Cultured Cell Sublines Highly Susceptible to Prion Infection

Prions are the transmissible pathogens that cause a class of neurodegenerative diseases in mammals including Creutzfeldt-Jakob disease in humans and scrapie in sheep. The only known component of the prion is an abnormal isoform of the prion protein (PrP) designated PrPSc. The precursor of PrPSc is PrPC, a glycophosphatidylinositol-anchored membrane protein of unknown function that is highly expressed in neurons of the central nervous system. Prion diseases can be transmitted to hamsters and mice, which are used extensively in laboratory studies of these conditions. However, the cost and complexity of prion studies in vivo have prompted many attempts to establish prion-infected cell lines. For unknown reasons, most cell lines are resistant to prion infection (4, 21). Stable propagation of prions has been established in only a few lines. The most successful of these have been derivatives of the C-1300 mouse neuroblastoma line, particularly the neuro-2A (N2a) subline (4, 20). Prion-infected N2a cells are traditionally referred to as ScN2a. Recently, a mouse hypothalamic cell line, GT1, has been infected and produces stable high titers of prions (ScGT1) (25). Although it is not widely used, the rat pheochromocytoma-derived line PC12 also has been reported to be infectible with mouse, but not rat, prions (21, 22). Typical N2a cultures exposed to prions produce only low levels of infectious prions, apparently because only a small percentage of the cells become infected (19, 20). In order to obtain cultures that produce sufficient quantities of PrPSc for biochemical analysis, prion-exposed N2a cultures must be subcloned and the most highly infected sublines must be selected (4, 19). This method reliably produces ScN2a lines that generate large amounts of PrPSc and prion titers of about 105 50% infective dose (ID50) units/107 cells (4). About 80 to 90% of cells in these cloned, infected sublines are prion infected (reference 19 and data below). To date, ScN2a has proved the most useful cell line for studying the cell biology of prion replication. This line has been used to determine the kinetics of PrPSc and the subcellular location of PrPSc formation (2, 6, 10, 12, 29, 30). These cells have also been used to seek inhibitors of scrapie formation, laying the groundwork for the development of antiprion therapeutic agents (5, 28). Recent studies using ScN2a cells transfected with mutant PrP sequences have mapped out regions of the PrP polypeptide responsible for dominant negative inhibition of prion propagation (13). A number of studies have compared properties of uninfected and prion-infected cells to look for prion infection-specific alterations in cellular metabolism (7, 15, 32, 33). Such investigations may suffer from the fact that ScN2a cells are subcloned from a population of cells to which they are then compared. Tumor cells in culture are known to have heterogeneous properties due at least in part to genetic instability (16). Therefore, the observed differences between infected and uninfected cell lines do not necessarily arise as a consequence of prion infection. The observed differences might represent purely fortuitous artifacts of cloning or might be due to a selection artifact; i.e., properties specific to ScN2a cells may be those which render the cells susceptible to prion infection and might be found in only a minority of cells in the parent population. We sought to generate prion-infected and uninfected cell lines in which artifactual differences between the lines would be eliminated. We first developed a method based on the cell blot technique for the rapid and sensitive detection of de novo prion infection in multiple independent cell cultures. Using this technique, we derived prion-susceptible cell lines from which we were able to generate prion-infected cultures without further subcloning. We also used these susceptible sublines and our modified cell blot method to develop a rapid and sensitive bioassay for one prion strain and to demonstrate the resistance of N2a to infection with other strains.