Genetic transformation of Drosophila willistoni using piggyBac transposon and GFP

BIOLOGICAL AND APPLIED SCIENCES

Genetic transformation of Drosophila willistoni using piggyBac transposon and GFP

Manuela Finokiet^I; Beatriz Goni^II; Élgion Lúcio Silva LoretoI, III, ^*

^ICurso de Pós Graduação em Biodiversidade Anima; Universidade Federal de Santa Maria;97105-900; [e-mail]; Santa Maria - RS - Brasil

^IISección Genética Evolutiva; Facultad de Ciências; Universidad de la República; Iguá 4225; 11400, [e-mail]; Montevideo - Uruguay

^IIIDepartamento de Biologia; [e-mail]; Universidade Federal de Santa Maria; Santa Maria - RS - Brasil

ABSTRACT

Studies were carried out on the use of piggyBac transposable element as vector and the green fluorescent protein (EGFP) from the jellyfish, Aquorea victoria, as a genetic marker for the transformation of Drosophila willistoni. Preblastoderm embryos of D. willistoni white mutant were microinjected with a plasmid containing the EGFP marker and the piggyBac ITRs, together with a helper plasmid containing the piggyBac transposase placed under the control of the D. melanogaster hsp70 promoter. G₀ adults transformants were recovered at a frequency of approximately 67%. Expression of EGFP in larvae, pupae and adults was observed up to the third generation, suggesting that this transposon was not stable in D. willistoni. Transformed individuals displayed high levels of EGFP expression during larvae and adult stages in the eye, abdomen, thorax and legs, suggesting a wide expression pattern in this species than reported to other species of Drosophilidae.

Key words: Transposable elements, insect transgenesis, germ-line transformation, fluorescent protein marker, microinjection

RESUMO

Descrevemos neste trabalho a transformação genética de Drosophila willistoni empregando o elemento transponível piggyBac como vetor e o gene EGFP (green fluorescent protein ) retirado da água-viva Aquorea victoria, como marcador de transformação. Embriões de D. willistoni em estágio pré-blastoderme, mutantes para o gene white, foram microinjetados com plasmídio contendo o marcador EGFP e as regiões ITRs do transposon piggyBac concomitantemente com um plasmídio auxiliar possuindo o gene da transposase de piggyBac sobre o controle do promotor do gene hsp70 de Drosophila melanogaster. Adultos transformantes Go foram gerados em uma taxa de 67%. A expressão de GFP em larvas, pupas e adultos foi observada somente até a terceira geração, sugerindo que este transposon não é estável em D. willistoni. Os indivíduos transformados exigem um alto nível de expressão de EGFP durante os estágios de larva e, também em adultos o gene marcador é expresso nos olhos, abdome, tórax e patas, mostrando um padrão de expressão mais amplo nesta espécie do que o registrado para outros drosofilídeos.

INTRODUCTION

Genetic manipulations of insects and other arthropods are an important tool for the study of the molecular basis of development and the evolutionary process (Horn and Wimmer, 2000; O'Brochta and Atkinson, 1998). The use of these methodologies is also being considered as a possible solution to certain medical and agricultural problems caused by some insects, through the cautious releasing in nature of insects with desirable genetic alterations (Kidwell and Wattam, 1998; Irvin et al., 2004). A widely method employed for insect germ-line transformation utilizes transposon-based vectors in a binary vector/helper system. The vector contains the parts of a transposon necessary for the transposition; however it is unable to produce the transposase, the enzyme required for the transposition. Furthermore, it can carry exogenous DNA fragments of interest that can be implanted in the genome together with the transposable element. The helper plasmid codifies a transposase. Even though it does not contain the ITR's (inverted terminal repeats), the sequences present at the ends of the transposon are essential for transposition. In this way, the helper plasmid provides the enzyme to vector transposition but cannot insert itself in the host genome (Handler, 2001; Miller and Capy, 2004).

The germ-line transformation was first demonstrated more than 20 years ago in Drosophila melanogaster by the use of the transposable P element (Rubin and Spradling, 1982). However, studies in many different species showed that this element was non-functional outside the Drosophilidae. The P-element system is dependant on the presence of several host factors specific to this species, or to closely related species. This has restricted the P mediated transformation to a few species of subgenus Sophophora of the genus Drosophila (Handler et al., 1993). The first transposon-mediated transformation of a non-drosophilid insect was registered 13 years later in the Mediterranean fruit fly, Ceratitis capitata, using the minos-mediated transformation (Loukeris et al., 1995).

