Figure 1. Verification of transgene and transcription in eggs of S. mansoni following transduction with lentivirus constructs.

(a) Lentivirus construct encoding shRNAmir/mCherry expression cassette under the control of a CMV promoter and containing a packaging signal (Ψ) as well as a long terminal repeat (5′-LTR) and a self-inactivating (3′-SIN) LTR sequence. The shRNAmir structure is indicated. (b) Genomic DNA (gDNA) and RNA were isolated from eggs 10 days after transduction. Lentiviral DNA was detected in gDNA by direct PCR amplification of transgene regions, using the gene ipse as a positive control; cDNA was produced by reverse transcription from total RNA from eggs. Transcription of the transgene was demonstrated by direct PCR of mCherry transgene regions from cDNA, employing the actin-β (act-b) coding region as a positive control (cf. Supplementary Fig. 1 for full gel images). (c) Representative Southern blot analysis of gDNA from eggs of S. mansoni (3 days after transduction) confirming genomic integration of the lentivirus by specific hybridization (vertical bar). For this analysis, gDNA was isolated from eggs 3 days after lentivirus transduction, digested with both restriction endonucleases SpeI and EcoRI (the plasmid vector contains only one site for each enzyme); 5 μg of digested DNA were resolved in an agarose gel (0.8%) and subjected to specific hybridization using a probe (see inset) directed to the transgene encoded by the lentivirus. The expected sizes of the empty vector (EV; plasmid) control and any non-integrated viral DNA were 3.5 and 5.5/2.1 kb, respectively. Lanes: M, molecular weight marker in bp; WT, wildtype; EV, EV control; I, shRNAmir-511+shRNAmir-557 to omega-1; II, shRNAmir-195+shRNAmir-384 to ipse; III, shRNAmir-616+shRNAmir-638 to kappa-5; shRNAmir, microRNA-adapted short hairpin RNA.