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* Department of Tropical Medicine, and Biomedical Sciences Program, Tulane University Health Sciences Center, New Orleans, Louisiana, USA;
Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Brisbane, Queensland, Australia;
Department of Biological Sciences, University of Notre Dame, South Bend, Indiana, USA;
Centre for Animal Biotechnology, Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria, Australia; and
|| Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
1Correspondence: Department of Tropical Medicine, Tulane University, Health Sciences Center, 1430 Tulane Ave., New Orleans, LA 70112, USA. E-mail: paul.brindley{at}tulane.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: schistosome • gene manipulation • electroporation • parasitic helminth • functional genomics • reverse genetics
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. Advances in molecular genetics and immunology hold the promise to control the spread of schistosomiasis and to guide development of new tools to combat this neglected tropical disease. At present, control of schistosomiasis relies largely on chemotherapy with praziquantel, but wide-scale use of this medication has led to concerns about the development of drug resistance (4)
.
Schistosomes have comparatively large genomes, estimated at 270 to 330 megabase pairs for the haploid genome of Schistosoma mansoni, arrayed on seven pairs of autosomes and one pair of sex chromosomes (5
, 6)
. This is about the same size as that of the puffer fish, Takifugu rubripes, 2- to 3-fold those of Arabidopsis thaliana or Caenorhabditis elegans, 10-fold the size of the Plasmodium falciparum genome, and one-tenth the size of the human genome. Schistosoma hematobium and Schistosoma japonicum, the other major schistosome species parasitizing humans, probably have a genome size similar to that of S. mansoni based on the similarity of their karyotypes (5)
. The genome is AT-rich (60–70% AT in the euchromatin) and replete with repetitive sequences, including retrotransposons. The 
17,000 protein-encoding genes include several to numerous introns ranging from small to very large in size. Single nucleotide polymorphisms occur, trans-splicing of a subset of the transcriptome takes place, and alternative splicing is known to occur with some genes, expanding the complexity of the proteome (reviewed in refs. 6
, 7
). It is anticipated that draft genome sequences of the nuclear genomes of S. mansoni and S. japonicum will be reported within the next year, following the release recently of reasonably comprehensive descriptions of their transcriptomes (8
, 9)
and proteome (10)
. Despite this abundance of sequence data, functional analysis of potential intervention target genes will not be routine until reliable methods for reverse genetics in schistosomes become available (6)
. Methods and tools for gene manipulation of schistosomes remain in their infancy compared with established transgenesis systems, tools, and strategies in parasitic protozoans and mosquito vectors of pathogens (see refs. 11
, 12
).
The fundamental nature of the blood flukes—their complex developmental cycles, large size, multicellular tissues, and complex organization—along with the absence of immortalized cell lines and our inability to rear the entire life cycle in vitro has hindered development of tractable transgenesis models (13)
. Nonetheless, loss-of-function procedures involving RNA interference targeting several discrete schistosome genes in several developmental stages have been reported (e.g., refs. 14
15
16
17
). Furthermore, gain-of-function approaches involving reporter gene activity following introduction of mRNA or circular plasmid DNA into schistosomes have been described, although the procedures and constructs used would not facilitate chromosomal integration of the transgenes (18
19
20
21
22)
. Here we report piggyBac transposon-mediated transgenesis of S. mansoni, which to our knowledge represents the first report of movement of an exogenous transposon into schistosome chromosomes. This somatic transgenesis was accomplished using square wave electroporation of a linearized piggyBac donor cassette and helper transposase mRNA into cultured schistosomules. Integration of the piggyBac transgenes was mediated at numerous sites in the parasite genome, and reporter luciferase activity driven by an endogenous actin gene promoter was detected in tissues of the transformed schistosomules.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Schistosomula were washed three times in wash medium and cultured at 37°C under 5% CO2 in air in modified Basch’s medium supplemented with small quantities of washed human erythrocytes (24)
. Culture media were changed every second day. For most procedures described here, schistosomules were cultured in vitro for 15 days before transformation with transgenes (22)
.
