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Abstract. The efficiency of transposon-mediated germline transformation is dependent on the transposon mobility in the host embryo, and on the detectability of ...

Dev Genes Evol (2000) 210:623–629

© Springer-Verlag 2000

S H O R T C O M M U N I C AT I O N

Carsten Horn · Brigitte Jaunich · Ernst A. Wimmer

Highly sensitive, fluorescent transformation marker for Drosophila transgenesis

Received: 12 July 2000 / Accepted: 27 July 2000

Abstract The efficiency of transposon-mediated germline transformation is dependent on the transposon mobility in the host embryo, and on the detectability of the used transformation marker. Therefore, high susceptibility of the transformation marker to position effect suppression is a disadvantage. Here we present data that the eye-specific expression of green fluorescent protein, driven by the 3xP3-EGFP marker, outperforms the commonly used “mini”-white transformation marker in Drosophila germline transformation experiments: 3xP3EGFP is more sensitive than “mini”-white in identifying transgenic individuals and reacts differently to position effect suppression. Therefore, 3xP3-EGFP offers an ideal marker for applications in functional genomics where as many gene loci as possible should be targeted in the genome of a specific organism, for example, as intended in the Drosophila gene disruption project. Furthermore, we give a detailed description of the embryonic and larval expression mediated by the 3xP3-EGFP marker. These pre-adult expression patterns, and the potentially universal applicability of the transformation marker also offer additional advantages for selecting transgenic individuals in organisms other than Drosophila. This will be of great interest to the field of evolutionary developmental biology and to modern pest management programs. Keywords Germline transformation · Green fluorescent protein · Pax-6 · Transposable elements · Transposon mutagenesis

Edited by C. Desplan C. Horn · B. Jaunich · E.A. Wimmer (✉) Lehrstuhl für Genetik, Universität Bayreuth, Universitätsstrasse 30 NWI, 95447 Bayreuth, Germany e-mail: [email protected] Tel.: +49-921-555813, Fax: +49-921-552710

Introduction In developmental biology, transposable elements provide effective methods for correlating genetic and molecular information. In Drosophila melanogaster, the P-element transposon (Engels 1996) has proven useful in manipulating the genome as a simple gene vector for introducing DNA-constructs (Spradling and Rubin 1982) as well as in cloning genes by using applications such as enhancer trapping (O’Kane and Gehring 1987) and transposon tagging (Cooley et al. 1988). Despite considerable efforts, this excellent P-element tool box seems restricted to some Drosophila species (O’Brochta and Handler 1988). However, recent success in transforming the medfly Ceratitis capitata (Loukeris et al. 1995; Handler et al. 1998), the housefly Musca domestica (Hediger et al. 2000), the mosquitoes Aedes aegypti (Coates et al. 1998; Jasinskiene et al. 1998; Pinkerton et al. 2000) and Anopheles stephensi (Catteruccia et al. 2000), the silkworm Bombyx mori (Toshiki et al. 2000), and the flour beetle Tribolium castaneum (Berghammer et al. 1999) with a series of more promiscuous transposable elements encourages the view that these powerful methodologies will soon also be available for a series of other arthropod species. The ability to use the same transformation system in a series of different organisms will be instrumental to comparative functional approaches in the emerging field of evolutionary developmental biology. Moreover, the application of different transposable elements might also be useful in Drosophila research itself, since transposable elements do not integrate into the genome completely at random, and with reasonable effort only about 85% of all genes can be targeted by P-elements (Spradling et al. 1999). As different transposable elements have distinct insertion specificities, alternative elements could be employed for disrupting previously untargeted genes (Smith et al. 1993). However, position effects in certain genomic regions can suppress marker gene expression necessary for the identification of the insertion (Hazelrigg et al. 1984). Therefore, genes in these regions could still be missed despite being inser-

