The purpose of this study was to investigate the development of bovine nuclear transfer (NT) embryos cultured in serum-free conditions. Bovine NT embryos cultured in various culture conditions were compared blastocyst development, total cell number and apoptosis using TUNEL assay. In experiment 1, blastocyst rates of NT embryos were significantly higher (P<0.01) in FBS (22.0％) and BSA (26.6％) groups than in PVA (6.3％) group. Total cell number was significantly higher in FBS (78.4±19.4) and BSA (90.9±29.1) groups than in PVA group (46.0±0.0). Apoptotic cell number was significantly fewer in FBS (3.1±1.4) and BSA (1.7±1.4) groups than in PVA group (7.0±20.0) However, all of results were not different between the FBS and BSA group. In experiment 2, blastocyst rates of NT embryos were significantly higher (P<0.05) in fatty acid free-BSA (FAF-BSA) group (26.8％) than in fraction V-BSA group (11.2％). Total cell number were somewhat higher in FAF-BSA group (89.8±30.7) than in fraction V-BSA group (88.1±19.3). Apoptotic cell number were somewhat fewer in FAF-BSA (1.7±1.5) group than in fraction V-BSA group (4.2±2.9). These findings suggest that serum free condition were effective for the in vitro development of bovine NT embryos. Therefore, we concluded that fatty acid free-BSA has beneficial effect in development bovine NT embryos and can be use as a serum substitute.

Open clusters are useful tools to investigate the structure and evolution of the Galactic disk. We have started a long-term project to obtain UBVI CCD photometry of open clusters which were little studied before, using the Doyak 1.8 m telescope of Bohyunsan Optical Astronomy Observatory in Korea. The primary goals of this project are (1) to make a catalog of UBVI photometry of open clusters, (2) to make an atlas of open clusters, and (3) to survey and monitor variable stars in open clusters. Here we describe this project and report the first results based on preliminary analysis of the data on four open clusters in the survey sample: Be 14, Cr 74, Biu 9, and NGC 2355. Isochrone fitting of the color-magnitude diagrams of the clusters shows that all of them are intermediate age to old (0.3-1.6 Gyrs) open clusters with moderate metallicity.

Controllable transgenic expression systems in transgenic animal model are valuable to the development of therapeutic approaches in human medical fields. The aim of this study was to 1) produce a transgenic cloned dog using inducible tetracycline vector system, and 2) investigate whether the transgenic cloned dog could be induced the transgene expression using doxycycline (Doxy). Canine fetal fibroblasts were infected with retroviral vectors designed to express the enhanced green fluorescent protein (eGFP) gene under the control of tetracycline-inducible promoter. For somatic cell nuclear transfer (SCNT), nucleus of an in vivo matured oocyte was removed and an eGFP expressed cell cultured with 1 ㎍/㎖ of Doxy was injected. After electrical fusion and chemical activation, the reconstructed embryos were transferred to a recipient and pregnancy diagnosis was performed by ultrasonography. Experiment I evaluated the mean fluorescence intensity (MFI) of infected cells while the cells were cultured in the presence of 1 ㎍/㎖ of Doxy for 5 days, and then in the absence of Doxy for 7 days using fluorescence-activated cell sorter. Experiment II was designed to produce an eGFP controllable transgenic cloned dog via SCNT. For verification of transgenic dog, experiment III was performed Southern Blot analysis and observation in vivo regulation of eGFP expression in the cloned dog treated with 100 ㎎/㎏ of Doxy every 2 days for 2 weeks under ultraviolet light. In experiment IV, western blot was used to detect eGFP increase and decrease in skin tissues of transgenic dog under the presence or absence of Doxy. In the results of experiment I, the MFI for infected cells was rapidly increased to approximately 42.3 times after 3 day-treatment compared to pre-treatment and quickly decreased 3 days after ceasing the treatment. In experiment II, a total of 203 embryos were transferred to nine recipients and three pregnant delivered three pups (Tet-on eGFP 0, Tet-on eGFP 1, and Tet-on eGFP 2) by C-sec and Tet-on eGFP 2 among them is still alive. All cloned pups were genetically identical to the donor cell. Tet-on eGFP 2 showed an apparent in vivo eGFP expression on her body after Doxy administration in experiment III. The result of Sothern blotting showed that the transgene insertion was detected from the three cloned dogs and all organs of Tet-on eGFP 1. Experiment IV indicated that a robust eGFP expression in skin tissue of Tet-on eGFP 2 rapidly increased after Doxy treatment and gradually decreased to basal level on 9 weeks after ceasing the treatment. In conclusion, we report here for the first time an inducible transgenic system in canine species and it can stably induce the transgene expression at intended time. This study has demonstrated the capacity to generate transgenic model dog which could regulate the transgene and it would contribute to human medical research fields.