PCR amplification, plasmid constructions
A 3.4 kb and a 2.5 kb fragment containing the Photinis pyralis (firefly) luciferase coding sequence under the control of the promoter of the S. mansoni actin 1.1 gene (22)
or the S. mansoni HSP-70 gene (20)
and the SV40 termination sequence were amplified from plasmid pJC1 (22)
and the plasmid HSP-70-pL3 Basic (20)
, respectively. PCRs using high-fidelity polymerase (Invitrogen, Carlsbad, CA, USA) employed primers Sm-Act-F 5'-ccggaattcTATGGGTAAGC-GTTGTTCAC and SV-40-R 5'-ccggaattcCATCGGTCGACGGATCCTTATC for the 3.5 kb SmAct-Luc-SV40 fragment, and primers SmHSP70-F 5'-ccggaattcTAATAATGTACAACTCGGTG and SV-40-R 5'-ggaagatctGGCATCGGTCGACGGATCC for the 2.5 kb SmHSP70-Luc-SV40 fragment. The PCR conditions included an initial 1 min denaturation at 94°C followed by 38 cycles of 30 s at 94°C, 30 s at 50°C, 4 min at 68°C, and a final extension at 72°C for 7 min. These amplified reporter gene sequences were ligated within the inverted terminal repeats of the piggyBac transposon in the vector pXL-BacII (25)
to construct donor plasmids pXL-BacII-SmAct-Luc and pXL-BacII-SmHSP70-Luc (Fig. 1
).
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Helper transposase mRNA, donor plasmids
To synthesize piggyBac transposase and firefly luciferase mRNAs, DNA template were prepared by PCR from pBSII-IE1-orf (25)
and pGL3-Basic (Promega, Madison, WI, USA) templates, respectively, with the T7 promoter mRNA-pB-Trnas-F 5'-taatacgactcactatagggATCCTATATAATAAA-ATGGG and mRNA-pB-Trnas-R, 5'-CCAAATTCAACAAACAATTT primers, and primers for the luciferase gene as described (21)
. In vitro transcriptions of capped RNAs from PCR DNA templates were accomplished using the mMessage mMachine T7 Ultra kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Subsequently, LiCl-precipitated RNAs were dissolved in nuclease-free water and quantified by spectrophotometry.
Two piggyBac donor plasmids were linearized by digestion with BssS I (New England BioLabs, Ipswich, MA, USA) at 37°C for 18 h. BssS I cleaves twice within the plasmid backbone but does not cleave within the transposon cassettes (25)
so that BssS I digestion releases two fragments: one contains the transposon cassette, of 6732 bp from pXL-BacII-SmAct-Luc and 5171 bp from pXL-BacII-SmHSP70-Luc, and the other a 1390 bp fragment of the plasmid backbone (supplemental Fig. S1). BssS I-digested donor plasmid DNAs were recovered by precipitation with ethanol/sodium acetate precipitation (twice) to ensure the purity of the linearized donor plasmid DNA, after which it was dissolved in schistosomule wash medium. Linearized donor transposons were delivered to schistosomules by electroporation at a concentration of 12 µg plasmid/100 µl schistosomule wash medium.
Electroporation of schistosomules
For electroporation of donor plasmids and helper as mRNA, the schistosomula were removed from culture 15 days after cercarial transformation. Electroporation was performed as described previously (22)
. In brief, at the time of electroporation, piggyBac transposase mRNA was added to the DNA at a final concentration of 6 µg mRNA/100 µl. Electroporations were accomplished in 4 mm gap cuvettes (BTX, San Diego, CA, USA) with 20,000 parasites resuspended in 100 µl of wash medium containing 12.0 µg donor plasmid and 6.0 µg of helper mRNA using the BTX ElectroSquarePoratorTM ECM830. We introduced either intact circular or linearized (donor) plasmids, with or without transposase mRNA, into schistosomules by square wave electroporation (125 V, 20 ms, 4 mm). Immediately after electroporation, parasites were transferred to prewarmed Basch medium and cultured for up to 7 days as described above. Subsequently schistosomula were washed three times with prewarmed wash medium and treated with DNase for 1 h at 37°C to remove any residual donor plasmids. Parasites were then washed three more times with wash media to remove DNase and stored at –80°C.
