Download Transposon mediated genetic transformation and more Essays (university) Biotechnology in PDF only on Docsity!
Transposon - mediated genetic transformation
Genetic Transformation of Drosophila willistoni Using piggyBac
Transposon and GFP
1. 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 trans-
formation 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 preexisting 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 germline 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 factoractivated, artificial promoter, containing three tandem repeats of the P 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
3. 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%. Figure 1 - Survival percentages in larvae, pupae and adult, and percentage of established and transformed lineages. 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 fluor- escence 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 genetransfer 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 pre-
sent 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. Figure 2 - Pattern of 3xP3-EGFP gene expression in D. willistoni transgenic larvae. (A) Control (left) and transformed (right) larva. (B) Yellowish auto fluorescence (left) and the green fluorescence expression of EGFP (right) in the whole larvae. Figure 3 - Pattern of 3xP3-EGFP gene expression in D. willistoni transgenic flies. (A) Control (top) and transformed (bottom) flies. (B) Pattern of expression in the eyes was the most intense, however, fluorescence could also be observed in the abdomen, thorax and legs. (C) Eyes fluorescence detected at low time of film exposition.
Berghammer, A.J., Klingler, M. and Wimmer, E.A. (1999), A universal marker for transgenic insects. Nature, 402, 370-371. Callaerts, P., Halder, G. and Gehring, W.J. (1997), PAX-6 in development and evolution. Annu. Rev. Neurosci., 20, 483-532. Cary, L.C., Goebel, M., Corsaro, H.H., Wang, H.H., Rosen, E. and Fraser, M.J. (1989), Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP insertions within the FP-Locus of nuclear polyhedrosis viruses. Virology, 161, 8-17. Catteruccia, F., T. Nolan, T.G., Loukeris, C., Blass, C., Savakis, F.C., Kafatos and Crisanti. A. (2000), Stable germline transformation of the malaria mosquito Anopheles stephensi. Nature, 405, 959-962. Deprá, M., Sepel, L.M.N. and Loreto, E.L.S. (2004), A low-cost methodology to Drosophila transformation with GFP. Genet. Mol. Biol., 27, 70-73. Fraser, J., Smith, G.E. and Summers, M.D. (1983), Acquisition of host-cell DNA sequences by baculoviruses relationship between host DNA insertions and FP mutants of Autographa- californica and Galleria-mellonella nuclear polyhedrosis viruses. J. Virol., 47, 287-300. Fujioka, M., Jaymes, J. B., Bejsovec, A. and Weir, M. (2000), Production of transgenic Drosophila. Methods Molec. Biol., 136, 353-363. Garcia, R.N. Variabilidade Genética e ecológica de Drosophila willistoni (DIPTERA, DROSOPHILIDAE): uma abordagem molecular através do isolamento e caracterização de fragmentos heterogêneos de DNA. PhD Thesis, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. Goñi, B., Parada, C., Rohde, C. and Valente, V.L.S. (2002), Genetic characterization of spontaneous mutations in Drosophila willistoni. I. Exchange and non-disjunction of the X chromosome. Dros. Inf. Serv., 85, 80-84. Hanazono, Y., Yu, J.M., Dunbar, C.E. and Emmons, R.V. (1997), Green fluorescent protein retroviral vectors: low titer and high recombination frequency suggest a selective dis- advantage. Hum. Gene Ther., 8, 1313-1319. Handler, A.M. (2001), A current perspective on insect gene transformation. Insect Biochem Molec. Biol 31, 111-128. Handler A.M. and Gomez SP. (1995), The hobo transposable element has transposase- dependent and - independent excision activity in drosophilid species. Mol. Gen. Genet., 247, 399-408. Handler, A.M. and Harrel, R.A. (1999), Germline transformation of Drosophila melanogaster with the piggyBac transposon vector. Insect Molec. Biol., 8, 449-457.
Handler, A.M. and Harrel, R.A. (2001), Transformation of the Caribbean fruit fly with a piggyBac transposon vector marked with polyubiquitin-regulated GFP. Insect Biochem. Molec. Biol., 31, 201-207. Handler, A.M. and McColombs, S.D. (2000), The piggyBac transposon mediates germline transformation in the Oriental fruit fly and closely related elements exist in its genome. Insect Molec. Biol., 9, 605-612. Handler, A.M., Gomez, S.P. and O’Brochta, D.A. (1993), A functional analysis of the P- element genetransfer vector in insects. Arch. Insect Biochem. Physiol., 22, 373-384. Handler, A.M., McColombs, S.D., Fraser, M.J. and Saul, S.H. (1998), The lepidopteran transposon vector, piggyBac, mediates germline transformation in the Mediterranean fruitfly. Proc. Natl. Acad. Sci. USA, 95, 7520-7525. Horn, C. and Wimmer, E.A. (2000), A versatile vector set for animal transgenesis. Dev. Genes Evol., 210, 630-637. Horn, C., Offen, N., Nystedt, S., Hacker, U. and Wimmer, E. (2003), piggyBac-based inser- tional mutagenesis and enhancer detection as a tool for functional insect genomics. Genetics, 163, 647-661. Horn, C., Schmid, B.G.M., Pogoda, F.S. and Wimmer, E.A. (2002), Fluorescent trans- formation markers for insects transgenesis. Insect Biochem. Molec. Biol., 32, 1221-1235. Irvin, N., Hoddle, M.S., O’Brochta, D.A, Carey, B. and Atkinson, P.W. (2004), Assessing fit- ness costs for transgenic Aedes aegypti expressing the GFP marker and transposase genes. Proc. Natl. Acad. Sci. USA, 101, 891-896. Kidwell, M. G. and Wattam, A. R. (1998), An important step forward in the genetic mani- pulation of mosquito vectors of human disease. Proc. Natl. Acad. Sci. USA, 95: 3349-3350. Klein, C.C., Essi, L., Golombiesk, R.M. and Loreto, E.L.S. (1999), Disgenesia do híbrido em poulações naturais de Drosophila melanogaster. Ciência e Natura, 21, 7-20. Lorenzen, M. D., Berghammer, A.J., Brown, S.J., Denell, R.E., Klingler, M. and Beeman, R.W. (2003), piggyBac-mediated germline transformation in the beetle Tribolium castaneum. Insect Molec. Biol., 12, 433-440. Loukeris, T.G., Livadaras, I., Arca, B., Zabalou, S. and Savakis, C. (1995), Gene transfer into the Medfly, Ceratitis capitata, with a Drosophila hydei transposable element. Science, 270, 2002-2005. Miller, W.J. and Capy, P. (2004), Mobile Genetic Elements. Methods in Molecular Biology, vol. 260. Humana Press Inc., Totowa. New York. O’Brochta, D.A. and Atkinson, P.W. (1997), Recent developments in transgenic insect technology. Parasitology, 13, 99-104.
