Proceedings of 6th International Fruit Fly Symposium 6–10 May 2002, Stellenbosch, South Africa pp. 419–425

piggyBac-based transposon and 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 -wide trapping of cis-regulatory elements. This system is based on the controlled mobiliza- tion of the broad-range piggyBac. A ‘jumpstarter’ element expressing the piggyBac 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 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 ; 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 .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 ,both the production of males than coding regions. Regulatory regions from only (based on a female-specific lethal transgene; 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). 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 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- 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 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 might be caused when the yellow fever mosquito (Aedes aegypti; Kokoza et al. novel 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 cation of successful germline transformation. transposable element Hermes (Warren et al. 1994) Ideally, different phenotypically discernible trans- this could be a problem, since endogenous formation markers are used in order to facilitate cross-reactivity can cause instability of Hermes- tracking multiple transgenes during crossing based transgene insertions (Sundararajan et al. schemes. 1999). Horn et al.: piggyBac-based transposon mutagenesis and enhancer trapping 421

Fig. 1. Crossing scheme for identifying new insertions and adult enhancer traps. The use of separable fluorescent transformation markers allows the different jumpstarter (ECFP), mutator (EYFP), and reporter (DsRed) elements to be followed. To demonstrate the presence of the different elements, fly heads were photographed with ECFP-, EYFP-, or DsRed-specific filters (Horn et al. 2002). Of G2-males carrying the mutator on the X and a jumpstarter on an autosome, all male progeny showing EYFP fluorescence must carry new mutator insertions on autosomes. Some of them also show adult enhancer traps, like the male showing segmental fluorescence in the abdomen.