Plasmid excision assay
Around 20,000 schistosomules were cultured for 15 days before transfection as described previously. Twelve micrograms of donor plasmid and 6 µg of mRNA were transfected by square wave electroporation into schistosomules. Seven days later, residual donor plasmid DNA was isolated along with schistosome gDNAs using the QIAprep® Spin Miniprep kit method described by Ziegler et al. (26)
, then used as template for PCR with the following primers: 5'-GCTGGCGAAAGGGGGATGTG and 5'-CGGCTCGTATGTTGTGTGGAA (27)
to detect the presence of donor plasmids that had undergone excision. The PCR included an initial step at 94°C for 1 min, then 38 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, followed by a final extension step at 72°C for 5 min, after which products were visualized in ethidium-stained agarose gels (1%).
Southern hybridizations
Total gDNA was isolated from transformed or control schistosomes using the Aquapure system from Bio-Rad (Hercules, CA, USA). Genomic schistosome DNA and donor plasmid pXL-BacII-SmAct-Luc were cleaved with SphI, separated by electrophoresis through 1% agarose, and the fragments were transferred by capillary action to nylon membranes (Zeta-probe, Bio-Rad) (28)
. A luciferase gene probe was amplified from pGL3 Basic (Promega) with primers Luc-F 5'-ATGGAAGACGCCAAAAACAT and Luc-R 5'-TACACGGCGATCTTTCCGCC, and cycling conditions of 1 min at 94°C followed by 38 cycles of 30 s at 94°C, 30 s at 50°C, 2 min at 68°C, and a final extension at 72°C for 7 min. The 1.6 kb product was isolated by agarose gel electrophoresis, eluted from the gel, and labeled with 32P.dCTP using random oligomer priming (RadPrime DNA Labeling System, Invitrogen). Southern blots were hybridized to the labeled luciferase gene probe for 18 h, then washed at high stringency (29)
. Hybridization signals were detected by autoradiography on X-ray film (Biomax, Kodak).
Retrotransposon-anchored PCR
We developed an anchored PCR-based approach we term RAP (=retrotransposon anchored PCR) in order to investigate piggyBac integrations into the schistosome genome. In brief, RAP used a primer specific for the luciferase (luc) transgene from the donor piggyBac cassette in tandem with a second primer specific for endogenous retrotransposons known to be present at high copy number in the genome of natural populations of S. mansoni (30)
. Specifically, the primers included sequences specific for the retrotransposons SR1, SR2, Boudicca, fugitive, or the SINE-like retroposon SM alpha (31
32
33
34
35
36)
. Sequences of the oligonucleotide primers and nested primers specific for luciferase and the retrotransposons are shown in supplemental Table S1 and supplemental Fig. S2. For SR1, SR2, and fugitive, discrete sets of primers were designed to amplify from either direction of the mobile genetic element (Supplemental Fig. S2). The RAP primers were used with 100 ng template gDNA from populations of transformed schistosomules and PCR SuperMix High Fidelity (Invitrogen). The RAP cycling conditions were 94°C for 30 s, followed by 38 cycles of 94°C for 30 s, 55°C for 30 s, and 68°C for 10 min, with a final extension at 72°C for 10 min. For nested PCR, the primary RAP products were diluted 1:10 and nested PCR was performed under the same cycling conditions using a second set of primers, one targeting the schistosome retrotransposon and the other the luciferase gene. Primary and nested RAP products were analyzed by ethidium-stained agarose gel (1%) electrophoresis, after which the products were transferred to nylon (Zeta-probe) and hybridized under stringent conditions to labeled luciferase probe, as above. From the nested RAP products that appeared to be positive in the Southern hybridizations, fragments ranging from 7 kb to 2 kb in size were isolated from a crystal violet gel. These RAP products were isolated from the gel and cloned into plasmid pCR-XL TOPO (Invitrogen) according to the manufacturer’s instructions to generate libraries of integration junction fragments. Minipreps of plasmids from randomly selected colonies from the libraries were isolated using the GenEluteTM; Plasmid Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA), digested with EcoR I to release the inserts from pCR-XL TOPO, sized by agarose gel electrophoresis, the fragments transferred to nylon, and the membranes probed with the labeled luciferase probe, as above. Nucleotide sequences of the inserts of positive clones were determined.
