Mannheimia haemolytica is the most important bacterial pathogen of bovine respiratory disease (BRD) which costs over $1 billion in annual losses to the U.S. cattle industry. M. haemolytica produces an exotoxin (leukotoxin) that binds to the intact signal peptide of CD18 on bovine leukocytes, and lyses them. We have previously shown that the signal peptide of ruminant CD18 is not cleaved due to the presence of ‘cleavage-inhibiting’ glutamine (Q), instead of ‘cleavage-conducive’ glycine (G), at amino acid position 5 upstream of the cleavage site. Site-directed mutagenesis of Q to G results in cleavage of the signal peptide and abrogation of cytolysis of transfectants expressing bovine CD18 with the Q(-5)G mutation. We hypothesized that genetically engineered cattle that produce leukocytes expressing CD18 containing the Q(-5)G mutation will be resistant to leukotoxin, and hence will be less susceptible to BRD. As the first step towards developing calves resistant to leukotoxin, fibroblast cell lines were developed from two 45 day-old fetuses. Fibroblasts were co-transfected with an oligonucleotide (120bp) carrying the Q(-5)G mutation, and mRNA of the zinc finger nuclease (ZFN) custom-designed to bind and induce double-strand break of the DNA in the vicinity of the amino acid to be mutated. Subsequently, the extracted genomic DNA was tested for the presence of a ZFN cut using a surveyor mutation detection assay (CEL-1). At one week post-transfection, cells were cloned by limiting dilution. Genomic DNA was extracted from single clones and tested for the presence of the Q(-5)G mutation by junction PCR. Five clones with bi-allelic modification of CD18 were obtained. Somatic cell nuclear transfer (SCNT) was performed with two selected clones. Fourteen embryos resulting from SCNT have been transferred to surrogate cows. If successful, this technology will provide a means of developing cattle resistant to M. haemolytica leukotoxin, and hence less susceptible to BRD.