We will demonstrate the feasibility of using a novel genome editing technology (termed GenEdit) to modify the bovine PrP gene (boPrP) in primary bovine fibroblasts (BF). The modified BFs may then be used with nuclear transfer/cloning technologies to generate cattle resistant to bovine spongiform encephalophy (BSE or """"""""mad cow disease""""""""). The genome editing technology, GenEdit, will be employed using short DNA fragments (SDF) to introduce two nucleotide changes in the boPrP gene. The success of this phase I (outlined below) and the eventual phase II proposal will have an immediate medical impact, reducing the potential human health risk associated with consuming BSE-infected beef and bovine by-products, strengthening the consumer's confidence in the safety of beef products, and circumventing possible catastrophic affects on the beef industry as a consequence of an eventual BSE outbreak. Moreover, the eventual introduction and breeding of BSE-resistant cattle into the beef and dairy industries will also be a major benefit to the medical fields, including drug and cosmetic industries that rely heavily on bovine by-products. Similarly, BSE-resistant cattle will reduce the need for the extensive monitoring programs and regulatory measures currently being employed to assure the safety of these products against BSE. Last year Europe spent over 500 million dollars on BSE monitoring.
Our specific aims for this proposal are the following:
Aim # 1) clone and characterize the boPrP gene sequence in the specific breeds we wish to introduce the resistant trait, and generate SDF molecules to introduce nonsense and missense mutations in the boPrP gene.
Aim # 2) Develop a rapid gene-editing detection assay.
Aim # 3) Establish optimal conditions for delivering SDF molecules to BF nuclei.
Aim # 4) Establish the frequency on gene editing events in BF cells using a gene-editing reporter system, LX-MIR.
Aim # 5) Modify the boPrP gene using GenEdit in primary BF cells and isolate a cell clone for the eventual use in nuclear transfer/cloning for generating cattle carrying this modified allele.
