Generation of Porcine PTEN Targeting Cell-Line as a Research Model for Human Cancer

Recently, there is an increasing interest in pig as a non-rodent research animal model for the study of human diseases. Because of their anatomical and physiological similarities to human, pigs are considered to be a useful animal model for human diseases such as cancer. With recent developments in genome editing technology using CRISPR/Cas9 system, genetically modified pigs can be generated at a higher efficiency. In this study, we have generated porcine cell lines which carry defective phosphatase and tensin homolog (PTEN) gene by using CRISPR/Cas9 system. The gene is a known tumor suppressor gene, thus modification of the gene will allow us to monitor cancer development in pigs. First, specific CRISPR/Cas9 systems to target porcine PTEN were designed and generated to induce a double-strand break on Exon2. The plasmids encoding for CRISPR/Cas9 system were transfected into porcine fetal fibroblasts by electroporation. After transfection, mutations generated through the CRISPR/Cas9 systems were identified by sequencing PCR amplicons flanking CRISPR/Cas9 target regions. The sequencing results confirmed that we have successfully generated PTEN knock-out cell lines carrying biallelic modification; efficiency of mutant targeting was 24/72 (33.3%). These fibroblasts were used as donor cells for somatic cell nuclear transfer, followed by embryo transfer to generate PTEN knock-out pigs. Pregnancy was established in one surrogate and the animals are expected to farrow in the near future. The genetically modified piglets produced from the surrogates could be useful resources for human cancer research and a potential model to develop novel cancer therapies.