Luciferase activity assay
Parasites transformed with luciferase mRNA were harvested 3 h after electroporation. The worms were washed three times and stored as pellets at –80°C until needed. Parasites transformed with donor piggyBac plasmids with or without helper transposase mRNA were harvested from 24 h to 7 days after electroporation. Luciferase activity in extracts of these schistosomules was monitored using Promega’s luciferase assay reagent system and a Sirius luminometer (Berthold, Pforzheim, Germany), as described (21)
. In brief, pellets of cultured schistosomules were subjected to sonication (5x5 s bursts, output cycle 4, Heat Systems-Ultrasonics, Plainview, NY, USA) in 250 µl CCLR lysis buffer (Promega). Aliquots of 100 µl of sonicate were injected into100 µl luciferin substrate (Promega) at room temperature, mixed, and the relative light units (RLUs) were determined in the luminometer 10 s later. Duplicate samples were measured, with results presented as the average of the duplicate readings. Recombinant luciferase (Promega) was included as a positive control.
Immunolocalization of luciferase, confocal microscopy
Schistosomules were processed in 1.5 ml microcentrifuge tubes with centrifugation for 2 min at 10,000 g between each wash. The worms were washed in PBS (3 x each, 1–2 min) to remove culture media, etc., then fixed in 10% formalin in PBS for 20 min at 23°C or overnight at 4°C. Schistosomule tissues were permeabilized by incubation in 0.2% Triton X-100 in PBS for 15 min, washed in PBS twice to remove the detergent, and incubated in 0.5% nonfat milk in PBS for 15 min at 23°C to block unbound antibody sites. After two further PBS washes, schistosomules were incubated in primary antibody (goat antiluciferase, 1:250; Promega) for 18 h at 4°C or for 45 min at 37°C. After removal of the primary antibody (PBS, 3 x washes), schistosomules were incubated in secondary antibody (Alexa 594 donkey anti-goat [red] (Molecular Probes, Eugene, OR, USA), 1:250) for 45 min 37°C and washed three times in PBS. Samples were mounted in 50% glycerol/PBS and viewed with an inverted microscope (Nikon, Eclipse TS100) fitted with a red filter (Texas Red, 540–580 nm excitation, 595 nm emission). Images were captured with a digital camera (CoolPix 5700, Nikon). In addition, confocal laser micrographs were obtained on similarly processed samples using a Leica SP2 AOB Microscopy System with a 543 nm He/Ne laser, as described (37)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Seven days later, preparations of plasmid DNAs together with genomic DNA of the schistosomes were isolated from the transformed schistosomules and the DNA preparation was used as template for PCRs with plasmid backbone specific primers that flank the piggyBac donor cassette. An excision-dependent PCR product with size 700 bp was detected, suggesting that successful excision of the piggyBac cassette had occurred (Supplemental Fig. S3A, B). The results suggested that piggyBac was active in schistosomule tissues (which was subsequently confirmed when integration junctions between piggyBac and schistosome chromosomes were cloned and sequenced; see below).
