Knockdown of the bovine prion gene PRNP by RNA interference (RNAi) technology

Abstract

Background: Since prion gene-knockout mice do not contract prion diseases and animals in which production of prion protein (PrP) is reduced by half are resistant to the disease, we hypothesized that bovine animals with reduced PrP would be tolerant to BSE. Hence, attempts were made to produce bovine PRNP (bPRNP) that could be knocked down by RNA interference (RNAi) technology. Before an in vivo study, optimal conditions for knocking down bPRNP were determined in cultured mammalian cell systems. Factors examined included siRNA (short interfering RNA) expression plasmid vectors, target sites of PRNP, and lengths of siRNAs.

Results: Four siRNA expression plasmid vectors were used: three harboring different cloning sites were driven by the human U6 promoter (hU6), and one by the human tRNAVal promoter. Six target sites of bovine PRNP were designed using an algorithm. From 1 (22 mer) to 9 (19, 20, 21, 22, 23, 24, 25, 27, and 29 mer) siRNA expression vectors were constructed for each target site. As targets of siRNA, the entire bPRNP coding sequence was connected to the reporter gene of the fluorescent EGFP, or of firefly luciferase or Renilla luciferase. Target plasmid DNA was co-transfected with siRNA expression vector DNA into HeLaS3 cells, and fluorescence or luminescence was measured. The activities of siRNAs varied widely depending on the target sites, length of the siRNAs, and vectors used. Longer siRNAs were less effective, and 19 mer or 21 mer was generally optimal. Although 21 mer GGGGAGAACTTCACCGAAACT expressed by a hU6-driven plasmid with a Bsp MI cloning site was best under the present experimental conditions, the corresponding tRNA promoter-driven plasmid was almost equally useful. The effectiveness of this siRNA was confirmed by immunostaining and Western blotting.

Conclusion: Four siRNA expression plasmid vectors, six target sites of bPRNP, and various lengths of siRNAs from 19 mer to 29 mer were examined to establish optimal conditions for knocking down of bPRNP in vitro. The most effective siRNA so far tested was 21 mer GGGGAGAACTTCACCGAAACT driven either by a hU6 or tRNA promoter, a finding that provides a basis for further studies in vivo.

Figure 1

Effects of siRNA expression vectors on bPRNP. A and B, effects of different lengths of siRNAs expressed by different vectors. A: piGENE hU6-EGFP was a positive control siRNA vector for EGFP. Numerals after vectors indicate the lengths of siRNA; 19, 23, 25, 27, and 29 correspond to No. 6, 10, 12, 13, and 14 in Table 2, respectively. B: piGENE Fluc is a positive control siRNA vector against firefly luciferase. Numerals after vectors indicate the lengths of siRNA; 19, 20, 21, and 22 correspond to No. 6, 7, 8, and 9 in Table 2, respectively. C and D, effects of different target sites and expression vectors on siRNA activities. C: numerals after vectors indicate the start sites of siRNA; 132, 372, 616, and 725 correspond to No. 2, 4, 9, and 16 in Table 2, respectively. D: as for numerals after vectors, see the legend to C. E: comparison of vector activities. Vectors expressed 19 mer, and the sequence corresponds to No. 6 in Table 2.

Figure 2

Silencing of b PRNP by siRNA as revealed by immunostaining. HeLaS3 cells were co-transfected with pbPrP- FLAG DNA and the control vector DNA of piGENE tRNA (A) or siRNA expression vector DNA of piGENE tRNA-616-21, the sequence of which is No. 8 in Table 2 (B). Cells were stained with SAF32 antibody. HeLaS3 cells were co-transfected with pbPrP- FLAG DNA and the control vector DNA of piGENE hU6 (C) or siRNA expression vector DNA of piGENE hU6-616-21(D). Cells were stained with P6488 antibody. HeLaS3 cells were co-transfected with pshort-bPrP- FLAG DNA and the control vector DNA of piGENE hU6 (E) or siRNA expression vector DNA of piGENE hU6-616-21 (F). Cells were stained with P6488 antibody.

Figure 3

Silencing of b PRNP by siRNA as revealed by Western blot analysis. Top, protein size markers (Bio-Rad, lane 1), proteins from HeLaS3 cells (lanes 2–4), proteins from cells co-transfected with pbPrP- FLAG DNA and the control vector DNA of piGENE hU6 (lanes 5–7) or siRNA expression vector DNA of piGENE hU6-616-21, the sequence of which is No. 8 in Table 2 (lanes 8–10), proteins from cells co-transfected with pbPrP- FLAG DNA and the control vector DNA of piGENE tRNA (lanes 11–13) or siRNA expression vector DNA of piGENE tRNA-616-21(lanes 14–16), and protein size markers (150, 100, 75, 35, 25, and 15 kDa from the top, GE Healthcare). Amounts of proteins applied were 1.25 (lanes 2, 5, 8, 11, 14), 2.5 (lanes 3, 6, 9, 12, 15), and 5.0 (lanes 4, 7, 10, 13, 16) μg/lane. The gel was stained with SAF32 antibody. Bottom, actin staining.