3×P3-EGFP: a universal transformation marker ‘master regulator’of eye-development (Halder etal. For the purpose of transgene identification in 1995),guiding regulatory circuits highly conserved a different set of insect species, universal transfor- in evolution (Callaerts et al.1997).A set of universal mation marker genes are advantageous over transformation vectors based on the 3×P3-EGFP species-specific ones. 3×P3-EGFP relies on the marker and the piggyBac, Hermes and mariner eye-specific expression of the gene encoding transposable elements has been established (Horn the enhanced green fluorescent protein (EGFP; & Wimmer 2000). 3×P3-EGFP-mediated transgene Berghammer et al. 1999; Horn et al. 2000). Eye- identification has been reported for dipteran specificity of EGFP expression is mediated by the (C. capitata; B. Savakis, pers. comm.; M. domestica, 3×P3 promoter element that comprises an artificial Hediger et al. 2001), lepidopteran (B. mori; Thomas trimerization of an ideal P3-binding site which is et al. 2002) and coleopteran (T. castaneum, responsive to the transcription factor Pa×6 (Sheng Berghammer et al. 1999) species. Moreover, differ- et al. 1997).Pa×6 has been demonstrated to be the ent spectral variants of the enhanced green fluo- 422 Proceedings of the 6th International Fruit Fly Symposium rescent protein, the yellow (EYFP) or cyan (ECFP) Mutator elements fluorescent proteins, and the red fluorescent As the mobilizable part of the system, the protein DsRed can be independently detected and mutator contains the 3×P3-EYFP transformation are unambiguously discernible (Horn et al. 2002). marker gene flanked by the piggyBac ITRs. Addi- These three fluorescent marker genes therefore tionally, we provided our mutators with a primary fulfill the requirement to develop complex genetic reporter gene which is sensitive to enhancer multi-component systems for transposon muta- activities since it includes a basal promotor. The genesis. primary reporter gene encodes a heterologous transcriptional activator protein, either the yeast ELEMENTS OF THE PIGGYBAC-BASED transactivator Gal4 (Brand & Perrimon 1993) or the MUTAGENESIS SYSTEM tetracycline-repressible transactivator tTA (Bello In general, mutagenic transposons offer several et al.1998).Both transactivators possess the poten- advantages over chemical or physical mutagenesis tial to stimulate transcription from respective cis- strategies: molecular information on the exact elements: UAS (upstream activating sequence) for genomic location of the novel mutator insertion Gal4 (Brand & Perrimon 1993) and TRE (tTA respon- can be retrieved by inverse PCR (Ochman et al. sive element) for tTA (Bello et al. 1998). Therefore, 1990), which facilitates cloning of the mutated Gal4/UAS and tTA/TRE-systems provide binary gene or cis-regulatory elements. Transposon- expression systems that can be used for directed mutagenesis is an ideal tool to identify genes gene expression studies (see responder elements). based on tissue-specific expression, since cis- In contrast to the constitutively active Gal4, the regulatory sequences (enhancers) acting on tTA protein is subject to conditional regulation nearby gene promoters can also act on a reporter because its DNA-binding affinity and correspond- gene included within the mutator element. A ing transactivation potential can be abolished by mutator modified in this way can mirror the - addition of the tetracycline.This offers an specific expression pattern and serve as a trap for additional temporal dimension of regulation. enhancer activity controlling e.g. female-specific, Particularly for the purpose of transgenic SIT appli- testis-specific,or transient early embryonic expres- cations, conditionality is useful when further sion. Moreover, if this primary reporter gene analysing candidate tTA-enhancer traps by cross- encodes a transcriptional activator protein, the ing to TRE-controlled lethality-mediating effector locus-specific expression can be mediated to a transgenes (Horn & Wimmer 2003). secondary reporter gene, which is provided by a different transgenic element, the responder. As a Responder elements result, the enhancer trap can later be utilized as an We created transgenic elements responsive to ectopic expression tool, i.e. to mediate stage-, the primary reporter proteins Gal4 or tTA by inte- tissue-, or sex-specific expression. We integrated gration into DsRed marked transformation vectors these features into the transposon mutagenesis of a secondary reporter gene under control of UAS system by developing different versions of mutator and TRE, respectively. To trace the expression and corresponding responder-elements. pattern of tTA or Gal4 mutator insertions in vivo,we employed responders with the enhanced yellow Jumpstarter elements fluorescent protein gene eyfp as secondary reporter. Jumpstarter elements need to provide transposase For embryonic detection of enhancer traps, we enzyme in order to induce the mobilization of its used lacZ (encoding E. coli $-galactosidase) as a counterpart, the mutator element. We placed this secondaryreportergene,whichallowsinsituanaly- piggyBac transposase under sis of gene expression by immunohistochemical or control of the constitutively active a-tubulin immunofluorescent staining procedures (Roberts promoter sequence (Theurkauf et al. 1986). To 1998). ensure that the integrated atub-piggyBac fusion gene cannot remobilize itself, we introduced Integration of a responder within the mutator the construct into the genome with elements element from different transposon superfamilies. With Theversatilityoftheeyfpgene,applicablebothas both Hermes- and mariner-based transformation transformation marker and as in vivo reporter, vectors, which were marked by ECFP (Horn & prompted us to integrate both features in a single Wimmer 2000),we could obtain stable jumpstarter mutator element. This UAS-Gal4-mutator carries lines. theprimaryreportergenegal4,andtheeyfpgeneis Horn et al.: piggyBac-based transposon mutagenesis and enhancer trapping 423 placed both under the control of the 3×P3-pro- stronger binary expression system than tTA/TRE. moter (transformation marker function) and under Further details on statistical interpretations, the UAS control (secondary reporter function). This enhancer traps observed, and the characterization makes it possible to directly observe trapped of lethal insertions have been described elsewhere enhancer activity in vivo without the need of cross- (Horn et al. 2003). ing to a separate responder strain. CONCLUSIONS A pilot enhancer-detection screen in To construct a transposon mutagenesis and Drosophila melanogaster enhancer-detection system that is widely applica- To demonstrate the functionality of constructed ble in insects, we took advantage of non- jumpstarter, mutator, and responder elements, as species-specific components like broad-range well as to assess the efficiency of the different transposons, potentially universal transformation types of mutators (tTA, Gal4, and UAS-Gal4) for markers and heterologous transactivators or enhancer detection,we performed a pilot screen in reporter genes. This system is advantageous over D. melanogaster. As illustrated in Fig. 1, the screen classical chemical or physical mutagenesis strate- comprises two crossing steps: first, an EYFP- gies, since it facilitates the characterization of marked mutator strain (mutator element located at targeted genes: molecular data on the mutator the X chromosome) was crossed to an ECFP- insertion reveal the identity of the locus and local enhancer activity is indicated by a fluorescent marked jumpstarter strain (generation G1).Second, reporter gene in vivo. The implementation of this from the progeny of this cross (generation G2) males that carry both transgenes were subse- system in germline-transformable insect pest quently crossed to DsRed-marked virgins of a specieswillenablegenome-widemutagenesisand respective responder strain (UAS-EYFP,TRE-EYFP,or enhancer detection thereby leading to the identifi- in case of the UAS-Gal4 mutator to white). In this cation of novel regulatory sequences. Identified step, single G2 males were crossed to maximize enhancers can subsequently be analysed for func- identification of independent transposition events tional integration into female- or embryo-specific lethality systems, which will help to establish in the progeny (generation G3). Finally, G3 males were analysed for the presence of EYFP eye fluores- transgenic SIT in insects of economic or medical cence which indicates novel mutator insertions. importance. Since the original mutator insertion was on the X chromosome, which is not paternally inherited, all ACKNOWLEDGEMENTS male G3 progeny showing the mutator element This work is supported within the priority marker must carry novel insertions. This scheme programme ‘International Research into the Devel- allows for the easy identification of transposition opment of Sustainable Agriculture and Forestry’ events. Furthermore, when G3 males are selected by the Robert Bosch Foundation, providing a ju- that lack the jumpstarter transgene, the novel nior professorship and independent research insertion is stable. group to E.A.W.; C.H. is a fellow of the Fonds der The jumping rate,measured in the percentage of Chemischen Industrie. fertile G2 crosses producing at least one EYFP-fluo- rescing son, was on average 72% (Gal4-UAS), REFERENCES ATKINSON, P.W., PINKERTON, A.C. & O’BROCHTA, D.A. 80% (tTA),or 92% (Gal4), respective to the mutator 2001. Genetic transformation systems in insects. element used. Novel mutator insertions were Annual Review of Entomology 46: 317–346. analysed for the presence of enhancer traps which BELLO, B., RESENDEZ-PEREZ, D. & GEHRING, W.J. 1998. Spatial and temporal targeting of gene expression in are reported by responder elements in the G3 generation. We observed enhancer traps in differ- Drosophila by means of a tetracycline-dependent transactivator system. 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