Luciferase activity in piggyBac transformed schistosomules
Next we investigated luciferase activity in transformed schistosomules. Schistosomules were transformed by electroporation with a piggyBac donor construct, linearized pXL-BacII-SmAct-Luc, in the presence of in vitro-transcribed helper piggyBac transposase mRNA (Fig. 1
; Supplemental Fig. S1). Control groups of somules were transformed with the donor construct but without the helper transposase mRNA. An additional control group included schistosomules transformed with luciferase mRNA only. The somules were cultured from 1 to 7 days after electroporation, then their tissues were examined for luciferase activity. With somules cultured in vitro for 15 days before transformation by electroporation, the highest levels of luciferase activity were seen 48 h after electroporation. Luciferase activity had waned substantially by 5 days after electroporation (Fig. 2
A). Pearce and co-workers (22)
reported a similar time course of luciferase activity in schistosomules cultured in vitro, driven by the S. mansoni actin 1.1 gene promoter in plasmid pGL3; after electroporation of somules with circular plasmid, luciferase activity was relatively constant from 24 to 72 h but was significantly reduced on day 7 compared with day 2. Luciferase protein was also detected by immunofluorescence in somules exposed to linearized donor constructs only or to luciferase mRNA (Fig. 2B-F
). Together, these findings directly demonstrated that the electroporation procedures had introduced luciferase mRNA into the somules, as reported (21)
. Further, luciferase activity detected in schistosomules transformed with the linearized donor cassette plasmid (with or without transposase mRNA) indicated successful transcription of luciferase from the donor piggyBac plasmid and/or integrated piggyBac cassette transgenes and translation to active luciferase enzyme. The linearized plasmid was introduced into schistosomules by square wave electroporation, a nucleic acids delivery method for these parasites pioneered by Pearce and co-workers (15
, 21)
. Earlier reports had described reporter gene activity after mRNA or circular plasmid were introduced into schistosomes (18
, 21)
. Therefore, this may be the first demonstration of activity of linearized DNA transgenes in schistosomes.
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Southern blot analysis indicates integrations of piggyBac into schistosome chromosomes
Because the outcome of the plasmid excision assay and translation of active enzyme after delivery of mRNA by electroporation both suggested that mobilization of piggyBac was feasible in schistosome tissues, we designed a Southern hybridization experiment to investigate integration of piggyBac into schistosome chromosomes. Genomic DNA (gDNA) isolated from 20,000 schistosomules (somules) transformed by electroporation with piggyBac (linearized donor plasmid pXL-BacII-SmAct-Luc with or without helper transposase mRNA) was digested with SphI, after which fragments were resolved by electrophoresis, transferred to nylon, and hybridized to a labeled luciferase gene probe. SphI cuts twice within pXL-BacII-SmAct-Luc, including once within the luciferase gene (luc) (Fig. 3
A, B), releasing a fragment of 2.5 kb from the donor cassette that is expected to hybridize to the luciferase probe. Hybridization to gDNA from somules electroporated with linearized pXL-BacII-SmAct-Luc and transposase mRNA was apparent (Fig. 3C
, lane 3). By contrast, a single weak signal at 2.5 kb was seen in gDNA from somules electroporated with the donor plasmid alone (without helper mRNA) (Fig. 3C
, lane 4). This 2.5 kb band in Fig. 3C
, lane 4 indicated the presence of residual, nonintegrated donor plasmid. SphI digestion of the (BssS I) -linearized pXL-BacII-SmAct-Luc plasmid would release two fragments, both 2.5 kb in length (Supplemental Fig. S1) and capable of hybridization to the luc probe. No hybridization was seen to gDNA isolated from nontransformed control schistosomes (Fig. 3C
, lane 2). The hybridization signals in Fig. 3C
, lane 3 in fragments of gDNA from somules exposed to donor piggyBac plasmid and helper mRNA ranged from <1 kb to >12 kb in length. Signals of variable length larger than 2.5 kb likely represented SphI fragments of gDNA into which the piggyBac transposon had been inserted and that included the 5'-region of the luc transgene to which the radiolabeled probe had hybridized (Fig. 3B
). The smear-like appearance of the hybridization signals in the group exposed to both donor and helper can likely be explained by the large population of individual somules transformed by electroporation with piggyBac, the large size of the schistosome genome, and the numerous potential integration sites. The absence of a dominant signal at 2.5 kb in Fig. 3C
, lane 3 was enigmatic since a SphI fragment of this size was expected to be released from each piggyBac integrant, although longer exposure of the autoradiograph revealed a comparatively stronger band at 2.5 kb (not shown).
