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, molecular analyses, and germline transmission. F0 showed no behavioral abnormalities over a long-term observation of 4 years and 9 months and no histological defects. All F0 tissues possessed 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 wildtype 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) of PRNP mutated 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.