Pigs are important livestock for food and have been used in various biomedical studies, particularly translational research, as experimental animals because of their anatomical and physiological similarity to humans. The recent development of genome editing techniques, such as ZFN, TALEN, and CRISPR/Cas9, has rapidly expanded the use of genome editing tools in a variety of animals, resulting in the relatively easy and efficient generation of gene knock-out pigs. In the past few years, there has been a sustained increase in reports describing the development of genetically modified pigs. This chapter introduces our workflow for establishing the genetically modified cells (nuclear donor cells) necessary to create gene knock-out pigs using somatic cell nuclear transfer and focuses on the actual generation of gene knock-out pigs using a cytoplasmic injection method.