Fig 1.
Lentiviral transduction of activated L3s.
(A) Schematic of lentiviral transduction of cells for delivery of a mCherry expression cassette under the control of the CMV promoter following binding of viral particles to the LDL receptor. A microRNA-adapted short hairpin (shRNAmir) targeting the gene of interest is encoded in the 3´-UTR of mCherry transcripts. (B) Detection of LDL receptor orthologues (lrp-1 and lrp-2) by RT-PCR from different stages of N. brasiliensis. Idhg-1 (Isocitrate dehydrogenase subunit) was amplified to control for cDNA integrity. L3A, activated L3s; NT, no template control. C) Uptake of DiD-labeled lentiviral particles. Activated L3s were exposed to DiD-labeled lentiviral particles (DiD-virus) or left untreated (wild type, WT) and analysed for fluorescence by confocal microscopy after 24 h. A heat–inactivated virus control (h.i. DiD-virus) was included. Fluorescence was detected at the intestinal epithelium of larvae exposed to viable DiD-labeled lentiviral particles alone. Autofluorescence in the green channel (blue laser) served as counterstain. Scale bar: 100 μm.
Fig 2.
Detection of proviral DNA in transduced L3s.
Genomic DNA was isolated from lentivirus-exposed (LV) or unexposed (wild type, WT) L3s after three days and analysed for proviral DNA by PCR. (A) The presence of proviral DNA in worm gDNA was confirmed by PCR of the transgene expression cassette resulting in a 1.4 kb amplicon (CMV-miR30). Tubulin alpha chain (tuba) was amplified to control for gDNA integrity. The absence of residual plasmid DNA in gDNA preparations was confirmed by PCR with primers binding the WPRE and SV40P region (WPRE-SV40 P). Amplification of lentivirus-encoding plasmid DNA served as a positive control (Plasmid CTRL). NT, no template control. (B) Detection of episomal circular 1-LTR viral DNA by Nested PCR confirms successful reverse transcription of viral genomic RNA in transduced worms. The location of primer pairs used for nested PCR step one (white arrows) and two (red arrows) is indicated. (C) Example of viral integration site. Split read showing alignment with lentiviral DNA and N. brasiliensis genomic DNA.
Fig 3.
Functionality of the expression cassette and viability of transduced L3s.
(A) Detection of mCherry transcripts over a time course of 96 hrs post-exposure to lentivirus encoding mCherry under the control of the CMV promoter. Activated L3s were exposed to lentivirus with (miR) or without (EV, ‘empty vector’) a complete shRNAmir structure or left untreated (wild type, WT) and analysed for presence of the transgene in cDNA. Tuba (tubulin alpha chain) was amplified to control for DNA integrity. NT, no template control. (B) Single worm RT-PCR to detect proportion of larvae expressing mCherry. Individual L3s either transduced with lentivirus (LV worm) or unexposed (WT worm) were used to amplify virus-encoded mCherry or control parasite idhg-1 transcripts as described in Materials and Methods. (C). Viability of L3 larvae was assessed by ATP assay 72 hrs after transduction. Bars represent the mean ± SEM of ATP content for three biological replicates (100 worms per sample). (D) Recovery of transduced worms from infected host animals. Activated L3s were exposed to lentivirus (LV) for 6 hrs or left untreated (wild type, WT). Worms were washed extensively before infection of host animals (rats). Adult worms were recovered from the intestines 6 days post-infection and analysed for presence of the mCherry transgene by PCR of genomic DNA.
Fig 4.
Detection of proviral DNA in the F1 generation.
Eggs and L3s were recovered from the F1 generation following infection of rats with larvae transduced with or unexposed to lentivirus. (A) The presence of proviral DNA in eggs was confirmed by PCR of mCherry and miR-30, detected in F1 eggs following infection with transduced parasites (LV) but not those unexposed to virus (WT). Amplification of N. brasiliensis eif-3C was confirmed in all samples to control for gDNA integrity (eif-3C). The absence of residual plasmid DNA was confirmed by PCR with primers to the WPRE and SV40P region (WPRE-SV40). Amplification of lentivirus-encoding plasmid DNA (pDNA) served as a positive control. NT, no template control. (B) The presence of proviral DNA in L3s was confirmed by PCR of mCherry, detected in F1 L3s following infection with parasites transduced with lentivirus but not those unexposed to virus (WT). LVI and LVII represent F1 generation L3s derived from infection of 2 different rats with transduced parasites. Amplification of N. brasiliensis eif-3C was confirmed in all samples to control for gDNA integrity. NT, no template control. Approximately 2,000 L3s or eggs were used for PCR reactions following the single worm PCR protocol described in Materials and Methods.
Fig 5.
