Utility and Efficiency of Homologous Recombination for Introducing Targeted Modifications to the Pig Genome

The production and utilization of genetically modified animals greatly improves their utility in agriculture, as biomedical research models of human disease, for the production of recombinant pharmaceutical proteins, and for production of organs with greater potential for xenotransplantation. While numerous strategies have been utilized in the production of transgenic large animals, cell-based transgenesis followed by somatic cell nuclear transfer (SCNT) is currently the most widely applied method. We constructed a targeting vector with 2774 and 1890 bp arms of homology flanking a neomycin resistant gene. Following delivery of the DNA targeting vector into the porcine fetal fibroblasts by electroporation and selection with G418, PCR and sequencing confirmed one of 547 transgenic clonal colonies contained the targeted introduction of the neomycin resistance gene. Genetically modified fibroblast donor cells can be utilized for SCNT for the development of genetically modified pigs to study biological mechanisms important for animal agriculture and to create biomedical models