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