Bovine spongiform encephalopathy is an emerging prion disease of cattle. Mounting evidence indicates that BSE is transmissible to humans in the form of a new, deadly variant of Creutzfeldt-Jakob disease (vCJD). Consumption of BSE-tainted beef has been implicated as the most likely mode of transmission. BSE thus represents a threat to human health via the food supply and other bovine-derived products. As no vaccine, diagnostic test or therapy exists for either vCJD or BSE, protection depends on preventative measures. The pathogenesis of prion disease requires expression of host-encoded prion protein. In mice, priori gene knockout of confers resistance to priori disease. Knockout of the prion gene in cattle should similarly render the bovine resistant to BSE. The long-term goal of this work is to test the hypothesis that cattle bearing bi-allelic prion knockouts are resistant to BSE. The current objective is to generate male and female founder cattle bearing mono-allelic pdon knockouts. Accordingly, the following Specific Aims are proposed: 1) generate bovine male and female primary somatic cell lines with mono-allelic prion gene knockouts, and 2) generate founder cattle from these cell lines using nuclear transfer technology.
For Aim 1, a promoter-trap targeting vector will be employed to insert a disruptive green fluorescent protein (GFP) selectable marker into the prion coding sequence. Homologous recombination arm will be generated by PCR from isogenic DNA, or amplified from a bovine BAC library. Fluorescence activated cell sorting will be used to isolate GFP-expressing cells, which will then be cloned and screened by PCR and Southern blot for targeting.
In Aim 2, cattle bearing prion knockouts will be generated by reconstructing oocytes with targeted cell nuclei, artificially activating oocytes to obtain pre-implantation stage embryos, and transferring the embryos to bovine surrogates for the balance of gestation. Offspring with mono-allelic prion knockouts will be bred in future work to generate cattle with bi-allelic prion knockouts.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Exploratory/Developmental Grants (R21)
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National Institute of Neurological Disorders and Stroke Initial Review Group (NSD)
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Nunn, Michael
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Virginia Polytechnic Institute and State University
Veterinary Sciences
Schools of Veterinary Medicine
United States
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Peralta, Oscar A; Huckle, William R; Eyestone, Willard H (2012) Developmental expression of the cellular prion protein (PrP(C) ) in bovine embryos. Mol Reprod Dev 79:488-98
Peralta, Oscar A; Huckle, William R; Eyestone, Willard H (2011) Expression and knockdown of cellular prion protein (PrPC) in differentiating mouse embryonic stem cells. Differentiation 81:68-77
Peralta, Oscar A; Eyestone, Willard H (2009) Quantitative and qualitative analysis of cellular prion protein (PrP(C)) expression in bovine somatic tissues. Prion 3:161-70