We propose to decipher the molecular basis of the species barrier by using transgenic (Tg) mice expressing chimeric human (Hu)/mouse (Mo) PrP genes. Earlier studies showed that mice expressing a chimeric PrP transgene designated MHu2M were susceptible to Hu prions. After systematically reverting some of the nine Hu residues to Mo at the N- and C-terminal ends of the "Hu2" insert, we identified Tg(MHu2M,S96N) mice with prolonged incubation times and Tg(MHu2M,M165V, E167Q) mice with abbreviated incubation times. The C-terminal residues 165 and 167 appear to be important in the binding of chimeric PrP to an auxiliary protein, provisionally designated protein X, that features in prion replication, while the N-terminal residues appear to bind to PrPSc in the inoculum. Our findings define a limited but feasible study of residues 96, 165 and 167 as well as the remaining six Hu residues in the Hu2 insert. Within the constraints posed by the need to construct Tg mice, we propose to introduce a substantial number of changes at the positions of the nine Hu residues in the Hu2 insert. Individual substitutions as well as combinations in the form of doublets and triplets will be made. We also plan to develop a cell culture system for the bioassay of chimeric PrP genes in ScN2a and ScGT1 cells by disrupting both copies of the MoPrP gene in these cells as described in Project 13. Such an assay would lift the constraints posed by the current need for Tg mice to test each chimeric PrP gene construct. From our studies of Tg mice, we should be able to determine the effects each residue in the Hu2 insert on incubation times for Hu prions and apply this knowledge to the development of rapid Tg mouse models for both Hu prion diseases and Chronic Wasting Disease (CWD) in cervids.