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RAP locates piggyBac integrated within schistosome chromosomes
Similar preparations of gDNA isolated somules transformed with linearized donor piggyBac plasmid (pXL-BacII-SmAct-Luc or pXL-BacII-SmHSP70-Luc) in the presence of helper transposase mRNA were used as templates for an anchored PCR-based approach that we term RAP (=retrotransposon anchored PCR). In brief, RAP uses a primer specific for the luciferase (luc) transgene from the donor piggyBac cassette in tandem with a second primer specific for endogenous retrotransposons known to be present at high copy number in the genome of natural populations of S. mansoni (see ref. 30
). Specifically, the primers included sequences specific for the retrotransposons SR1, SR2, Boudicca, fugitive, or the SINE-like retroposon SM alpha (see ref. 35
). Figure 4
A provides a schematic representation of the RAP protocol. We analyzed patterns of the resulting PCR products in ethidium-stained gels and by Southern hybridization analysis of the RAP products to a labeled luciferase gene probe (Fig. 4B
). The patterns of hybridization indicated the presence of amplicons representing integration events of piggyBac into S. mansoni chromosomes. Subsequently we cloned some Southern hybridization-positive PCR bands into pCR-XL-TOPO. Figure 4C
presents a representative example of the results we obtained, in this example showing positive RAP fragments amplified using SR2 retrotransposon-specific RAP primers. When the nucleotide sequences of the inserts of the positive pCR-XL-TOPO clones were determined, it was apparent that piggyBac had integrated at numerous loci in the schistosome chromosomes. We cloned and sequenced 19 discrete integrations, the details of which are described in Fig. 5
and supplemental Table S2. These integration events included integrations of pXL-BacII-SmAct-Luc and pXL-BacII-SmHSP70-Luc donor cassettes. They were recovered with primers specific for three of the five endogenous mobile genetic elements targeted: retrotransposons SR1, SR2, and Boudicca.
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piggyBac integrations are TTAA-site-specific and widespread
The characteristic insertion site footprint TTAA was in place at the junction of the piggyBac insertions into all 19 of the piggyBac integration sites within the schistosome chromosomes (Fig. 5)
. These integration sequences have been assigned GenBank accession nos. E1211024 to E1211042. The GenBank accessions include annotations of the genomic locations of the transgenes as determined by Blast analyses of the NCBI (www.ncbi.nlm.nih.gov/BLAST) and Sanger Institute (www.sanger.ac.uk/cgi-bin/blast/submitblast/s_mansoni/omni) databases. In brief, piggyBac was found to have inserted in the vicinity of endogenous mobile genetic elements, including the retrotransposons SR1, SR2, Boudicca, and Perere-5 and the transposon, Merlin (e.g., accession nos. E1211028, E1211035, E1211025, E1211037, and E1211024). piggyBac also inserted near to or within the introns of protein encoding genes, including those encoding adenylosuccinate lyase, glutathione peroxidase 1, and glutathione S transferase (E1211038, E1211040, and E1211031). The findings definitively document successful somatic transgenesis of the S. mansoni genome with the piggyBac transposon and indicate a widespread distribution of piggyBac integrations within the schistosome genome.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 39)
. piggyBac is 2472 bp long with identical short-terminal inverted repeats 13 bp in length and additional asymmetric 19 bp internal repeats (40
, 41)
. It targets the tetranucleotide TTAA (40
, 42)
and, upon excision, reconstitutes the TTAA target sequence without insertion or deletion mutations (43
, 44)
. piggyBac has been used to transform the germline and establish lines of transgenic insects of >10 species, including mosquitoes (see refs. 45
, 46
), and will also mobilize in mammalian cells (47
48
49)
and the malaria parasite P. falciparum (50)
. Furthermore, it is active in the planarian Girardia tigrina (51)
, which though not a parasite is, like S. mansoni, a flatworm and a member of the phylum Platyhelminthes. In the present report, we investigated the potential of piggyBac to mobilize in schistosomes.
