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Transposon-mediated chromosomal integration of transgenes in the parasitic nematode Strongyloides ratti and establishment of stable transgenic lines.

ABSTRACT: Genetic transformation is a potential tool for analyzing gene function and thereby identifying new drug and vaccine targets in parasitic nematodes, which adversely affect more than one billion people. We have previously developed a robust system for transgenesis in Strongyloides spp. using gonadal microinjection for gene transfer. In this system, transgenes are expressed in promoter-regulated fashion in the F1 but are silenced in subsequent generations, presumably because of their location in repetitive episomal arrays. To counteract this silencing, we explored transposon-mediated chromosomal integration of transgenes in S. ratti. To this end, we constructed a donor vector encoding green fluorescent protein (GFP) under the control of the Ss-act-2 promoter with flanking inverted tandem repeats specific for the piggyBac transposon. In three experiments, free-living Strongyloides ratti females were transformed with this donor vector and a helper plasmid encoding the piggyBac transposase. A mean of 7.9% of F1 larvae were GFP-positive. We inoculated rats with GFP-positive F1 infective larvae, and 0.5% of 6014 F2 individuals resulting from this host passage were GFP-positive. We cultured GFP-positive F2 individuals to produce GFP-positive F3 L3i for additional rounds of host and culture passage. Mean GFP expression frequencies in subsequent generations were 15.6% in the F3, 99.0% in the F4, 82.4% in the F5 and 98.7% in the F6. The resulting transgenic lines now have virtually uniform GFP expression among all progeny after at least 10 generations of passage. Chromosomal integration of the reporter transgenes was confirmed by Southern blotting and splinkerette PCR, which revealed the transgene flanked by S. ratti genomic sequences corresponding to five discrete integration sites. BLAST searches of flanking sequences against the S. ratti genome revealed integrations in five contigs. This result provides the basis for two powerful functional genomic tools in S. ratti: heritable transgenesis and insertional mutagenesis.

SUBMITTER: Shao H

PROVIDER: S-EPMC3415448 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Transposon-mediated chromosomal integration of transgenes in the parasitic nematode Strongyloides ratti and establishment of stable transgenic lines.

Shao Hongguang H Li Xinshe X Nolan Thomas J TJ Massey Holman C HC Pearce Edward J EJ Lok James B JB

PLoS pathogens 20120809 8

Genetic transformation is a potential tool for analyzing gene function and thereby identifying new drug and vaccine targets in parasitic nematodes, which adversely affect more than one billion people. We have previously developed a robust system for transgenesis in Strongyloides spp. using gonadal microinjection for gene transfer. In this system, transgenes are expressed in promoter-regulated fashion in the F1 but are silenced in subsequent generations, presumably because of their location in re ...[more]

PMID: 22912584

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Project description:Heme is an essential molecule for vast majority of organisms serving as a prosthetic group for various hemoproteins. Although most organisms synthesize heme from 5-aminolevulinic acid through a conserved heme biosynthetic pathway composed of seven consecutive enzymatic reactions, nematodes are known to be natural heme auxotrophs. The completely sequenced Caenorhabditis elegans genome, for example, lacks all seven genes for heme biosynthesis. However, genome/transcriptome sequencing of Strongyloides venezuelensis, an important model nematode species for studying human strongyloidiasis, indicated the presence of a gene for ferrochelatase (FeCH), which catalyzes the terminal step of heme biosynthesis, whereas the other six heme biosynthesis genes are apparently missing. Phylogenetic analyses indicated that nematode FeCH genes, including that of S. venezuelensis (SvFeCH) have a fundamentally different evolutionally origin from the FeCH genes of non-nematode metazoa. Although all non-nematode metazoan FeCH genes appear to be inherited vertically from an ancestral opisthokont, nematode FeCH may have been acquired from an alpha-proteobacterium, horizontally. The identified SvFeCH sequence was found to function as FeCH as expected based on both in vitro chelatase assays using recombinant SvFeCH and in vivo complementation experiments using an FeCH-deficient strain of Escherichia coli. Messenger RNA expression levels during the S. venezuelensis lifecycle were examined by real-time RT-PCR. SvFeCH mRNA was expressed at all the stages examined with a marked reduction at the infective third-stage larvae. Our study demonstrates the presence of a bacteria-like FeCH gene in the S. venezuelensis genome. It appeared that S. venezuelensis and some other animal parasitic nematodes reacquired the once-lost FeCH gene. Although the underlying evolutionary pressures that necessitated this reacquisition remain to be investigated, it is interesting that the presence of FeCH genes in the absence of other heme biosynthesis genes has been reported only for animal pathogens, and this finding may be related to nutritional availability in animal hosts.

Project description:The infectious form of many parasitic nematodes, which afflict over one billion people globally, is a developmentally arrested third-stage larva (L3i). The parasitic nematode Strongyloides stercoralis differs from other nematode species that infect humans, in that its life cycle includes both parasitic and free-living forms, which can be leveraged to investigate the mechanisms of L3i arrest and activation. The free-living nematode Caenorhabditis elegans has a similar developmentally arrested larval form, the dauer, whose formation is controlled by four pathways: cyclic GMP (cGMP) signaling, insulin/IGF-1-like signaling (IIS), transforming growth factor β (TGFβ) signaling, and biosynthesis of dafachronic acid (DA) ligands that regulate a nuclear hormone receptor. We hypothesized that homologous pathways are present in S. stercoralis, have similar developmental regulation, and are involved in L3i arrest and activation. To test this, we undertook a deep-sequencing study of the polyadenylated transcriptome, generating over 2.3 billion paired-end reads from seven developmental stages. We constructed developmental expression profiles for S. stercoralis homologs of C. elegans dauer genes identified by BLAST searches of the S. stercoralis genome as well as de novo assembled transcripts. Intriguingly, genes encoding cGMP pathway components were coordinately up-regulated in L3i. In comparison to C. elegans, S. stercoralis has a paucity of genes encoding IIS ligands, several of which have abundance profiles suggesting involvement in L3i development. We also identified seven S. stercoralis genes encoding homologs of the single C. elegans dauer regulatory TGFβ ligand, three of which are only expressed in L3i. Putative DA biosynthetic genes did not appear to be coordinately regulated in L3i development. Our data suggest that while dauer pathway genes are present in S. stercoralis and may play a role in L3i development, there are significant differences between the two species. Understanding the mechanisms governing L3i development may lead to novel treatment and control strategies.

Dataset Information

Transposon-mediated chromosomal integration of transgenes in the parasitic nematode Strongyloides ratti and establishment of stable transgenic lines.

Publications

Transposon-mediated chromosomal integration of transgenes in the parasitic nematode Strongyloides ratti and establishment of stable transgenic lines.

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