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tion tagged. Since diverse marker genes might react differently to position effects, not only will various transposable elements be necessary for targeting all genomic regions, but also several diverse marker genes need to be applied to identify novel insertions at different gene loci. Berghammer et al. (1999) presented a novel eye-specific and potentially universal transformation marker that is based on the enhanced green fluorescent protein (EGFP; Tsien 1998), and an artificial promoter that is responsive to the evolutionary conserved transcription factor Pax-6 (Sheng et al. 1997). Here, we show that this transformation marker, 3xP3-EGFP, can serve as an excellent tool to further harness Drosophila developmental genetics: the marker 3xP3-EGFP is highly sensitive, reacts to position effects differently than the commonly used marker “mini”-white and is detectable during embryonic, larval and adult stages.

Materials and methods Plasmid constructions The EGFP coding region and SV40-polyA was cloned as a 1 kb SalI-ClaI fragment from pHermes[EGFP] (Pinkerton et al. 2000) into SalI and ClaI digested pSL1180 (Pharmacia), which resulted in pSL-EGFP. Three tandem copies of the Pax-6 homodimer binding site (P3; Wilson et al. 1993, 1995; Czerny and Busslinger 1995) plus the TATA-homology of hsp70 (–40 to +70) (3xP3; Sheng et al. 1997) forming a 240 bp EcoRI-SalI fragment were cloned into EcoRI and SalI digested pSL1180 and pSL-EGFP, generating the plasmids pSL-3xP3 and pSL-3xP3-EGFP, respectively. pHer{3xP3-EGFP} was created by introducing the 260 bp EcoRI-ApaI fragment from pSL-3xP3 into EcoRI and ApaI cut pHermes[EGFP]. pMos{3xP3-EGFP} and pBac{3xP3-EGFP} were generated by introducing the 1.3 kb EcoRI-NruI fragment (Klenow blunted) from pSL-3xP3-EGFP into pMos1 (Medhora et al. 1991) opened with SalI (Klenow blunted) or into p3E1.2 (Cary et al. 1989) digested with HpaI, respectively. For both constructs, insert orientations were chosen in order that the disrupted transposase gene and the 3xP3-EGFP transgene were transcribed in opposite directions. pP{w+mC, 3xP3-EGFP} was cloned by introducing the 1.3 kb EcoRI-NruI fragment (Klenow blunted) from pSL-3xP3-EGFP into StuI digested pCaSpeR4 (Thummel and Pirrotta 1992) so that the 3xP3 promoter and the “mini”-white promoter were oriented in the same direction, but separated from each other by the EGFP marker gene and the SV40-polyA signal. All DNA preparations (including preparations for microinjection) were performed using Qiagen plasmid kits. Germline transformation Germline transformation experiments were performed by microinjection of DNA-constructs into a D. melanogaster white (w) strain as described by Rubin and Spradling (1982). Prior to injection, plasmids were co-precipitated and dissolved in injection buffer (5 mM KCl, 0.1 mM KH2PO4/Na2HPO4). P-element derived transformation vectors were at final concentrations of 300 ng/µl construct (pP{w+mC, 3xP3-EGFP}) and 50 ng/µl helper . mariner, Hermes and piggyBac derived transformation vectors were at final concentrations of 500 ng/µl construct (pMos{3xP3-EGFP}, pHer{3xP3-EGFP}, pBac{3xP3-EGFP}) and 300 ng/µl helper [pKhsp82MOS (Coates et al. 1997), pKhsp82Hermes (Sarkar et al. 1997), pBac∆Sac (Handler et al. 1998), phsp-pBac (Handler and Harrell 1999)], respectively. Since in the mariner, Hermes and piggyBac helpers, the transposase gene is driven by heat shock promoters, a 1–1.5 h 37°C heat shock was usually performed

