63 ESTABLISHMENT OF GREEN FLUORESCENT PROTEIN EXPRESSED DOG CELL LINES CONTROLLED BY DOXYCYCLINE

An inducible gene expression system in transgenic animals has been widely used in biomedical science. The aim of this study was to establish green fluorescent protein (GFP) inducible dog cell line and evaluate the system in embryos using interspecies somatic cell nuclear transfer (iSCNT). Canine fetal fibroblasts were transfected with retroviral vector containing GFP, rtTA, and TRE and designated Gteton cell line. For iSCNT, bovine ovaries were collected from a local slaughterhouse and COCs were matured for 24 h. The denuded oocytes were enucleated, injected with Gteton cells, treated with 24 h of doxycycline (DOX), and electrically fused (NEPA GENE, 34 V, 15 μs, 2 pulses). The reconstructed oocytes were activated and then cultured in modified SOF medium. To verify the stability of the Gteton cells, 2 experiments were designed. Experiment 1 was designed to compare the cell size and viability of Gteton and nontransfected cells. Countness™ (Invitrogen, version 1.0, Carlsbad, CA, USA) was used for analysis. In experiment 2, the control of GFP gene expression was observed when the cells were cultured with 1 mg mL-1 of DOX. The cells were also cultured without DOX after 24 h of DOX treatment. Photographs were taken of cultured cells every 12 h. The intensity of GFP expression was analyzed by using Image J freeware (U.S. National Institutes of Health, version 1.42, NIH, Bethesda, MD, USA). To evaluate the reprogramming ability of the Gteton cells in embryos, another 2 experimental designs were planned. Experiment 3 estimated GFP expression in iSCNT embryos when they were cultured with and without DOX. Experiment 4 assessed the development of the iSCNT embryos under microscopy. Data were analyzed using statistical analysis system program (version 9.1, SAS Institute, Cary, NC, USA). In experiment 1, there was no significance (P < 0.05) in average viable cell size (13.7 v. 13.2 μm) or viability (97.0 v. 98.7%). In experiment 2, the GFP intensity increased steadily when cultured in medium containing DOX. The intensity was increased approximately two times after 24 h compared with 12 h of treatment. The intensity after 24 h of DOX treatment decreased to the basal level after 5 days. In experiment 3, the GFP intensity of iSCNT embryos cultured in mSOF containing DOX was increased approximately two times in 16-cell stage compared with 2-cell stage. In experiment 4, the cleavage rate was not significantly different between the 2 groups. In conclusion, we dtermined that the inducible system of Gteton cell line was established in a stable manner. Furthermore the results from iSCNT may indicate the possibility to produce GFP-expressed transgenic puppies controlled by doxycyline. This study was supported by Korean MEST through KOSEF (grant # M10625030005-09N250300510) and BK21 program, RNL BIO, and Natural Balance Korea.