Long-term viability and stable germline transmission of prion-free cattle over multiple generations

Previously, we reported the generation of founder (F0) cattle and F1 offspring harboring a targeted mutation in the prion protein gene (PRNP) using an all-in-one vector system. This study aimed to assess the long-term viability and physiological normality of F0 through comprehensive monitoring, systemic histological examinations, and molecular analyses. F0 showed no phenotypical or behavioral abnormalities over a long-term observation of 4 years and 9 months. All F0 tissues p106ossessed the PRNP-targeted mutation, and protein analysis confirmed the absence of PRNP expression in the obex region. Furthermore, germline transmission (F2) of F1 frozen-thawed sperm and wild-type oocytes was confirmed through in vitro fertilized blastocysts. PRNP mutations were successfully transmitted to F2-generation blastocysts, as evidenced by germline transmission analysis. Overall, to our knowledge, these data present the longest monitoring of PRNP-mutated cattle (F0), including comprehensive organ analyses and germline transmission (F2) in PRNP-knockout cattle (F1), thereby establishing the first stable PRNP-mutated cattle line worldwide. This cattle line can serve as an invaluable biological resource for understanding bovine prion pathobiology and food security related to bovine spongiform encephalopathy.