Letter | Published:

Production of cattle lacking prion protein

Nature Biotechnology 25, 132–138 (2007) | Download Citation

Subjects

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

Rent or Buy

All prices are NET prices.

References

  1. 1.

    Detecting mad cow disease. Sci. Am. 291, 86–93 (2004).

  2. 2.

    et al. Normal development and behavior of mice lacking the neural cell-surface PrP protein. Nature 356, 577–582 (1992).

  3. 3.

    et al. 129/Ola mice carrying a null mutation in PrP that abolishes mRNA production are developmentally normal. Mol. Neurobiol. 8, 121–127 (1994).

  4. 4.

    et al. Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proc. Natl. Acad. Sci. USA 90, 10608–10612 (1993).

  5. 5.

    et al. Mice devoid of PrP are resistant to scapie. Cell 73, 1339–1347 (1993).

  6. 6.

    et al. Sequential targeting of the genes encoding immunoglobulin-μ and prion protein in cattle. Nat. Genet. 36, 775–780 (2004).

  7. 7.

    , & Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 411, 810–813 (2001).

  8. 8.

    . Scrapie in Sheep: Clinical Features and Differential Diagnostic, VIth International Workshop on the Diagnosis of Spongiform Encephalopathies, Weybridge, UK, November 25–29, 2002 (Veterinary Laboratories Agency, Weybridge, UK, 2002).

  9. 9.

    et al. Detection and clinical evolution of scrapie in sheep using third eyelid biopsy. J. Vet. Intern. Med. 20, 187–193 (2006).

  10. 10.

    Prion protein and the transmissible spongiform encephalopathy diseases. Neuron 24, 503–506 (1999).

  11. 11.

    Prions. Proc. Natl. Acad. Sci. USA 95, 13363–13383 (1998).

  12. 12.

    & Prions and the blood and immune systems. Haematologica 90, 542–548 (2005).

  13. 13.

    Prion disease, blood and the immune system: concerns and realty. Haematologica 85, 3–10 (2000).

  14. 14.

    & The normal cellular form of prion protein modulates T cell responses. Immunol. Lett. 96, 147–150 (2005).

  15. 15.

    , , & T-lymphocyte activation and the cellular form of the prion protein. Immunology 92, 161–165 (1997).

  16. 16.

    , , & In vitro generation of infectious scrapie prions. Cell 121, 195–206 (2005).

  17. 17.

    , & Cyclic amplification of protein misfolding and aggregation. Methods Mol. Biol. 299, 53–65 (2005).

  18. 18.

    , & in Methods and Tools in Bioscience and Medicine (eds. Lehmann, S. & Grassi, J.) 198–213, (Birkhauser Verlag, Basel, 2004).

  19. 19.

    , & RNA molecules stimulate prion protein conversion. Nature 425, 717–720 (2003).

  20. 20.

    et al. Sulfated glycans and elevated temperature stimulate PrPSc-dependent cell-free formation of protease-resistant prion protein. EMBO J. 20, 377–386 (2001).

  21. 21.

    et al. Prion protein gene (PRNP) variants and evidence for strong purifying selection in functionally important regions of bovine exon 3. Proc. Natl. Acad. Sci. USA 101, 15142–15147 (2004).

  22. 22.

    , & Detection of prions in blood. Nat. Med. 11, 982–985 (2005).

  23. 23.

    et al. Loss of cerebellar Purkinje cells in aged mice homozygous for a disrupted PrP gene. Nature 380, 528–531 (1996).

  24. 24.

    et al. Ataxia in prion protein (PrP)-deficient mice is associated with upregulation of the novel PrP-like protein doppel. J. Mol. Biol. 292, 797–817 (1999).

  25. 25.

    et al. Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain. EMBO J. 20, 694–702 (2001).

  26. 26.

    et al. Doppel is an N-glycosylated, glycosylphosphatidylinositol-anchored protein. Expression in testis and ectopic production in the brains of Prnp0/0 mice predisposed to Purkinje cell loss. J. Biol. Chem. 275, 26834–26841 (2000).

  27. 27.

    , , , & Transmission of prions. Proc. Natl. Acad. Sci. USA 99 Suppl 4, 16378–16383 (2002).

  28. 28.

    et al. Identification of a novel gene encoding a PrP-like protein expressed as chimeric transcripts fused to PrP exon 1/2 in ataxic mouse line with a disrupted PrP gene. Cell. Mol. Neurobiol. 20, 553–567 (2000).