16–18 h after the injection. However, this heat shock procedure is not required, since omission of the heat shock does not significantly reduce transformation efficiency. Individual male G0 injection survivors were mated with four virgin w females, and pools of four G0 females, were mated with five w males. Progeny from these crosses (G1-generation) was screened for the presence of green fluorescing eyes by epifluorescence microscopy. G1 individuals showing green eye fluorescence were used to establish transgenic fly lines and analyzed for chromosomal location of the transgene by crossing to the double-balanced marker strain wR135 (w; Sp/CyO; MKRS/TM2). 3xP3-EGFP marker gene detection by epifluorescence microscopy All EGFP fluorescence observations were conducted employing the Leica MZ FLIII fluorescence stereomicroscope and the GFP2 (GFP Plus) filter set (excitation filter: 480/40 nm, barrier filter: 510 nm). Flies were observed with the planachromatic 0.5× objective, whereas embryos and larvae were observed with the planapochromatic 1.6× objective. To analyze embryos for 3xP3-EGFP expression, a dechorionation step was performed (3 min in 1.5% sodium hypochlorite) followed by rinsing twice with PBX (140 mM NaCl, 7 mM Na2HPO4, 3 mM KH2PO4, 0.1%Triton-X). Embryos were mounted on glass slides in 96% glycerol and immediately inspected. Larvae were collected, rinsed in water, anesthetized with diethyl-ether, covered with water and directly analyzed by epifluorescence microscopy. Remobilization of inserted P-element constructs Male individuals of Dm[P{w+mC/3xP3-EGFP}] transformants showing no visible red eye pigmentation, but strong eye fluorescence, were selected and individually mated to virgins of a Drosophila P-element “jumpstarter” strain (Bc Elp / CyO H{P∆2–3}). Male progeny of these crosses showing a mosaic eye pigmentation phenotype was mated to w virgins and progeny was analyzed for the presence of red eye pigmentation and green fluorescence.

Results and discussion 3xP3-EGFP presents a highly sensitive transformation marker that reacts to position effects differently than “mini”-white To construct a potentially universal transformation marker, we made use of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria which has been shown to be active across the animal and plant kingdoms (Cubitt et al. 1995). Since GFP has recently been “engineered” to further enhance brightness (EGFP; Tsien 1998), and filter sets have been optimized to distinguish the EGFP signal from abundant autofluorescence (Plautz et al. 1996), identification of transgenic organisms should be fairly simple. However, GFP requires a strong promoter to ensure reliable detection of single-copy insertions. In addition, activation in a spatial pattern is preferred to allow distinction of the transgene signal from common autofluorescence. The transcription factor Pax-6/Eyeless behaves as the master regulator of eye development throughout the metazoa (Callaerts et al. 1997). After multimerization, an artificially derived ideal Pax-6 homodimer binding site (P3; Wilson et al. 1993, 1995; Czerny and

625 Fig. 1A–H Comparison between the transformation markers 3xP3-EGFP and “mini”white. A–C, G Illumination with cold light source, observation without filters. D–F, H Illumination and observation through the GFP2 filter set. A, D Female w control. B, E Female transgenic for P{w+mC/3xP3-EGFP} with red eye pigmentation and green fluorescence. Note that eye pigmentation starts to quench green fluorescence which can only be detected in ommatidia pointing towards the observer (E). C, F Female transgenic for P{w+mC/3xP3-EGFP} without red eye pigmentation but strong green fluorescence (F). G, H After remobilization of a Pelement insertion originally showing no detectable eye pigmentation but strong green fluorescence (G0 control in upper left hand corner), G2 progeny shows different amounts of red eye pigmentation which are probably due to diverse new insertions. One new insertion shows very high levels of red eye pigmentation (lower left hand corner) that strongly quenches the green fluorescence, which however is still detectable in the ocelli (arrow in H). Note that there is no correlation between the levels of red eye pigmentation and green fluorescence

