Proceedings of 6th International Fruit Fly Symposium 6–10 May 2002, Stellenbosch, South Africa pp. 419–425 piggyBac-based transposon mutagenesis and enhancer trapping as a tool for functional insect genomics Carsten Horn*, Nils Offen & Ernst A. Wimmer‡ Lehrstuhl Genetik, Universität Bayreuth, Universitätsstrasse 30, NW 1, 95447 Bayreuth, Germany Transgene-based approaches to the sterile insect technique need to provide a combination of conditional female-specific lethality and male sterility.The identification of appropriate cis-regula- tory sequences that mediate female- or testis-specific expression is of key importance to develop such transgenic approaches. Here, we present a transposon mutagenesis system that is suitable for genome-wide trapping of cis-regulatory elements. This system is based on the controlled mobiliza- tion of the broad-range transposable element piggyBac. A ‘jumpstarter’ element expressing the piggyBac transposase gene is used to mobilize a non-autonomous piggyBac-based ‘mutator’ element. This mutator element carries a heterologous transactivator gene that serves as a primary reporter of enhancer activities.The heterologous transactivator then activates a secondary reporter within a ‘responder’ element, which is used for the visible detection of the enhancer activity. To be able to independently follow the different elements, they were marked with discernible spectral variants of the green fluorescent protein or DsRed under control of the widely applicable eye- specific promoter 3×P3.In a pilot screen carried out in Drosophila melanogaster,we observed trans- position events in the progeny of about 72–92% of single male crosses. Owing to the use of broad- range components, this system should allow for large-scale, genome-wide mutagenesis and enhancer trapping in different genetically tractable insects, including pest species of economic importance such as the Mediterranean fruit fly, Ceratitis capitata. INTRODUCTION dominant embryonic lethal transgene; Horn & The sterile insect technique (SIT) provides an Wimmer, 2003) have been successfully achieved in environmentally friendly method of area-wide Drosophila melanogaster.These systems rely on the insect pest management.SIT applications in Medi- conditional expression of a lethality-mediating terranean fruit fly, Ceratitis capitata, eradication or effector gene that is controlled by the binary suppression programmes benefit particularly from ‘TetOff’expression method (Gossen & Bujard 1992), the development of a ‘genetic sexing strain’ (GSS) which is based on the tetracycline-repressible that currently allows for the worldwide production transactivator protein, tTA. The tissue-, develop- of over 1000 million males per week (Franz 2002). mental stage- or sex-specific pattern of cytotoxicity However, the generation of these state-of-the-art is determined by the enhancer/promoter se- Medfly GSSs has been labour-intensive and time- quence controlling tTA gene expression.Therefore consuming. Comparable efforts are faced when cis-regulatory sequences play a crucial role in considering the implementation of analogous GSS transgene-based SIT systems. systems for other insect pest species. However, the approaches described so far have Transgene technology offers an opportunity to been established only in the extensively studied accelerate the development of efficient SIT strains. and experimentally highly tractable insect model, With the development of widely applicable D. melanogaster. It is questionable, whether the transposable elements and transformation markers, employed transgene constructs (which are based the genetic manipulation of economically important on Drosophila cis-regulatory elements) can be insect pest species has become feasible (Handler directly transferred to other insect pest species, 2001; Atkinson et al. 2001; Horn et al. 2002). By since cis-regulatory elements are far less conserved genetic engineering,both the production of males than coding regions. Regulatory regions from only (based on a female-specific lethal transgene; genes for yolk proteins from the house fly, Musca Heinrich & Scott 2000; Thomas et al. 2000) as well domestica, and the blowfly, Calliphora erythro- as male sterility (based on the inheritance of a cephala, mediate correct tissue specificity when *To whom correspondence should be addressed. Present employed in Drosophila melanogaster, but do not address: Max-Planck-Institut für Molekulare Genetik, Abteilung show sex-specificity (Tortiglione & Bownes 1997). Vertebrate Genomics, Hessische Str. 3-4, D-10115 Berlin, Germany. E-mail: [email protected] Similarly, a 2.1 kb Aedes aegypti vitellogenin gene ‡Present address: Abteilung für Entwicklungsbiologie, Institut für promoter fragment neither directs