Efficient and specific down-regulation of prion protein expression by RNAi
Section snippets
Materials and methods
Two regions of 21 nt (named 1 and 2) were chosen within the ovine VRQPrP cDNA sequence as target for RNAi (Fig. 1). They were inserted in both sense and antisense orientations in pBluescript/U6 plasmid (supplied by Dr. Shi) and separated by a HindIII restriction site [21]. To assess the potential of these two constructs to down-regulate Prnp gene expression in vivo, these vectors or the empty pBluescript/U6 plasmid were co-transfected by the Lipofectamine method (GIBCO/BRL) into rabbit kidney
Results and discussion
Efficient inhibition of expression of ovine PrP was observed in cells co-transfected with the plasmids carrying either the 1 or the 2 RNAi sequences, as compared with cells co-transfected with the empty U6 plasmid (Fig. 2A). The observed average percentages of inhibition (mean ± SD) were of 81 ± 7% and 88 ± 6% (n=3) with RNAi 1 and 2, respectively. This down-regulation was associated with a concomitant reduction of the steady state level of expression of the Prnp mRNA (Fig. 2C) suggesting that, as
Acknowledgments
We are most grateful to Dr. Shi (Harvard Medical School, Boston, USA) for the kind gift of the pBluescript/U6 plasmid. This work was supported by a grant from the Groupement d’Intérêt Scientifique ‘Infections à Prion.’
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These two authors contributed equally to this work.