorescent protein reporters and/or transposases. Such expression systems have many uses other than for jumpstarters, for example the production of fluorescent sperm for ecological and mating studies (and SIT), and these other applications will be explored as time and success allow. 1. Krafsur, E. J. Agric. Entomol. 15, 303-317 (1998). 2. Alphey, L. Insect. Biochem. Mol. Biol. 32, 1243-1247 (2002). 3. Handler, A. Insect Biochem. Mol. Biol. 32, 1211-20 (2002). 4. Horn, C., Schmid, B., Pogoda, F. & Wimmer, E. Insect Biochem. Mol. Biol. 32, 1221-35 (2002). 5. Handler, A. & McCombs, S. Insect Mol. Biol. 9, 605-12 (2000). 6. Dafa'alla, T. H., Condon, G. C., Condon, K. C., Phillips, C. E., Morrison, N. I., Jin, L., Epton, M. J., Fu, G., and Alphey, L. (2006). Nature Biotechnology 24, 820-821