C. elegans-derived promoter and 3´-UTR enhances transgene expression in transduced L3s.
Activated L3s were exposed to lentivirus encoding mCherry under the control of different promoter constructs and analysed for transgene expression 72 hrs later. (A) Schematic of the three expression cassettes tested. The mCherry sequence was optimised for N. brasiliensis codon usage (Nb_mCherry) and expression placed under the control of the CMV promoter or the C. elegans-derived hlh11 promoter in conjunction with the 3´-UTR of the C. elegans tbb-2 gene. (B) Relative transcription of mCherry under the control of the different promoter constructs at different multiplicity of infection (MOI) was assessed by qPCR relative to the CMV promoter construct at a MOI of 10. Transcription was normalised to the geometric mean of Ct values for rbd-1 and idhg-1. Data are from a representative experiment with 1,000 worms per sample group. (C). Worms were transduced at a MOI of 400 and analysed after 72 hrs for red fluorescence by confocal imaging. Autofluorescence in the green channel (blue laser) served as counterstain. Arrows indicate localisation of mCherry in transduced worms. Scale bar: 100 μm.
Fig 6.
Transcriptional knockdown of beta-tubulin 1 and 2 in L3s following transduction with dsRNA constructs. (A) Detection of dcr-1, rde-1, rrf-1 and sid-1 orthologue transcripts in two different samples of activated L3s and adult worms of N. brasiliensis by RT-PCR. Amplification of idhg-1 confirms cDNA integrity. NT, no template control; NoRT, no reverse transcription control. (B) Schematic of lentiviral expression cassettes encoding a target gene-specific shRNAmir placed immediately downstream of the mCherry coding region and upstream of the tbb2 3´-UTR, or a ~400 nt long hairpin structure (lhp) consisting of a 186 or 145 bp stem and 64 or 55 nt loop sequence (tbb-1 or tbb-2, respectively). (C) Down-regulation of tbb-1 or (D) tbb-2 transcription in activated L3s three days after transduction. L3s were exposed to a cocktail consisting of four shRNAmir lentiviruses targeting either tbb-1 or tbb-2 (Table 1) or with lentivirus encoding a lhp targeting tbb-1 or tbb-2 transcripts. Control worms were left untreated (wild type, WT) or transduced with virus encoding mCherry lacking a hairpin sequence (empty vector, EV). Transcription of tbb-1 or tbb-2 in L3s was assessed by RT- qPCR three days after transduction relative to the wild type control worms and normalised against the geometric mean of Ct values of reference genes eif-3C, idhg-1 and rbd-1. Box plot representing the median and upper/lower quartile of a data pool of 4 (tbb-1) or 3 (tbb-2) independent experiments with 3–4 biological replicates consisting of ~2,000 worms each. Whiskers indicate the highest or lowest value. Treatment groups were analysed for significant differences with the Kruskal-Wallis test and Dunns post-hoc test in relation to the wild type or empty vector control group. Statistical significance: ns = not significant, p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****).
Table 1.
Mature shRNAmir sequences targeting tbb-1, tbb-2 or ache-b transcripts.
The number indicates the position of the first nucleotide of the target sequence.
Fig 7.
Transcriptional knockdown of secreted acetylcholinesterase B and processing of dsRNA in L3s following transduction with dsRNA constructs.
(A) Down-regulation of secreted ache-b transcripts in activated L3s three days after transduction. L3s were exposed to a cocktail consisting of two shRNAmir lentiviruses (Table 1) or with lentivirus encoding a 250 bp lhp targeting ache-b. Control worms were left untreated (wild type, WT) or transduced with virus encoding mCherry lacking a hairpin sequence (empty vector, EV). L3 transcripts were assessed by RT-qPCR three days after transduction relative to wild type control worms and normalised against the geometric mean of Ct values of reference genes eif-3C and idhg-1. Box plot representing the median and upper/lower quartile of a data pool of 6 (ache-b) or 3 (ace-1,2,3/4) independent experiments with 2–4 biological replicates consisting of ~2,000 worms each. Whiskers indicate the highest or lowest value. Treatment groups were analysed for significant differences with the Kruskal-Wallis test and Dunns post-hoc test in relation to the wild type or empty vector control group. Statistical significance: p<0.05 (*), p<0.01 (**), p<0.001 (***) (B) Detection of mature miRNA in transduced worms. Total RNA was isolated from transduced worms at 48 or 72h post exposure to virus and analysed for presence of mature miRNAs generated from shRNAmir_468 using miScript detection system (Qiagen) and miRNA specific forward primer, resulting in a ~85 bp amplicon. (C) Detection of secondary siRNAs (22-G RNAs) generated from dsRNA in adult worms. L3 worms were transduced with lentivirus and introduced into host animals to complete their life cycle. Adult worms were recovered 5 days post infection. Small RNA libraries were prepared from total RNA and analysed following deep sequencing. The y axis shows 22G-RNAs to ache-b reads per million (rpm) total small RNAs. (D) Quantitation of transcripts for components of the exogenous RNAi pathway in activated L3s and adult worms was assessed by RT-qPCR. Interleaved scatter plot with bars and mean ± SEM of 4 biological replicates.