To determine whether piggyBac might be active in schistosomes, we undertook plasmid excision assays. Recovery of excised plasmids from tissues of transformed schistosomules indicated that piggyBac was active in schistosomes. We also investigated the activity of helper plasmid DNA encoding the piggyBac transposase. Evidence of mobility of the piggyBac cassette out of the plasmid backbone and/or integration into schistosome chromosomes were not apparent (not shown). By contrast, introduction of transposase mRNA (rather than helper plasmid) as the source of help for the linearized donor plasmid constructs facilitated mobilization of piggyBac in schistosomules. Integration into schistosome chromosomes from supercoiled plasmid constructs was not apparent by Southern hybridization or by RAP (not shown). Provision of mRNA as the source of transposase has been reported to mediate transposon insertion and expression in other systems, including Sleeping Beauty in mammalian cells (52
, 53)
and piggyBac in insects (27)
. Not only might in vitro-transcribed piggyBac transposase be more efficient for mobilization of the transposon in schistosome cells, its use also precludes potential problems with integration and continued expression of the transposase when codelivered as a DNA (rather than RNA) molecule. Although we did not titrate optimal ratios of donor plasmid and helper transposase in order to define maximal transposition activity, molar ratios of transposon and transposase may not be critical in the piggyBac system for schistosomes, because piggyBac does not appear to exhibit the overproduction inhibition phenomenon exhibited by Sleeping Beauty and other transposons (49)
. In any event, in vitro-transcribed mRNA clearly was an effective source of transposase for piggyBac-mediated transposition in schistosomes.
Of the methods reported to date for transformation of schistosomes with exogenous transgenes, most would not reliably direct integration of the transgene into schistosome chromosomes (18
, 19
, 54)
. Transduction of schistosomes with pseudotyped murine leukemia virus apparently leads to integration of transgenes into schistosome chromosomes (55)
although it is not yet clear how suitable or proficient this system is for schistosomes given, for example, the unusual lipid bilayer at the surface of the parasite’s tegument (56)
and reporter gene silencing of proviral retrovirus transgenes in some other contexts (57)
. However, integration of reporter transgenes into schistosome chromosomes clearly is a desirable feature for the development of gene manipulation vectors for schistosomes. The genome of S. mansoni is AT-rich, and since piggyBac exhibits specificity for TTAA target sites (44)
, we hypothesized that piggyBac might be capable of integration into the schistosome genome. On the other hand, given the large size of the schistosome genome, the abundant potential integration sites, and the fact that the gDNA preparations were likely to be contaminated with excess donor plasmids, locating piggyBac integrations was not straightforward. Nonetheless, Southern hybridization analysis of gDNAs digested with SphI from populations of schistosomules transformed with donor constructs plus helper transposase mRNA detected a luciferase-positive signal at 2.5 kb and a smear of other variable length signals. The luciferase gene-positive SphI bands all represented potential piggyBac transposon insertions. The intensities of these signals were similar, which suggested insertions at chromosomal loci in multiple cells of the population of transfected schistosomules.