Busslinger 1995) mediates photoreceptor specific gene expression without providing receptor-subtype specificity (Sheng et al. 1997). Therefore, the multimerized P3 binding site presents the ideal choice to express EGFP in photoreceptors of a wide variety of organisms. In our experiments, we used three tandem repeats of the P3 site placed in front of the TATA homology of hsp70 (3xP3). The small size of the artificial 3xP3-TATA promoter (240 bp) and the EGFP coding region (730 bp) keeps the amount of exogenous DNA in the transposon constructs extremely small, which allows high transformation efficiencies. We first tested the applicability of this potentially universal transformation marker (Berghammer et al. 1999) in Drosophila melanogaster by inserting 3xP3-EGFP in a P-element vector carrying “mini”-white as its primary transformation marker (Thummel and Pirrotta 1992). When screening the G1 offspring for transformants, we checked both for eye pigmentation due to integration of the “mini”-white marker, and for green eye fluorescence to determine the functionality of our 3xP3-EGFP marker (Fig. 1). Each G0 cross with transgenic offspring gave

rise to a series of transformants with varying levels of eye pigmentation, which indicated that multiple insertion events had taken place. All transgenic flies identified by eye pigmentation also showed green fluorescence in compound eyes and ocelli (Fig. 1B, E). However, based on screening for fluorescence, we detected additionally about 20% of transgenic flies in the G1 offspring that had no detectable eye pigmentation, or only some sparsely pigmented ommatidia which normally would not have allowed identification as transformants (Fig. 1C, F). This showed not only that 3xP3-EGFP can indeed serve as a transformation marker, but also indicated that 3xP3EGFP is actually a more efficient marker for transgenesis than “mini”-white. Since many of the phenotypically white-eyed transformants showed rather high levels of green fluorescence, it seemed unlikely that the greater amount of transformants identified with 3xP3-EGFP was simply due to the higher sensitivity of our screening process for fluorescence. It appeared rather that position effect suppression had a different and more prominent influence on “mini”-white than on 3xP3-EGFP. To further address

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this question we chose several independent, phenotypically white eyed, but strongly fluorescing transformants and remobilized the P-element construct by single crosses to a “jumpstarter” line providing P-transposase activity. After outcross of the jumpstarter chromosome, we recovered again a complete spectrum of different eye pigmentation phenotypes from each cross (Fig. 1G), which indicated that the P-element construct was intact, and that the original white eye phenotype must have been caused by position effect suppression. Like the original, all new P-element insertions also mediate green fluorescence (Fig. 1H). As no correlation between the level of eye pigmentation and the level of green fluorescence is detected, position effects appear to affect “mini”-white differently and probably more severely than 3xP3-EGFP. A similar difference in the efficiency of Drosophila “mini”-white and another EGFP-based transformation marker has recently been shown by Handler and Harrell (1999). In piggyBac mediated germline transformation experiments, they compared “mini”-white with nuclear localized EGFP driven by the polyubiquitin promoter and concluded that less than 40% of the transformants identified by fluorescence also showed eye pigmentation. This indicates that not even half of all insertions in the Drosophila genome can be identified with “mini”white as a transformation marker. This seriously questions its further use in transposon mutagenesis and enhancer trap screens. Thus, for the isolation of novel gene functions in so far untargeted gene loci, EGFP-based transformation markers will be much more useful. A further advantage to the use of EGFP-based markers is the simple detection under fluorescence equipped stereomicroscopes. We can screen for transformant flies while they are still in the fly vials. We only need to empty out the vials to isolate the transformants once we have detected them which significantly speeds up sorting through single G0 matings. 3xP3-EGFP is applicable in mariner, Hermes, and piggyBack constructs P-element based constructs can be applied in few Drosophila species only (O’Brochta and Handler 1988) and their preference for hotspots and warmspots hampers mutational saturation aimed for by the Drosophila gene disruption project (Spradling et al. 1999). Hence, we introduced the 3xP3-EGFP marker into a diverse set of unrelated but more promiscuous transposable elements. We chose the hAT element Hermes derived from Musca domestica (Warren et al. 1994), the TTAA-specific element piggyBac from Trichoplusia ni (Cary et al. 1989), and the Tc1/mariner element Mos1 from D. mauritiana (Medhora et al. 1988). By introduction of 3xP3-EGFP as the sole transformation marker, the open reading frame of the respective transposase genes became interrupted, rendering the constructs non-autonomous. Germline transformation was carried out by the use of helper plasmids serving as transient transposase sources

