Lentiviral transduction facilitates RNA interference in the nematode parasite Nippostrongylus brasiliensis
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.