Fig 2: miR-based shRNA included in the intronic region of a gamma-retroviral vector leads to the best knock-down efficiencies, regardless of the target. Flow cytometry histograms of one representative titration experiment are shown upon transduction of C1498. Empty histograms demonstrate the staining upon transduction with miR-based shRNA control (“miRCTRL”) while solid histograms depict transduction with miR-based shRNA against the protein of interest (“miRPOI”). The Thy1.1 histogram (that is matched to the H-2Db/H-2Kb experiment) reveals Thy1.1 expression together with transduction efficiency for each strategy used. For H-2Kb/H-2Db, CD25, CD44, CD45, and CD54 histograms, only Thy1.1+ cells were analyzed. Bar graphs display pooled percent knock-down efficiency + SEM from three independent experiments (depicted by symbols). ns: not significant; *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 (repeated measures one-way ANOVA, Tukey’s post hoc test). KD: knock-down.

Fig 3: Antisense cassettes can accommodate introns and miR-based shRNAs with various promoters without a massive drop in viral titers(A) Analysis of GFP expression in 293T cells upon plasmid transfection. 293T cells were transfected with the RMS vector containing an antisense cassette (“ϕ”) that includes the GFP driven by UBC (α-UBC), PGK (α-PGK), CMV (α-CMV), RSV (α-RSV), or EF-1α (α-EF-1α) promoters. In parallel, 293T cells were transfected with plasmids comprising the same cassettes in which an intron containing an inverted polyadenylation site was added (“int”). Results show the mean GFP expression ±SEM from four independent experiments (symbols) 48 h after plasmid transfection. ∗∗p < 0.01 (Student’s paired t test; for each promoter, GFP expression comparison between the presence and the absence of the intron/α-SV40pA insert). (B) Viral titer determination. 293T cells were transfected with the RMS vector containing an antisense cassette (“ϕ”) that includes the GFP driven by UBC (α-UBC), PGK (α-PGK), CMV (α-CMV), RSV (α-RSV), and EF-1α (α-EF-1α) promoters. In parallel, these cells were transfected with an additional plasmid driving NoV B2 expression (“NoV B2”) or were transfected with plasmids in which an intron containing an inverted and premature pA site was added (“int”). 48 h after transfection, viral supernatants were used to determine viral titers upon transduction of C1498 cells. Results show the mean viral titer + SEM from three independent experiments (symbols) 72 h after transduction. ns, not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (two-way ANOVA, Tukey post hoc test). (C) Representative scheme of an antisense cassette bearing a β-globin intron (“β-globint”) that includes a microRNA (miR)-based short hairpin RNA (shRNA; “miR”) in the RMS vector. During production of viruses the antisense cassette includes a premature pA signal preventing dsRNA generation. This premature pA is lost by splicing during viral genomic RNA production. Consequently, integrated viral genome leads to PGK-driven expression of GFP and to intronic pri-miR processing for target protein knockdown. W: Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). (D) Viral titer and GFP expression level analysis upon β-globin intron and miR-based shRNA inclusion. The set up was the same as in (B). In addition, an inverted β-globin intron without (“int+ β-globin intron”) or with a miR-based shRNA (“int+ β-globin intron+miR”) were inserted. Results show the mean viral titer (left graph) or mean GFP expression level (right graph, where less than 20% cells were transduced) + SEM from three independent experiments (symbols) 72 h after transduction. N/A, not assessed: β-globin intron was not added in the α-EF-1α condition, since the promoter already contains an intron. ns, not significant, ∗p < 0.05, ∗∗p < 0.01 (Student’s paired t test). (E) Knockdown efficiency depending on the promoter driving miR-based shRNA expression. β-globin intron and miR-based shRNA were inserted in antisense cassettes driven by UBC, PGK, and EF-1α promoters as in (D). Protein levels were analyzed by flow cytometry upon transduction of C1498 cells with miR-based shRNA sequences for various protein of interest (“miRPOI”): β2-microglobulin (a subunit needed for H-2Db/Kb expression), CD44, CD45, or CD54. Protein levels were compared to those obtained with a non-targeting miR-based shRNA control (“miRCTRL”). Representative flow cytometry histograms are shown depicting protein levels (from the reporter gene -expressing population) obtained upon infection with three doses of viral supernatants. Bar graph shows the mean knockdown efficiency + SEM from three independent experiments (symbols) 72 h after transduction where less than 20% cells were transduced. ns, not significant, ∗p < 0.05, ∗∗∗∗p < 0.0001 (two-way ANOVA, Tukey post hoc test).