female-specific Zoologie, Anthropologie und Entwicklungsbiologie, GZMB, Georg-August-Universität Goettingen, Justus-von-Liebig Weg 11, gene activation in the homologous nor in the D-37077 Goettingen, Germany. heterologous (Drosophila melanogaster) situation 420 Proceedings of the 6th International Fruit Fly Symposium (Kokoza et al. 2000, 2001a). While the loss of Transposon mutagenesis based on P elements sex-specificity in the latter case might be due to was first described for the insect model organism, insufficient cis-regulatory information within the D.melanogaster (Cooley et al. 1988) and since then 2.1 kb promoter region studied,the lack of conser- has become a valuable tool for forward genetics vation of sex-specific promoters across different (Berg & Spradling 1991) and enhancer detection species might reflect a general phenomenon: (O’Kane & Gehring 1987).As part of the Drosophila vitellogenesis is stimulated cooperatively both by genome project, considerable efforts have been the sex determination cascade and by hormonal undertaken to disrupt the substantial amount of regulatory circuits (Sappington & Raikhel 1998). 25% of essential genes (Spradling et al. 1999). Depending on physiological requirements, However, the direct transfer of P-based systems to species-specific adaptations might be reflected insect species of economic importance failed due in different modes of regulation,and an evolution- to host-specific requirements of P elements (Rio & ary highly conserved female-specific enhancer Rubin 1988). module might not exist. Therefore, cis-regulatory elements for the gener- UNIVERSAL TRANSPOSON MUTAGENESIS ation of transgenic SIT will probably have to be To overcome Drosophila-specific restrictions of derived from the targeted pest species itself. Here insertional mutagenesis and enhancer detection, we describe a transposon mutagenesis system based transposons with a broad-range mobility spectrum on controlled mobilization of piggyBac-derived and a widely applicable marker system are re- transgenes that can be used for genome-wide quired. searches of suitable cis-regulatory elements. The system is based on widely applicable transposable piggyBac: a broad-range transposon elements and transformation markers, which will The piggyBac transposable element was originally allow tagging of mutated loci and the identifica- isolated from a cabbage looper, Trichoplusia ni, cell tion of enhancer activities. line (Cary et al. 1989). piggyBac inserts specifically into the tetranucleotide sequence TTAA (Elick et al. The principle of transposon mutagenesis 1997). piggyBac-derived transformation vectors Transposons are mobile DNA elements that have been successfully applied to transform the encode a transposase enzyme which recognizes germline of a broad spectrum of dipteran,lepidop- specifically the inverted terminal repeats (ITR) teran and coleopteran species: Medfly (Ceratitis located at the transposon ends, and catalyses the capitata; Handler et al. 1998), oriental fruit fly excision of the element and subsequent reinser- (Bactrocera dorsalis; Handler & McCombs 2000), tion into new DNA target sites. As a result of this housefly (Musca domestica;Hediger et al.2001),the process, a mutation might be caused when the yellow fever mosquito (Aedes aegypti; Kokoza et al. novel insertion interrupts a gene function. To 2001b), two anopheline mosquito species (Anoph- employ transposons as a tool for mutagenesis, a eles gambiae, Grossman et al. 2001; Anopheles controlled mobilization step is required.Controlled stephensi,Ito et al.2002;Nolan et al.2002),silkworm mobilization is achieved in an artificial bipartite (Bombyx mori, Tamura et al. 2000), pink bollworm system,comprising two genetically modified insect (Pectinophora gossypiella, Peloquin et al. 2000), strains which are transgenic for non-autonomous and red flour beetle (Tribolium castaneum, Berg- parts of the original transposon: i) a jumpstarter hammer et al. 1999). Owing to this broad-range strain contains the transposase gene but omits the applicability and its documented mobility (Fraser functional transposon ends. This strain serves as a et al.1995),we chose piggyBac to develop a poten- source for stable transposase activity. ii) A mutator tially universal transposon mutagenesis and strain carries a transgene construct, including enhancer-detection system. The absence of de- functional transposon terminal repeats, but omit- tectable endogenous piggyBac transposase activ- ting the transposase gene, which is replaced by ity in the Drosophila genome (Lobo et al. 1999) some cargo DNA of choice. Both constructs are allows for testing the system in this experimentally provided with marker genes that allow for identifi- very accessible organism. For the broad-range