Figure 1. Targeting of PrP^C by lentivector-mediated RNAi.

(A) The HIV-based lentivector (LVshPrPC, top) used carries an H1 promoter–driven shRNA cassette and a PGK-EGFP expression cassette. Ψ, packaging signal; ppt, polypurine tract; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; triangle, self-inactivating mutation; arrows, direction of transcription. The lentivector-encoded anti-PrP^C shRNA (shPrPC, bottom) consists of 19- or 21-bp stems separated by a loop. (B) Representative bright-field (left) and fluorescence microscope (right) images of N2a cells 72 hours after transduction with LVshPrPC. (C) LVshPrPC-induced knock down of PrP^C in N2a cells. Control, uninfected cells. (D) PrP^C expression in N2a cells infected with a low (×1) and high (×10) concentration of LVsh512 and LVshscr. (E) Analysis of PrP^C expression in granule cells 72 hours after infection with LVsh512 or a control vector carrying only the EGFP expression cassette (LVEGFP). Western blots (C–E) were probed with the indicated antibodies. (F) Effect of LVsh512 on the accumulation of protease K–resistant (PK-resistant) PrP^Sc in ScN2a cells. Western blot analysis 72 hours after transduction with the indicated lentivectors. (G and H) Intracranial injection of LVshscr (G) and LVsh512 (H) in tga20 mice overexpressing PrP^C. Analysis of EGFP (left) and PrP^C (right) expression 3 weeks after injection. Arrows indicate the lentivector-transduced area.