Generation of transgenic pigs by additive gene transfer and gene targeting combined with SCNT. (A) Additive gene transfer. Cells are transfected with a vector containing the gene of interest (GOI) and a resistance gene. After antibiotic selection the single cell clones are mixed and transferred to an enucleated oocyte (SCNT) followed by an embryo transfer (ET) to synchronized gilts. After genotyping and gene expressing analysis of the born pigs the best expressing animal is re-cloned or used for a second round of transfection. (B) Gene targeting. In the first targeting round the cells are transfected with a targeting vector containing beside homologous regions of the target locus, a resistance gene (RG1). After selection and characterization of the generated single cell clones, heterozygous knockout cells are used for SCNT followed by the ET. For the targeting of the second allele cells isolated of the heterozygous knockout animals are used for a second round of transfection, using a second targeting vector with another resistance gene (RG2). All following procedures resemble the first round.