Transgenic (TG) pigs are currently regarded as an important animal model for various biomedical researches including a disease modeling and a regenerative medicine. We tried to develop a pig astrocyte-specific CreERT2-LoxP recombination system as a part of TG pig model of brain tumor arisen from astrocytic cell lineage. We designed two vector systems one retains pig glial fibrillary acidic protein (GFAP) promoter-CreERT2 transgene and the other has GFP gene flanked by LoxP sites which can be eliminated through the CreERT2-mediated recombination. Then, generated TG pigs through somatic cell nuclear transfer (SCNT) technique were analyzed whether the GFAP-CreERT2-LoxP recombination had been occurred. SCNT and embryo transfer were performed three times to just before ovulation state of the surrogate mothers. One of them was pregnant, and delivered five transgenic piglets to 115 days after pregnancy. Results from gDNA in skin tissues of TG piglets were confirmed that transgenes are introduced. It was confirmed that CreERT2 gene expression with the GFAP promoter was highly expressed only in the cerebrum by real-time PCR. After 3 months, we were oral administration of 15 mg/kg of tamoxifen during five days, and then euthanized after 7 days. As a result of PCR, was confirmed that the recombination was induced in the cerebrum. However, the other organ samples did not occur recombination. In addition, the TG neural cell line was established by primary culture of cerebrum and it was confirmed the induction of recombination by PCR. In conclusion, CreERT2 gene, expressed by the GFAP promoter, was found to exist in the specific part of the cerebrum. The observation in the study suggests GFAP-CreERT2-LoxP recombination system consisting of two vector construction works properly and specifically in vivo pig brain. This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A2A2A04008751), Republic of Korea.
11 Jul 2016 - 11 Jul 2016