Suppression of prion protein in livestock by RNA interference

• DOI: 10.1073/pnas.0600813103
• Publication Date: 2006-03-28T01:34:47Z
• Authors: Michael C. Golding; Charles R. Long; Michelle A. Carmell; Gregory J. Hannon; Mark E. Westhusin
• Keywords: 2. Zero hunger; 0303 health sciences; Prions; Cloning, Organism; Blotting, Western; Genetic Vectors; Lentivirus; Animals, Genetically Modified; 03 medical and health sciences; Animals, Domestic; Animals; RNA Interference; Transgenes

Given the difficulty of applying gene knockout technology to species other than mice, we decided explore utility RNA interference (RNAi) in silencing expression genes livestock. Short hairpin RNAs (shRNAs) were designed and screened for their ability suppress caprine bovine prion protein (PrP). Lentiviral vectors used deliver a transgene expressing GFP an shRNA targeting PrP into goat fibroblasts. These cells then nuclear transplantation produce cloned fetus, which was surgically recovered at 81 days gestation compared with age-matched control derived by natural mating. All tissues examined fetus expressed GFP, PCR analysis confirmed presence encoding shRNA. Most relevant, Western blot performed on brain comparing transgenic demonstrated significant (>90%) decrease levels. To confirm that similar methodologies could be applied bovine, recombinant virus injected perivitelline space ova. After vitro fertilization culture, 76% blastocysts exhibited expression, indicative they shRNAs PrP. Our results provide strong evidence approach described here will useful producing livestock conferring potential disease resistance effective strategy suppressing variety large-animal models.