Malcolm J. Fraser

Precise excision of TTAA-specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera

Transposon mutagenesis of baculoviruses provides an ideal experimental system for analysis of the movement of a unique family of mobile element identified from lepidopteran genomes. Members of this family of short‐inverted‐repeat elements are characterized by their extreme Specificity for TTAA target sites. This report describes the analysis of excision events for two representatives of this family, tagalong (formerly TFP3) and piggysac (formerly IFP2). These elements were tagged with a polyhedrin/lacZ reporter gene and inserted back into the virus genome either by homologous recombination or by transposition. Revertants were selected based on a white plaque phenotype. Both elements excise in a precise fashion from their positions in the baculovirus genome in either TN‐368 cells or IPLB‐SF21 AE cells. The precise excision of these elements in infected IPLB‐SF21 AE cells occurs in the absence of either tagalong or piggysac element encoded functions. The common characteristics of both insertion and excision for these elements provides further validation for their inclusion In a single family of unique transposons.

Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties

The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers.

Excision of the piggyBac transposable element in vitro is a precise event that is enhanced by the expression of its encoded transposase.

The piggyBac Lepidopteran transposable element moves from the cellular genome into infecting baculovirus genomes during passage of the virus in cultured TN-368 cells. We have constructed genetically tagged piggyBac elements that permit analysis of excision when transiently introduced on plasmids into the piggyBac-deficient Spodoptera frugiperda IPLB-SF21AE cell line. Precise excision of the element from these plasmids occurs at a higher frequency in the presence of a helper plasmid that presumably supplies the piggyBac transposase. The results suggest that the piggyBac transposon encodes a protein that functions to facilitate not only insertion, but precise excision as well. This is the first demonstration of piggyBac mobility from plasmid sources in uninfected Lepidopteran cells.

piggyBac internal sequences are necessary for efficient transformation of target genomes

A previously reported piggyBac minimal sequence cartridge, which is capable of efficient transposition in embryo interplasmid transposition assays, failed to produce transformants at a significant frequency in Drosophila melanogaster compared with full‐length or less extensive internal deletion constructs. We have re‐examined the importance of these internal domain (ID) sequences for germline transformation using a PCR strategy that effectively adds increasing lengths of ID sequences to each terminus. A series of these piggyBac ID synthetic deletion plasmids containing the 3xP3‐ECFP marker gene are compared for germline transformation of D. melanogaster. Our analyses identify a minimal sequence configuration that is sufficient for movement of piggyBac vectored sequences from plasmids into the insect genome. Southern hybridizations confirm the presence of the piggyBac transposon sequences, and insertion site analyses confirm these integrations target TTAA sites. The results verify that ID sequences adjacent to the 5′ and 3′ terminal repeat domains are crucial for effective germline transformation with piggyBac even though they are not required for excision or interplasmid transposition. Using this information we reconstructed an inverted repeat cartridge, ITR1.1k, and a minimal piggyBac transposon vector, pXL‐BacII‐ECFP, each of which contains these identified ID sequences in addition to the terminal repeat configuration previously described as essential for mobility. We confirm in independent experiments that these new minimal constructs yield transformation frequencies similar to the control piggyBac vector. Sequencing analyses of our constructs verify the position and the source of a point mutation within the 3′ internal repeat sequence of our vectors that has no apparent effect on transformation efficiency.

piggyBac transposon mediated transgenesis of the human blood fluke, Schistosoma mansoni

The transposon piggyBac from the genome of the cabbage looper moth Trichoplusia ni has been observed in the laboratory to jump into the genomes of key model and pathogenic eukaryote organisms including mosquitoes, planarians, human and other mammalian cells, and the malaria parasite Plasmodium falciparum. Introduction of exogenous transposons into schistosomes has not been reported but transposon‐mediated transgenesis of schistosomes might supersede current methods for functional genomics of this important human pathogen. In the present study we examined whether the piggyBac trans‐poson could deliver reporter transgenes into the genome of Schistosoma mansoni parasites. A piggyBac donor plasmid modified to encode firefly luciferase under control of schistosome gene promoters was introduced along with 7‐methylguanosine capped RNAs encoding piggyBac transposase into cultured schistosomula by square wave electroporation. The activity of the helper transposase mRNA was confirmed by Southern hybridization analysis of genomic DNA from the transformed schistosomes, and hybridization signals indicated that the piggyBac transposon had integrated into numerous sites within the parasite chromosomes. piggyBac integrations were recovered by retrotransposon‐anchored PCR, revealing characteristic piggyBac TTAA footprints in the vicinity of the endogenous schistosome retro‐transposons Boudicca, sri, and sr2. This is the first report of chromosomal integration of a transgene and somatic transgenesis of this important human pathogen, in this instance accomplished by mobilization of the piggyBac transposon.—Morales, M. E., Mann, V. H., Kines, K.J., Gobert, G. N., Fraser, M. J.,Jr., Kalinna, B. H., Correnti, J. M., Pearce, E. J., Brindley, P. J. piggyBac transposon mediated transgenesis of the human blood fluke, Schistosoma mansoni. FASEB J. 21, 3479–3489 (2007)

Transposition of the piggyBac element in embryos of Drosophila melanogaster, Aedes aegypti and Trichoplusia ni.