similar to P-element transformation (Rubin and Spradling 1982). Hermes-mediated transformation yielded a 50% transformation efficiency (the percentage of fertile injection survivors producing fluorescent offspring), which is at least as high as our P-element mediated transformation efficiencies. In several G0 crosses more than half of the G1 progeny showed green fluorescence. When we tried to genetically localize the different insertions, we found that many transformant lines had insertions on different chromosomes. This indicates that under our conditions Hermes-mediated transformation led to a high frequency of multiple insertions. For piggyBac-mediated transformation, we first used the helper pBac∆Sac (Handler et al. 1998) that uses the endogenous transposase promoter and only achieved a 2% transformation efficiency. All transformants were derived from injected female G0, as none of the injected male G0 crosses yielded transformed progeny. However, when we switched to the helper phsp-pBac (Handler and Harrell 1999) our transformation efficiency went up to about 35% from both male and female G0 crosses (Berghammer et al. 1999; Horn and Wimmer 2000). We genetically localized the insertions and found in the progeny of several G0 parents insertions on different chromosomes, which again indicates multiple insertions. mariner-mediated transformation yielded a 4% transformation efficiency. In most cases only one or two flies in the G1 progeny showed fluorescence, and when genetically mapping the insertions there was no indication of multiple insertions. Thus, mariner-mediated transformation seems to be an infrequent event and most transformant lines can probably be considered to be single insertions. However, molecular characterization of all the different insertions has not yet been carried out. Some improvement to the mariner-mediated transformation efficiency (up to 10%) could be gained by truncating the transposon backbone (Horn and Wimmer 2000). For all three transposon backgrounds, no sex-specific difference in fluorescence intensity could be detected, but there can be large differences in the fluorescence intensity between different fly lines. In the case of Hermes- and piggyBac-mediated transformation, this might be due to the difference in copy numbers of insertions. However, since these differences also occur in lines derived from mariner-mediated transformation experiments (Fig. 2), we believe that at least some of the differences are due to position effect suppression. Thus, the 3xP3-EGFP marker is also susceptible to position effects, although in a different manner than the “mini”white marker (Fig. 1 G, H). The use of different transposable elements in combination with diverse transformation markers in transposon mutagenesis and enhancer trap screens might indeed allow the isolation of novel gene functions through the identification of insertions in previously untargeted gene loci. Moreover, by using a novel transformation marker in combination with different transposable elements, transposon mutagenesis could even be carried out in the

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Fig. 2A, B 3xP3-EGFP expression as the only transformation marker in a Drosophila w background. Variations in fluorescence signal strength among different Dm[Mos{3xP3-EGFP}] lines. A Strong green fluorescence in both ocelli and compound eyes. B Barely detectable green fluorescence in compound eye. Differences in signal strength are likely to be due to position effects