Alternative transposable elements as minos, mariner, Hermes and piggyBac have recently been used for transformation in more than 15 different species of insects, including some that are important for human health and agriculture (Robinson, et al., 2004). The properties of the bacterial transposon TN5 have also been tested looking for improving insect transgenesis methods (Rowan et al., 2004).

The piggyBac element was originally identified by its association with a mutation in a baculovirus passed through the Trichoplusia ni cell line culture (Fraser et al., 1983; Cary et al., 1989). This transposon is naturally found in the genomes of some lepidopteran (Cary et al., 1989). The piggyBac element was first used to transform the medfly, C. capitata (Handler et al, 1998), and more recently it has been used for transformation of many other species (Horn and Wimmer, 2000; Handler and McColombs, 2000; Handler, 2001; Horn et al., 2002; Atkinson, 2002; Robinson et al., 2004; Irvin et al., 2004).

Other important component for the effective development of insect transformation technology is the use of genetic markers that allows recognition of the transformed individuals (O'Brochta and Atkinson, 1997). Eye color genes are quite often used as transformation markers in insects carrying eye color mutations allowing the identification of transformants by mutant-rescue selection. In these cases, the marker gene represents the wild-type allele of a gene that, when mutated, causes a recessive, visible but viable phenotype. However, the lack of suitable recipient mutant strains for most insects of medical and agricultural importance limits their application. Initially, the search for dominant-acting selection markers that could act independently of pre-existing mutant strains focused on genes which confer chemical or drug resistance. These marker systems showed low reliability and efficiency (Handler, 2001; Horn et al., 2002).

Recently, an interesting marker system was developed based on the properties of fluorescent proteins like GFP (green fluorescent protein) and their variants (Horn et al., 2002). The GFP gene from the jellyfish Aequorea victoria (Prasher et al., 1992) is well suited, since it is easily detectable in vivo and has proved to be functional in different tissues of many heterologous systems (Tsien, 1998). It has been used in several organisms as insects, plants, fish, mice and cells of mammals, (Amsterdam et al., 1995; Plautz et al., 1996; Bagis and Keskintepe, 2001). For the expression control of GFP several promoters have been used. Constitutive promoters active in all cells provide the advantage of allowing selection of transformants at all stages since GFP fluorescence can be scored in living embryos, larvae, and adults. The promoter polyubiquitin of D. melanogaster successfully employed by Handler and Harrel (1999) allowed the generation of a transformation marker and the identification of transgenic flies in D. melanogaster. Another commonly used constitutive promoter to drive EGFP is derived from the D. melanogaster actin5C gene tested in Aedes aegypti, Anopheles stephensi and others insects (Pinkerton et. al., 2000; Catteruccia et al, 2000). For the stable germ-line transformation of lepidopteran species, the Bombyx mori A3 cytoplasmic actin gene promoter (BmA3) was chosen to drive EGFP in the silkworm B. mori (Tamura et. al., 2000). At present, transformation markers based on constitutive promoters have only been applied to closely related species, and it is questionable if any such promoter can be functional across insect orders (Horn et al., 2002).

Other constructs already tested in D. melanogaster and Tribolium castaneum which have wide applicability usage as promoters are derived from the rhodopsin gene (3xP3) from Drosophila (Berghaman et al.1999). This is a transformation marker based on a single transcription factor-activated, artificial promoter, containing three tandem repeats of the P3 site in front of a TATA box to drive a strong, eye-specific expression of EGFP (Horn et al., 2002, 2003; Horn and Wimmer, 2000). An evolutionary conserved genetic circuit governs eye development of all metazoan animals, which is under the control of the transcriptional activator Pax-6/Eyeless (Callaerts et al., 1997). This marker construction (3xP3-EGFP) expresses most strongly in the brain, eyes, and ocelli in adults, but can also be observed from several structures in pupae and larvae (Handler et al., 1998). The evolutionary conserved "master regulator" function of Pax-6 in eye development of metazoan suggests that this marker should be applicable to all eye-bearing animals (Callaerts et al., 1997; Handler, 2001; Horn et al., 2002). Furthermore, the small size (1.3 kb) of the 3xP3-EGFP marker provides an additional advantage, as it allows small transposon constructs resulting in high transformation rates (Horn et al., 2002). This transformation system has been applied to generate transgenic insects in different orders as Diptera, Hymenoptera, Lepidoptera and Coleoptera (Horn et al.,2002; Lorenzen et al., 2003; Robinson et al., 2004; Irvin et al., 2004).

