Genetic modification of pigs has many agricultural and biomedical applications. Ectopic overexpression of foreign genes is necessary in many cases to generate transgenic pigs with favorable phenotypes1. However, random integration of foreign genes often leads to unpredictable expression and unstable phenotypes2. Rosa26 is ubiquitously expressed in embryonic as well as adult tissues3. Targeting genes to the Rosa26 locus is a desirable method to create transgenic animals consistently expressing foreign genes at a high level. Insertion of a reporter or toxin gene into the Rosa26 locus has been widely used to trace or ablate specific cell lineages4, and this approach plays a fundamental role in understanding cell differentiation in vivo. Rosa26 was first identified and targeted in mouse embryonic stem cells (ESCs) in 1990s3, and then in human ESCs in 20075. With the establishment of rat pluripotent stem cells, the Rosa26 locus was also successfully identified and targeted in rats recently6. However, Rosa26 has not been tackled in large animals due to unavailability of germline-competent pluripotent stem cells. By taking advantage of recently emerging technology of gene editing mediated by transcription activator–like effector nuclease (TALEN)7, here we characterized the porcine Rosa26 (pRosa26) locus and targeted a Cre-dependent reporter gene into the pRosa26 locus. Using this approach, we also created transgenic pigs stably overexpressing a gene of interest through recombinase-mediated cassette exchange (RMCE)5.

To generate transgenic pigs with stable and ubiquitous overexpression of exogenous genes and also reliable reporters that can be used for stem cell and human disease studies, we attempted to introduce a Cre-dependent EGFP reporter into the pRosa26 locus. As porcine ESCs for gene targeting are not available, we applied the targeting strategy to pig fetal fibroblasts (PFFs) and performed somatic cell nuclear transfer (SCNT) to generate transgenic pigs1. We first applied a traditional homologous recombination-mediated targeting strategy. We selected 425 colonies, but none of them was correctly targeted. Thus, the efficiency of the traditional strategy appeared too low for us to obtain targeted clones.

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