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. 1992 Aug;1(8):986–997. doi: 10.1002/pro.5560010804

Chimeric prion protein expression in cultured cells and transgenic mice.

M R Scott 1, R Köhler 1, D Foster 1, S B Prusiner 1
PMCID: PMC2142161  PMID: 1338978

Abstract

The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558-1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847-857; Prusiner, S.B., et al., 1990, Cell 63, 673-686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease-resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa-specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.

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Selected References

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  1. Alper T., Cramp W. A., Haig D. A., Clarke M. C. Does the agent of scrapie replicate without nucleic acid? Nature. 1967 May 20;214(5090):764–766. doi: 10.1038/214764a0. [DOI] [PubMed] [Google Scholar]
  2. Alper T., Haig D. A., Clarke M. C. The scrapie agent: evidence against its dependence for replication on intrinsic nucleic acid. J Gen Virol. 1978 Dec;41(3):503–516. doi: 10.1099/0022-1317-41-3-503. [DOI] [PubMed] [Google Scholar]
  3. Barany F. Two-codon insertion mutagenesis of plasmid genes by using single-stranded hexameric oligonucleotides. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4202–4206. doi: 10.1073/pnas.82.12.4202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barry R. A., Prusiner S. B. Monoclonal antibodies to the cellular and scrapie prion proteins. J Infect Dis. 1986 Sep;154(3):518–521. doi: 10.1093/infdis/154.3.518. [DOI] [PubMed] [Google Scholar]
  5. Basler K., Oesch B., Scott M., Westaway D., Wälchli M., Groth D. F., McKinley M. P., Prusiner S. B., Weissmann C. Scrapie and cellular PrP isoforms are encoded by the same chromosomal gene. Cell. 1986 Aug 1;46(3):417–428. doi: 10.1016/0092-8674(86)90662-8. [DOI] [PubMed] [Google Scholar]
  6. Bockman J. M., Prusiner S. B., Tateishi J., Kingsbury D. T. Immunoblotting of Creutzfeldt-Jakob disease prion proteins: host species-specific epitopes. Ann Neurol. 1987 Jun;21(6):589–595. doi: 10.1002/ana.410210611. [DOI] [PubMed] [Google Scholar]
  7. Borchelt D. R., Scott M., Taraboulos A., Stahl N., Prusiner S. B. Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. J Cell Biol. 1990 Mar;110(3):743–752. doi: 10.1083/jcb.110.3.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Boshart M., Weber F., Jahn G., Dorsch-Häsler K., Fleckenstein B., Schaffner W. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell. 1985 Jun;41(2):521–530. doi: 10.1016/s0092-8674(85)80025-8. [DOI] [PubMed] [Google Scholar]
  9. Bruce M. E., McConnell I., Fraser H., Dickinson A. G. The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis. J Gen Virol. 1991 Mar;72(Pt 3):595–603. doi: 10.1099/0022-1317-72-3-595. [DOI] [PubMed] [Google Scholar]
  10. Butler D. A., Scott M. R., Bockman J. M., Borchelt D. R., Taraboulos A., Hsiao K. K., Kingsbury D. T., Prusiner S. B. Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins. J Virol. 1988 May;62(5):1558–1564. doi: 10.1128/jvi.62.5.1558-1564.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Caughey B., Raymond G. J. The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive. J Biol Chem. 1991 Sep 25;266(27):18217–18223. [PubMed] [Google Scholar]
  12. Felgner P. L., Gadek T. R., Holm M., Roman R., Chan H. W., Wenz M., Northrop J. P., Ringold G. M., Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7413–7417. doi: 10.1073/pnas.84.21.7413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gabizon R., Prusiner S. B. Prion liposomes. Biochem J. 1990 Feb 15;266(1):1–14. doi: 10.1042/bj2660001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell. 1981 Jan;23(1):175–182. doi: 10.1016/0092-8674(81)90282-8. [DOI] [PubMed] [Google Scholar]
  15. Hay B., Barry R. A., Lieberburg I., Prusiner S. B., Lingappa V. R. Biogenesis and transmembrane orientation of the cellular isoform of the scrapie prion protein [published errratum appears in Mol Cell Biol 1987 May;7(5):2035]. Mol Cell Biol. 1987 Feb;7(2):914–920. doi: 10.1128/mcb.7.2.914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hsiao K. K., Scott M., Foster D., Groth D. F., DeArmond S. J., Prusiner S. B. Spontaneous neurodegeneration in transgenic mice with mutant prion protein. Science. 1990 Dec 14;250(4987):1587–1590. doi: 10.1126/science.1980379. [DOI] [PubMed] [Google Scholar]
