Suppression of prion protein in livestock by RNA interference

Golding, M. C., Long, C. R., Carmell, M. A., Hannon, G. J., Westhusin, M. E. (April 2006) Suppression of prion protein in livestock by RNA interference. Proc Natl Acad Sci U S A, 103 (14). pp. 5285-90. ISSN 0027-8424 (Print)

[img]
Preview
PDF (Paper)
Hannon PNAS 2006.pdf - Published Version

Download (1623Kb) | Preview
URL: https://www.ncbi.nlm.nih.gov/pubmed/16567624
DOI: 10.1073/pnas.0600813103

Abstract

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

Item Type: Paper
Uncontrolled Keywords: Animals Animals Domestic Animals Genetically Modified Blotting Western Cloning Organism Genetic Vectors Lentivirus genetics Prions antagonists inhibitors RNA Interference Transgenes
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNAi
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene silencing
organism description > animal > mammal > goat
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > green fluorescent protein
organism description > virus > lentivirus
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > shRNA
therapies > viral vectors
CSHL Authors:
Communities: CSHL labs > Hannon lab
Depositing User: CSHL Librarian
Date: 4 April 2006
Date Deposited: 14 Dec 2011 17:23
Last Modified: 09 Nov 2017 16:16
PMCID: PMC1459347
Related URLs:
URI: http://repository.cshl.edu/id/eprint/22801

Actions (login required)

Administrator's edit/view item Administrator's edit/view item
CSHL HomeAbout CSHLResearchEducationNews & FeaturesCampus & Public EventsCareersGiving