Among the Drosophila species, D. willistoni stands out for possessing a great genetic variability and ecological versatility, being one of most studied of the family Drosophilidae. The genome of this species was chosen to be completely sequenciated (http://bugbane.bio.indiana.edu:7151/). Despite the great variability and importance in evolutionary and genetic studies, D. willistoni has not been tested in experiments of genetic transformation. This work aimed to achieve the genetic transformation of a mutant white of D. willistoni using the binary system (vector/auxiliar) introducing a gene marker that codified the protein EGFP, through the microinjection of recombinant plasmids in pre-blastoderm embryos. The pattern of expression of the protein in the larva and adult, and the stability of the transformants were analyzed along six generations.

MATERIALS AND METHODS

Drosophila Stocks and Rearing Conditions

The D. willistoni white mutant strain EM1.00 described by Goñi et al. (2002) was used for genetic transformation. The strain were maintained in standard cornmeal medium (Klein et al., 1999) at constant temperature and humidity (20ºC +/- 2ºC; 60% r.h.).

Plasmids

The vector pBac [3xP3-EGFP] (Horn and Wimmer, 2000) containing the transposon piggyBac inverted terminal sequences (ITRs) necessary for mobility as well as the marker gene EGFP were used. As helper plasmid was used pBƒ´Sac containing the piggyBac functional transposase gene which possesses a deletion in the 5 ' ITR enabling it to transpose (Handler and Harrel, 1999). The plasmids were prepared according to the PEG (polyethylene glycol) purification protocol (Fujioka et al., 2000). The solution for microinjection had a final concentration of 100 µg/ml of each plasmid in ultra-pure water.

Microinjection and Cultural Treatments

The methodology used for microinjection and post-injection treatments is described in Deprá et al. (2004). The hatching larvae were maintained separately in banana medium consisting of 20% (w/v) banana and 1.5% (w/v) agar. After pupation, they were transferred to a standard cornmeal medium.

Analysis of Transformants

The adult flies outcoming from the microinjected eggs (Go) were backcrossed to white flies and the progeny and subsequent generations (G₁-G₅) were examined to identify the fluorescence expression. A diodo laser system was used for this analysis where a filter of emission of 510nm was adapted to stereomicroscopic lens. The observations were made in a dark room (Deprá et al. 2004). The stability of the transgenic lineages was analyzed by the transmission of the transgene to the next generation. During six generations, the individuals were routinely examined and the frequency of larvae and adults that manifested fluorescence was annotated.

RESULTS AND DISCUSSION

A total of 539 embryos (Go) were injected with a mixture of vector and helper plasmids, obtaining 15 larvae and 8 adults. Go adults were backcrossed to white flies, six of them were fertile allowing to establish six isolines. Expression of the fluorescent protein EGFP could be observed in the progeny of four of the isolines established (Fig. 1). The rate of genetic transformation of the Go adults was of 66.7%. This rate is high when compared with that obtained by other authors for other species. In a review about the use of different transposons and genetic markers, Handler and Harrel (1999) describe transformation rates, to piggyBac-GFP varying from 6 to 7%.

According to Horn et al. (2002), one of the obstacles for the use of fluorescent markers in insects is the auto fluorescence generated by many biological materials, as ingested foodstuff, malpighian tubules, the chitinous exoskeleton and necrotic tissue. Therefore, an appropriate emission filter was used to help distinguish the yellowish auto fluorescence of the fluorescence green resultant of the expression of EGFP in the flies. Using this method, it was possible to verify that all the transformed larvae expressed the protein GFP in the whole body, but they presented variation in the intensity among the lineages. The fluorescence of GFP easily could be differentiated of the auto fluorescence common in controls larvae (Fig.2). Although, for adults another pattern was observed, the expression was more intense in the eyes; however, it could also be observed in the abdomen, thorax and legs (Fig. 3).

