Knockdown of the bovine prion gene PRNP by RNA interference RNAi technologyReportar como inadecuado




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BMC Biotechnology

, 7:44

First Online: 26 July 2007Received: 12 November 2006Accepted: 26 July 2007

Abstract

BackgroundSince 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.

ResultsFour siRNA expression plasmid vectors were used: three harboring different cloning sites were driven by the human U6 promoter hU6, and one by the human tRNA 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.

ConclusionFour 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.

Electronic supplementary materialThe online version of this article doi:10.1186-1472-6750-7-44 contains supplementary material, which is available to authorized users.

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Autor: Shizuyo Sutou - Miho Kunishi - Toshiyuki Kudo - Pimprapar Wongsrikeao - Makoto Miyagishi - Takeshige Otoi

Fuente: https://link.springer.com/article/10.1186/1472-6750-7-44







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