presence of P-elements, like in FRT-based mosaic (Xu and Rubin 1993) or eye mis-expression screens (Karim et al. 1996). This would facilitate the isolation of previously unidentified gene functions in organogenesis or other late developmental processes. Transformation marker-mediated fluorescence during Drosophila development A further advantage of the 3xP3-EGFP transformation marker is its detectability in adult, pupal, and larval stages (Berghammer et al. 1999). In order to determine the exact spatiotemporal expression pattern mediated by the artificial 3xP3 promoter construct (Sheng et al. 1997), we analyzed EGFP fluorescence of several transgenic lines at different developmental stages. EGFP fluorescence could first be detected in embryos at stage 16 in the developing larval eye, the bolwig organ (BO; Fig. 3A). At stage 17, shortly before hatching, additional fluorescence starts in the central nervous system (CNS), the peripheral nervous system (PNS), the hindgut (HG) and the anal plates (AP). These fluorescence patterns are continuously observed throughout all three larval stages (Fig. 3B), and only cease at the pupal stage during metamorphosis. In late pupal stages, about a day before eclosure, fluorescence is detectable in the developing compound eye (CE; Fig. 3C). The described fluorescence patterns were detected in transgenic fly lines generated with constructs based on four different transposons: P-element (Rubin et al. 1982), mariner (Medhora et al. 1988), Hermes (Warren et al. 1994), and piggyBac (Cary et al. 1989). This confirms that all depicted patterns are indeed mediated by the 3xP3 promoter and do not result from enhancer traps

Fig. 3A–C 3xP3-EGFP transformation marker expression in preadult stages of Drosophila. A Dorsal view (anterior to the left) of a stage 16 embryo, showing the first detectable green fluorescence in the developing larval eyes (Bolwig organ, BO). Yellow autofluorescence of yolk remnants is detectable within the gut. B Second instar larva (lateral view, anterior to the left) showing typical larval expression: BO, central nervous system (CNS), peripheral nervous system (PNS), hindgut (HG) and anal plates (AP). Yellow autofluorescence within the gut is due to ingested fly food. C Late pupal stage (about 24 h before eclosure; anterior to the left, dorsal up) showing strong green fluorescence in the developing compound eye (CE)

or expression mediated by transposon backbones. However, not all transgenic lines showed all of the described expression pattern. Thus, in some lines, certain aspects of the fluorescence pattern are suppressed, which is most likely due to developmental stage- or tissue-specific position effects.

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The artificial 3xP3 promoter element was designed to bind three Pax-6 homodimers (Sheng et al. 1997). Since the two Drosophila Pax-6 homologs, eyeless (ey) and twin of eyeless (toy), are expressed in the BO, CNS, and CE, it was expected that the 3xP3 promoter would mediate fluorescence in these organs (Czerny et al. 1999; Hauck et al. 1999). The fluorescence in the developing CE of late pupae correlates especially well with the late pupal expression of ey (Sheng et al. 1997). However, not in all tissues where the Pax-6 homologs are expressed is fluorescence detectable. Thus, there are additional factors required to allow the 3xP3 promoter to work. Moreover, the fluorescence in the PNS, HG and AP must be driven by different transcriptional activators, as ey and toy are not expressed there. P3 is not specific to dimers of Pax-6 homeodomains but can also be bound by several other paired-class type homeodomains (Wilson et al. 1993). What other paired-class type homeodomain transcription factors are responsible for the fluorescence in PNS, HG and AP is not yet known. The ability to identify embryos and larvae that carry specific transgene constructs by the linked transformation marker will simplify Drosophila developmental research projects, but in species without marked balancer chromosomes this will actually be a fundamental prerequisite. Furthermore, the promiscuity of the described transposon vectors, and the evolutionary conserved “master regulator” function of Pax-6 in eye development (Callaerts et al. 1997), will allow the very same transformation system to be applied to a series of diverged organisms, which will make it a vital tool for the emerging field of evolutionary developmental biology. Acknowledgements We thank Andreas J. Berghammer, Martin Klingler, and Diethard Tautz for valuable discussions on the use of different transposable elements, Anna Wolf, Stefan Heidmann, and Joachim Reischl for critically reading the manuscript. We are indebted to Claude Desplan, Guojun Sheng, Paul Shirk, Malcolm Fraser, Al Handler, Kristin Michel, Alex Pinkerton, and Peter Atkinson for freely providing fly stocks and plasmids. We are thankful to Christian F. Lehner and the members of his group for support, encouragement, and discussions during the course of this work, which was supported financially by a grant from the Deutsche Forschungsgemeinschaft (DFG Le 987/2–1).

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