The results demonstrated that the piggyBac transposable element was an efficient gene-transfer vector system for Drosophila willistoni. Actually, it was much more efficient to D. willistoni genetic transformation, producing a higher frequency of transformants than in other dipterans so far investigated, as D. melanogaster and Ceratitis capitata (Handler et al., 1998). The transposition rate for each transposon varied amongst the tested species. The piggyBac transposon could be extremely active in D. willistoni. Other fact that could be an indicative of high transposition activity of piggyBac in this species was the observation that GFP expression was maintained for only three generations what could be explained by a lost, in the isoline, caused by mobilization of the transposon carrying the EGFP gene. While piggyBac was not present in the D. willistoni genome, some intrinsic transposase source present in the D. willistoni genome could be able to mobilize piggyBac. As illustration of this kind of cross-mobilization, was hobo transposable element from D. melanogaster that could be mobilized by intrinsic sources in a large number of Drosophilidae species (Handler and Gomez, 1995). Other hypothesis for the missing of EGFP expression in D. willistoni in the older generations could be the inactivation of the chromosomal region containing the transgene. Garcia (2004) demonstrated that D. willistoni presented a DNA methylation mechanism not present in D. melanogaster. This mechanism could be involved in gene inactivation, mainly in those regions with transposable elements.

A high mortality at the egg-larvae developmental stage, about 97.2% was observed, a similar rate was also described by several authors (Irvin et al., 2004). Usually, this high mortality was due to problems associated with the technique or to the specific stresses caused by manipulation. According to O'Brochta and Atkinson (2004), the preparation of embryos for injection, the insertion of needles in the eggs and the procedures post-injection were critical steps for the success in the development and maintenance of flies and it was characteristic for each species. Hanazono et al (1997) suggested that the high mortality at the beginning of the development could be associated with the possible toxicities of the GFP and its variants, when expressed in high levels.

According to Atkinson (2002), the transposable elements and the fluorescent proteins seemed to have general application in the production of transgenic insects. However, the use of appropriate promoters to command the expression of the reporter proteins, as well as the stability of the transgene, could depend, or be influenced by the species or the family of the insect to be transformed.

Horn and Wimmer (2000) and Horn et al. (2002, 2003) described the expression of the GFP under the tissue-specific artificial promoters control (3xP3) in several species of Drosophila and observed the reporters protein expression in the eyes and occelii of adult flies. However, some of the transformants of D. willistoni showed a wider expression that was previously described in other species. As suggested by Atkinson (2002) this promoter could have a diverse expression in different species as it happened in D. willistoni. Lorenzen et al. (2003) also obtained the most obvious expression in the eyes and brain with Tribolium castaneum. However, in several additional insertions novel EGFP expression patterns appeared, suggesting that the EGFP marker was influenced by chromosomal enhancers sequences near the sites of piggyBac integration. This was not surprising, since previous reports have shown that the 3xP3 promoter acted as an enhancer detector (Horn et al., 2002, 2003). As seen in the Figs. 2 and 3, the EGFP expression was not restricted solely to the eyes in adults. This was an interesting observation considering the modest phylogenetic distance splitting D. willistoni and D. melanogaster, and the fact that 3xP3 promoter worked precisely in the later species.

The results of the experiment presented here demonstrated that the transformation system composed by transposon piggyBac and marker EGFP was quite efficient to mediate the transformation in D. willistoni. Meanwhile, no stable isolines were obtained suggesting a high mobilization of the piggyBac in this species. The utilization of a concentration of a plasmid smaller that the one usually employed by other researchers (Handler et al., 1998) did not impede the successful genetic transformation of D. willistoni, being the species that showed the highest transformation rate using piggyBac as a vector.

ACKNOWLEDGEMENTS

We would like to thank Dr. Ernest Wimmer from the Universitat Bayreuth, Germany, for providing the pBƒ´Sac and pBac [3xP3-EGFP] plasmids, to Dr. Vera Lúcia Valente for suggestions and to Mr. Fernando Luces for the English revision of the manuscript. This study was partly supported by CSIC (Uruguay), FAPERGS, CNPq, and FIPE-UFSM (Brazil).

Received: March 14, 2005;

Revised: January 27, 2006;

Accepted: September 06, 2006.

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Author for correspondence

Publication Dates

• Publication in this collection
  30 May 2007
• Date of issue
  Jan 2007

History

• Accepted
  06 Sept 2006
• Reviewed
  27 Jan 2006
• Received
  14 Mar 2005