  17. Kascsak R. J., Rubenstein R., Merz P. A., Tonna-DeMasi M., Fersko R., Carp R. I., Wisniewski H. M., Diringer H. Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins. J Virol. 1987 Dec;61(12):3688–3693. doi: 10.1128/jvi.61.12.3688-3693.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kaufman R. J., Murtha P., Davies M. V. Translational efficiency of polycistronic mRNAs and their utilization to express heterologous genes in mammalian cells. EMBO J. 1987 Jan;6(1):187–193. doi: 10.1002/j.1460-2075.1987.tb04737.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kimberlin R. H., Cole S., Walker C. A. Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters. J Gen Virol. 1987 Jul;68(Pt 7):1875–1881. doi: 10.1099/0022-1317-68-7-1875. [DOI] [PubMed] [Google Scholar]
  20. Kimberlin R. H., Cole S., Walker C. A. Transmissible mink encephalopathy (TME) in Chinese hamsters: identification of two strains of TME and comparisons with scrapie. Neuropathol Appl Neurobiol. 1986 Mar-Apr;12(2):197–206. doi: 10.1111/j.1365-2990.1986.tb00050.x. [DOI] [PubMed] [Google Scholar]
  21. Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 1991 Nov;115(4):887–903. doi: 10.1083/jcb.115.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25;12(2):857–872. doi: 10.1093/nar/12.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kozak M. Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo. Nature. 1984 Mar 15;308(5956):241–246. doi: 10.1038/308241a0. [DOI] [PubMed] [Google Scholar]
  24. Oesch B., Westaway D., Wälchli M., McKinley M. P., Kent S. B., Aebersold R., Barry R. A., Tempst P., Teplow D. B., Hood L. E. A cellular gene encodes scrapie PrP 27-30 protein. Cell. 1985 Apr;40(4):735–746. doi: 10.1016/0092-8674(85)90333-2. [DOI] [PubMed] [Google Scholar]
  25. PATTISON I. H. RESISTANCE OF THE SCRAPIE AGENT TO FORMALIN. J Comp Pathol. 1965 Apr;75:159–164. doi: 10.1016/0021-9975(65)90006-x. [DOI] [PubMed] [Google Scholar]
  26. Pattison I. H., Jones K. M. Modification of a strain of mouse-adapted scrapie by passage through rats. Res Vet Sci. 1968 Sep;9(5):408–410. [PubMed] [Google Scholar]
  27. Pelletier J., Sonenberg N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature. 1988 Jul 28;334(6180):320–325. doi: 10.1038/334320a0. [DOI] [PubMed] [Google Scholar]
  28. Prusiner S. B., Cochran S. P., Groth D. F., Downey D. E., Bowman K. A., Martinez H. M. Measurement of the scrapie agent using an incubation time interval assay. Ann Neurol. 1982 Apr;11(4):353–358. doi: 10.1002/ana.410110406. [DOI] [PubMed] [Google Scholar]
  29. Prusiner S. B. Novel proteinaceous infectious particles cause scrapie. Science. 1982 Apr 9;216(4542):136–144. doi: 10.1126/science.6801762. [DOI] [PubMed] [Google Scholar]
  30. Rogers M., Serban D., Gyuris T., Scott M., Torchia T., Prusiner S. B. Epitope mapping of the Syrian hamster prion protein utilizing chimeric and mutant genes in a vaccinia virus expression system. J Immunol. 1991 Nov 15;147(10):3568–3574. [PubMed] [Google Scholar]
  31. Scott M. R., Butler D. A., Bredesen D. E., Wälchli M., Hsiao K. K., Prusiner S. B. Prion protein gene expression in cultured cells. Protein Eng. 1988 Apr;2(1):69–76. doi: 10.1093/protein/2.1.69. [DOI] [PubMed] [Google Scholar]
  32. Scott M., Foster D., Mirenda C., Serban D., Coufal F., Wälchli M., Torchia M., Groth D., Carlson G., DeArmond S. J. Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques. Cell. 1989 Dec 1;59(5):847–857. doi: 10.1016/0092-8674(89)90608-9. [DOI] [PubMed] [Google Scholar]
  33. Serban D., Taraboulos A., DeArmond S. J., Prusiner S. B. Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins. Neurology. 1990 Jan;40(1):110–117. doi: 10.1212/wnl.40.1.110. [DOI] [PubMed] [Google Scholar]
  34. Weissmann C. A 'unified theory' of prion propagation. Nature. 1991 Aug 22;352(6337):679–683. doi: 10.1038/352679a0. [DOI] [PubMed] [Google Scholar]

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