A single copy gene encodes both the scrapie (PrPSc) and cellular (PrPC) isoforms of the prion protein (PrP). Cultured cell lines were found to express the endogenous PrP mRNA at levels comparable to those observed in the brains of adult rodents; however, these cells were invariably found to express greatly reduced levels of PrP. In all the cell lines examined, PrP was undetectable by Western immunoblot analysis. These cells were also poor recipients for expression constructs linking the hamster PrP gene open reading frame to several strong eukaryotic promoters; stable clones derived by transfection of these expression vectors failed to show elevated expression of PrP. When extremely high levels of PrP mRNA were produced using either an insect baculovirus or a mammalian SV40 based vector, significant quantities of PrP were produced, although in both cases the proteins were apparently processed differently from the PrPC observed in brains. In an expression system using an SV40 late promoter vector in monkey COS-7 cells, a significant fraction of PrP was transported to the cell surface where PrPC is found in vivo. PrP synthesized by the baculovirus vector failed to induce scrapie in hamsters and did not possess the characteristics of the PrPSc isoform associated with infectivity. The SV40 late promoter vector system may permit experiments designed to elucidate the role of PrPSc during scrapie infection as well as the function of PrPC in normal metabolism.
Bibliographical noteFunding Information:
The authors thank Dr M.McKinley for gifts of RNA and Dr M.Summers for help with the baculovirus expression system, as well as L.Gallagher for manuscript production assistance. M.R.D.S. was supported by a fellowship from the John Douglas French Foundation, D.A.B. was supported by a San Francisco Foundation/UCSF Grant for Medical Students, D.E.B. was supported by a National Institutes of Health postdoctoral fellowship (NS08100), and K K.H. was supported by a National Institutes of Health postdoctoral fellowship (GM11354). This work was suported by research grants from the National Institutes of Health (AG02132 and NS14069) and a Senator Jacob Javits Center of Excellence in Neuroscience (NS 22786) as well as by gifts from Sherman Fairchild Foundation, RJR-Nabisco, Inc., The Fairleigh S.Dickinson, Jr Foundation, Inc. and the Arthur L Swim Foundation.
- Cultured cells
- Expression vectors
- PrP gene