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Production of cattle lacking prion protein

Abstract

Prion diseases are caused by propagation of misfolded forms of the normal cellular prion protein PrPC, such as PrPBSE in bovine spongiform encephalopathy (BSE) in cattle and PrPCJD in Creutzfeldt-Jakob disease (CJD) in humans1. Disruption of PrPC expression in mice, a species that does not naturally contract prion diseases, results in no apparent developmental abnormalities2,3,4,5. However, the impact of ablating PrPC function in natural host species of prion diseases is unknown. Here we report the generation and characterization of PrPC-deficient cattle produced by a sequential gene-targeting system6. At over 20 months of age, the cattle are clinically, physiologically, histopathologically, immunologically and reproductively normal. Brain tissue homogenates are resistant to prion propagation in vitro as assessed by protein misfolding cyclic amplification7. PrPC-deficient cattle may be a useful model for prion research and could provide industrial bovine products free of prion proteins.

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Figure 1: Generation of PRNP−/− cattle.
Figure 2: Histopathological analysis of obex and cerebellum of 14-month-old cattle.
Figure 3: Comparative analysis of immune system of PRNP−/− and wild-type (WT) control cattle at 12–13 months old.
Figure 4: In vitro propagation of PrPBSE and PrPTME in PRNP−/− and PRNP+/+ wild-type (WT) cattle brain homogenates.

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Acknowledgements

We thank the team at Transova Genetics for their efforts in embryo transfer and Todd Stahl, Rebecca Cuperus, Maria Diaz, Cliff Mazour for their assistance in calf delivery and care. We thank Melanie Nichols, Jason Griffin, Melissa Bien, Molly Ahlers, Rachael Paulson, Sarah Viet, Cassandra Voss for their assistance in gene targeting, cell culture and embryonic cloning. We thank Kevin Hassall for an additional western blotting. We also thank Ralph Kubo and Tomoyuki Tahara for their useful comments on the immunological study. Soto is part of the European Community project TSELAB and was supported in part by National Institutes of Health grants NS050349 and NS049173.

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Correspondence to Jürgen A Richt, James M Robl or Yoshimi Kuroiwa.

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Some authors are employees of Hematech Inc., Gemini Science Inc. or Kirin Co., Ltd. These companies funded the work.

Supplementary information

Supplementary Fig. 1

Fertility of PRNP−/− bovine sperm.

Supplementary Fig. 2

PRNP−/−IGHM−/− double knockout (DKO) cattle.

Supplementary Table 1

Serum chemistry values for PRNP cattle.

Supplementary Table 2

Hematology values for PRNP−/− and control cattle.

Supplementary Table 3

Comparison of lymphocyte populations between PRNP−/− and wild-type cattle.

Supplementary Table 4

Blastocyst development in IVF between sperm from the PRNP−/− bulls and oocytes from wild-type cows.

Supplementary Table 5

Blastocyst development from PRNP−/−fibroblast cell lines.

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Richt, J., Kasinathan, P., Hamir, A. et al. Production of cattle lacking prion protein. Nat Biotechnol 25, 132–138 (2007). https://doi.org/10.1038/nbt1271

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