Abstract The Lepidopteran transposable element piggyBac is being recognized as a useful vector for genetic engineering in a variety of insect species. This transposon can mediate transformation in the Dipteran species Ceratitis capitata, and can potentially serve as a versatile vector for transformation of a wide variety of insect species. Using a plasmid-based interplasmid transposition assay, we have demonstrated that this transposon, of the short inverted terminal repeat type, is capable of transposition in embryos of three different insect species, Drosophila melanogaster, the yellow fever mosquito Aedes aegypti, and its host of origin, Trichoplusia ni. This assay can confirm the potential utility of piggyBac as a gene transfer tool in a given insect species, and provides an experimental model for assessing molecular mechanisms of transposon movement.

Insect Transgenesis: Current Applications and Future Prospects

The ability to manipulate the genomes of many insects has become a practical reality over the past 15 years. This has been led by the identification of several useful transposon vector systems that have allowed the identification and development of generalized, species-specific, and tissue-specific promoter systems for controlled expression of gene products upon introduction into insect genomes. Armed with these capabilities, researchers have made significant strides in both fundamental and applied transgenics in key model systems such as Bombyx mori, Tribolium casteneum, Aedes aegypti, and Anopheles stephensi. Limitations of transposon systems were identified, and alternative tools were developed, thus significantly increasing the potential for applied transgenics for control of both agricultural and medical insect pests. The next 10 years promise to be an exciting time of transitioning from the laboratory to the field, from basic research to applied control, during which the full potential of gene manipulation in insect systems will ultimately be realized.

Germ line transformation of the yellow fever mosquito, Aedes aegypti, mediated by transpositional insertion of a piggyBac vector

Mosquito‐vectored diseases such as yellow fever and dengue fever continue to have a substantial impact on human populations world‐wide. Novel strategies for control of these mosquito vectored diseases can arise through the development of reliable systems for genetic manipulation of the insect vector. A piggyBac vector marked with the Drosophila melanogaster cinnabar (cn) gene was used to transform the white‐eyed khw strain of Aedes aegypti. Microinjection of preblastoderm embryos resulted in four families of cinnabar transformed insects. An overall transformation frequency of 4%, with a range of 0% to as high as 13% for individual experiments, was achieved when using a heat‐shock induced transposase providing helper plasmid. Southern hybridizations indicated multiple insertion events in three of four transgenic lines, while the presence of duplicated target TTAA sites at either ends of individual insertions confirmed characteristic piggyBac transposition events in these three transgenic lines. The transgenic phenotype has remained stable for more than twenty generations. The transformations effected using the piggyBac element establish the potential of this element as a germ‐line transformation vector for Aedine mosquitoes.

piggyBac is an effective tool for functional analysis of the Plasmodium falciparum genome

BackgroundMuch of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the Plasmodium genome.ResultsIn this study, we investigated the lepidopteran transposon, piggyBac, as a molecular genetic tool for functional characterization of the Plasmodium falciparum genome. Through multiple transfections, we generated 177 unique P. falciparum mutant clones with mostly single piggyBac insertions in their genomes. Analysis of piggyBac insertion sites revealed random insertions into the P. falciparum genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in P. falciparum with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development.ConclusionOur results clearly demonstrate that piggyBac is a novel, indispensable tool for forward functional genomics in P. falciparum that will help better understand parasite biology and accelerate drug and vaccine development.

Transposon mutagenesis of baculoviruses: analysis of TFP 3 lepidopteran transposon insertions at the FP locus of nuclear polyhedrosis viruses

We report the complete sequences of two representatives of the TFP3 transposable element family of the lepidopteran, Trichoplusia ni. These elements were isolated as insertions mobilized from the Lepidopteran host genome into two closely related nuclear polyhedrosis viruses (NPV) during infection. Both elements inserted within the 500-bp FP locus of the respective viral genomes (map units 36.0 to 37.0), causing a distinctive plaque morphology phenotype and the loss of a 25-kDa viral-specific protein. Both insertions occurred at the identical TTAA target site in the respective genomes, in the same relative orientation, and are flanked by 15-bp imperfect inverted repeats. The inserted elements interrupt the 25K open reading frame (ORF). One of these FP mutants undergoes spontaneous reversion. Sequence analysis at the excision site of a spontaneous revertant demonstrates that the TFP3 elements are capable of precise excision, restoring the expression of the 25-kDa protein. We also compare the sequences of the 25K genes of the Autographa californica and Galleria mellonella viruses (AcMNPV and GmMNPV, respectively). The 25K gene sequences diverge in five areas, resulting in an additional EcoRV and TaqI site within the GmMNPV 25K gene, and extension of the ORF for an additional 2 amino acids at the C-terminus of the predicted GmMNPV 25 kDa protein. The phenomenon of transposon mutagenesis of Baculovirus genomes provides a unique opportunity for analysis of transposon mobility.