  29. 29.

    et al. Prion protein is necessary for normal synaptic function. Nature 370, 295–297 (1994).

  30. 30.

    et al. Post-natal knockout of prion protein alters hippocampal CA1 properties, but does not result in neurodegeneration. EMBO J. 21, 202–210 (2002).

  31. 31.

    , & Sleep and sleep regulation in normal and prion protein-deficient mice. J. Neurosci. 17, 1869–1879 (1997).

  32. 32.

    et al. Altered circadian activity rhythms and sleep in mice devoid of prion protein. Nature 380, 639–642 (1996).

  33. 33.

    et al. Mice deficient for prion protein exhibit normal neuronal excitability and synaptic transmission in the hippocampus. Proc. Natl. Acad. Sci. USA 93, 2403–2407 (1996).

  34. 34.

    et al. Mice devoid of prion protein have cognitive deficits that are rescued by reconstitution of PrP in neurons. Neurobiol. Dis. 19, 255–265 (2005).

  35. 35.

    et al. Expression of amino-terminally truncated PrP in the mouse leading to ataxia and specific cerebellar lesions. Cell 93, 203–214 (1998).

  36. 36.

    , , & Distinct molecular phenotypes in bovine prion diseases. EMBO Rep. 5, 110–115 (2003).

  37. 37.

    et al. Atypical Proteinase K-resistant prion protein (PrPres) observed in an apparently healthy 23-month-old Holstein steer. Jpn. J. Infect. Dis. 56, 221–222 (2003).

  38. 38.

    et al. Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic Creutzfeldt-Jakob disease. Proc. Natl. Acad. Sci. USA 101, 3065–3070 (2004).

Download references

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.

Author information

    • Jürgen A Richt
    •  & Yoshimi Kuroiwa

    These authors contributed equally to this work.

Affiliations

  1. National Animal Disease Center, Agriculture Research Services, United States Department of Agriculture, 2300 Dayton Avenue, Ames, Iowa 50010, USA.

    • Jürgen A Richt
    • , Amir N Hamir
    •  & Francisco Vargas

  2. Hematech, Inc., 4401 S. Technology Drive, Sioux Falls, South Dakota 57106, USA.

    • Poothappillai Kasinathan
    • , Thillai Sathiyaseelan
    • , Janaki Sathiyaseelan
    • , Hua Wu
    • , Hiroaki Matsushita
    • , Julie Koster
    •  & James M Robl

  3. Department of Neurology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas, 77555-0646, USA.

    • Joaquin Castilla
    •  & Claudio Soto

  4. Pharmaceutical Division, Kirin Brewery Co., Ltd., 26-1, Jingumae 6-chome, Shibuya-ku, Tokyo, Japan.

    • Shinichiro Kato
    • , Isao Ishida
    •  & Yoshimi Kuroiwa

  5. Gemini Science Inc., 9420 Athena Circle, La Jolla, California 92109, USA.

    • Shinichiro Kato
    •  & Yoshimi Kuroiwa

Authors

  1. Search for Jürgen A Richt in:

  2. Search for Poothappillai Kasinathan in:

  3. Search for Amir N Hamir in:

  4. Search for Joaquin Castilla in:

  5. Search for Thillai Sathiyaseelan in:

  6. Search for Francisco Vargas in:

  7. Search for Janaki Sathiyaseelan in:

  8. Search for Hua Wu in:

  9. Search for Hiroaki Matsushita in:

  10. Search for Julie Koster in:

  11. Search for Shinichiro Kato in:

  12. Search for Isao Ishida in:

  13. Search for Claudio Soto in:

  14. Search for James M Robl in:

  15. Search for Yoshimi Kuroiwa in:

Competing interests

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

Corresponding authors

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

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Fertility of PRNP−/− bovine sperm.

  2. 2.

    Supplementary Fig. 2

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

Word documents

  1. 1.

    Supplementary Table 1

    Serum chemistry values for PRNP cattle.

  2. 2.

    Supplementary Table 2

    Hematology values for PRNP−/− and control cattle.

  3. 3.

    Supplementary Table 3

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

  4. 4.

    Supplementary Table 4

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

  5. 5.

    Supplementary Table 5

    Blastocyst development from PRNP−/−fibroblast cell lines.

To obtain permission to re-use content from this article visit RightsLink.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nbt1271

Further reading Further reading