Whereas the Southern hybridization analysis strongly indicated that piggyBac integrations had taken place, it did not definitively establish whether integration into schistosome chromosomes had occurred or, if so, where the integrations had taken place. These specific details can only be confirmed by molecular cloning and sequencing of piggyBac integration events. PCR-based procedures, including inverse PCR (e.g., ref. 58
), can be expected to facilitate recovery of the integration events. Likewise, anchored PCR procedures such as linker-mediated PCR have been used to locate exogenous transgenes (59
, 60)
. However, the utility of inverse or anchored PCR techniques is constrained by the requirement to digest the gDNA, which can affect the efficiency of the techniques to recover the integration events. We developed an alternative anchored PCR protocol to search for piggyBac integrations, a technique termed RAP that uses direct PCR targeting nondigested template DNA. RAP is similar to the Alu-PCR procedure that has been used to locate hepatitis C provirus within human chromosomes (e.g., ref. 61
). Alu-PCR relies on the presence of the 1.1 million copies of the Alu retroposon interspersed throughout the human genome (62)
.
Like Alu-PCR, RAP is likely to be convenient in schistosomes because there are numerous copies of each of five or more discrete retrotransposons within the genome of S. mansoni (30
, 35
, 63)
that might act as PCR anchors, providing piggyBac had integrated in the vicinity of a copy of one of the endogenous mobile genetic elements. Moreover, in human cells many piggyBac integrations are known to occur near endogenous retrotransposons (49)
. Of the five endogenous elements we harnessed, we recovered piggyBac integrations from RAPs anchored with the retrotransposons SR1, SR2, and Boudicca. The cloned RAP fragments ranged in size from 1.5 kb to 4 kb, and 16 of the 19 (84%) were amplified using SR2-specific primers. SR2 is present in the schistosome genome at a higher copy number than SR1 or Boudicca (33)
, which may account for the superior recovery rate with SR2. In addition, of the integrations recovered, most (17 of 19; 89%) involved the SmHSP70-Luc piggyBac cassette. Since the insert size of this is smaller (3180 bp) than that of the pXL-BacII-SmAct-Luc piggyBac (4080 bp) cassette, the pXL-BacII-SmHSP70-Luc cassette may be more efficiently mobilized, resulting in more frequent integrations. Nonetheless, piggyBac can deliver large transgenes of >10 kb in length in mammalian cells (47)
. In overview, by using RAP to locate transgene-schistosome chromosome junctions, we provide the first direct evidence of somatic transgenesis of schistosomes, or indeed of any parasitic helminth. We anticipate that RAP will be helpful for further delineating the mechanisms underlying piggyBac (or indeed other kinds of) transgene integration, including issues of target site preference, transgene copy number, and vertical transmission.
In conclusion, the piggyBac transposon can mediate transgenesis of the human blood fluke, S. mansoni. Somatic transgenesis of schistosomes was accomplished by electroporation of linearized donor piggyBac plasmid and helper transposase mRNA into cultured schistosomules. Integration of piggyBac took place at target TTAA sites. Reporter luciferase activity was evident in piggyBac transformed schistosomules driven by the S. mansoni actin gene promoter. To reiterate, this represents the first report of movement of an exogenous transgene into schistosome chromosomes and it expands the range of mobilization of the piggyBac transposon into a parasitic helminth of major public health significance. Further, the findings suggest a pathway forward toward reverse genetics, heritable transgenesis, and functional genomics of schistosomes. Our future studies with piggyBac-mediated transgenesis of schistosomes will include a focus on germ line transgenesis and enhanced reporter gene activity. Moreover, by using chimeric transposase, it might be feasible to direct piggyBac integration into specific sites on schistosome chromosomes (45)
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ACKNOWLEDGMENTS |
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Received for publication April 3, 2007. Accepted for publication May 24, 2007.
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