Stable transgenic lines of <i>S. ratti</i> are established by microinjection of nucleic acid constructs followed alternating rounds of host and culture passage, selecting on expression of a fluorescent reporter gene product.

<p><b>A</b>) Diagram of major steps in isolation of stable transgenic lines of <i>S. ratti</i>. Initial gene transfer into parental (P0) free-living females is by gonadal microinjection of donor vectors alone or in combination with helper vector or capped transposase encoding mRNA. F1 larvae are derived in culture and screened for GFP expression. Transgene expressing individuals are reared to infective L3 (L3i) and inoculated into rats to establish as parasitic females. F2 progeny released in the feces of these rats are screened for transgene expression and positive larvae reared in culture to free-living males and females and allowed to mate. F3 progeny arising from these crosses are reared to L3i in culture in inoculated into rats. F4 progeny arising in the feces are screened for reporter transgene expression and used for further alternating rounds of culture and host passage with continued selection on GFP. The timeline indicates the intervals in days following microinjection of parental worms in which each generation is isolated up to the F5 when transmission and expression are generally stabilized. <b>B</b>) Frequency of transgene expressing progeny by generation during selection of stable transgenic lines of <i>S. ratti</i>. Data are mean (± one standard deviation) percentages of reporter transgene-expressing individuals in each generation of alternating host and culture passage with selection on GFP (see panel A). Means are derived from three independently established lines (PV2, PV3 and PV4). Sample sizes (<b>n</b>) indicated below the abscissa are totals of worms examined from all three lines. Statistics: in panel B, 2-way ANOVA, performed using Prism ver. 5.0c (GraphPad Software, Inc., La Jolla, California, USA), revealed a highly significant effect due to generation (P<0.0001) but no significant effect (P = 0.2) due to donor plasmid.</p

Transgene specific sequences are widely distributed in restriction digests of genomic DNA from three stable lines of transgenic <i>S. ratti</i>.

<p>Southern hybridization of genomic DNA (gDNA) of <i>S. ratti</i> from stably transformed lines probed for the <i>gfp</i> coding sequence. <b>A</b>) Transgene diagram showing position of the probe used for Southern hybridization analysis and the single restriction site for BsrGI, the enzyme used for restriction digestion of gDNA. <b>B</b>) Southern blot of BsrGI digests of gDNA from pooled free-living adults from three independent integrated lines (PV2, PV3 and PV4). The positive control lane is a blot of a BsrGI digest of donor plasmid pPV356, and the negative control is a blot of a BsrGI digest of gDNA from non-transformed <i>S. ratti</i> free-living adults (<i>S.r.</i> gDNA). Note <i>gfp</i> hybridization signals in multiple restriction fragments in gDNA from each of the three integrated lines. A single hybridizing band of 8.1 kb appears as predicted in the positive control digest of pPV356. No <i>gfp</i>-specific signal is detected in the negative control digest of gDNA from non-transformed <i>S. ratti</i>. <b>C</b>) Gel analysis of PCR products from genomic DNA templates from non-transformed control parasites, parasites from each of three stable lines, PV2, PV3 and PV4, with integrated, stably expressed transgenes and F1 progeny of free-living female worms microinjected with donor plasmid pPV356 and helper plasmid pPV402. Also included are PCR products from control reactions with plasmids pPV356 and pPV254 as templates and from a reaction from which template was omitted. Upper gel image depicts 624 bp amplification products resulting from a forward primer hybridizing to the M13 reverse priming site in the vector and a reverse primer hybridizing within the transposon sequence. Lower gel image is a loading control showing the expected 554 bp amplification product from reactions with primers specific for the constitutively expressed cellular actin-encoding gene <i>Sr-act-2</i>.</p

Genomic integration sites for reporter transgenes and their flanking sequences in three stable transgenic lines of <i>Strongyloides ratti</i>.

<p>Contig identities, insertion locations within contigs and, where possible, chromosome numbers are derived from the draft <i>S.ratti</i> genome sequence.</p>a<p>Chromosome.</p>b<p>Not determined.</p

Analysis of relative transgene copy number via qRT-PCR for stable transgenic lines of <i>S. ratti</i> transformed with the <i>piggyBac</i> transposon system.

a<p>Calculated from three technical replicates within the assay.</p>b<p>Calculated from three replicate assays.</p>c<p>Differences between mean relative copy number for stable lines not significant by one-way ANOVA (P>0.05).</p>d<p>Wildtype <i>S. ratti</i>.</p

Parasitic and free-living adults from a stable transgenic line of <i>Strongyloides ratti</i> express GFP in the body wall-specific manner expected for the <i>Ss-act-2</i>prom::<i>gfp</i> transcriptional reporter.

<p>Typical patterns of GFP expression in parasitic female, free-living male and free-living female <i>S. ratti</i> expressing the integrated reporter transgene encoded in pPV356. (<b>A, B</b>) DIC and fluorescence images, respectively, of a parasitic female <i>S. ratti</i> from integrated line PV2. Note expression predominating in the body wall up to the level of the esophageal/intestinal boundary (<b>ei</b>). Position of the head is indicated (<b>h</b>). (<b>C, D</b>) DIC and fluorescence images, respectively, of a free-living female <i>S. ratti</i> from integrated line PV2. Note uniform expression throughout the body wall with additional loci of expression in the vulva (<b>v</b>) and rectum (<b>re</b>). (<b>E, F</b>) DIC and fluorescence images, respectively, of a free-living male <i>S. ratti</i> from transgenic line PV2. Note uniform expression in the body wall with additional expression in the cloaca (<b>cl</b>). (<b>G, H</b>) DIC and fluorescence images, respectively, of a non-transformed free-living male <i>S. ratti</i>. The fluorescence image in panel H was exposed for a period≥exposure times in panels B, D and F. Scale bar = 200 µm in all panels.</p

Vector constructs used to establish stable transgenic lines of <i>Strongyloides ratti</i> incorporate elements of the <i>piggyBac</i> transposon system.

<p><b>A</b>) Donor vector pPV254 incorporating a fluorescent reporter gene in which expression of GFP is driven by the <i>Ss-act-2</i> promoter and terminated by the <i>Ss era-1</i> 3′ UTR. The reporter transgene in pPV254 is flanked by the inverted terminal repeats (ITR) plus internal sequences common to <i>piggyBac</i> transposable elements. <b>B</b>) Donor vector pPV356, which is like pPV254 in all respects except that the coding sequence is flanked by the gypsy retroviral insulator sequences from <i>Drosophila</i>. <b>C</b>) Helper vector pPV402 in which expression of the <i>piggyBac</i> transposase gene is driven by <i>Ss-rps-21</i> promoter and terminated by the <i>Ss-era-1</i> 3′ UTR. <b>D</b>) Plasmid pPV257 for in vitro transcription of mRNA encoding the <i>piggyBac</i> transposase under the T7 promoter. In lieu of helper vector pPV402, this mRNA was capped, tailed and co-injected with donor vector pPV356 in Experiment 2.</p