This study was conducted to optimize the efficiency of cloning and to produce cloned mice. The majority of cloned mammals derived by nuclear transfer (NT) die during gestation and have enlarged and dysfunctional placentas. In this study, the optimized conditions were established to produce clone mice. The parthenogenetic oocytes were activated after 6 h regardless of cytochalasin B (CB) concentration. CB treatment (2 μg/ml) was found second polar body. Lower concentration of CB was decreased the activation rate, but the second polar body was the best highly increased during 6 h incubation. The small fragments were exhibited in the 5 μg/ml treatment of CB, but it was not found in lower concentration groups (> 2.5 μg/ml). To examine effects of SrCl2 on the adult cumulus cells, somatic cell NT oocytes were exposed during 0.5, 1 and 6 hrs. The second polar body was significantly greater in 0.5 h exposure group (6.6%) than 1, 6 hrs. Developmental rate from 2-cell to 4-cell was the lowest in 7.5 mM Strontium chloride (SrCl2) groups (84.1% and 64.3%) than 5, 10 m MSrCl2. The implantation rate was not significantly difference among 5, 7.5 and 10 m MSrCl2 group. Three live fetuses were produced by SCNT. SCNT placentas were remarkably heavier than IVF group (8 fetuses) (0.34, 0.34, 0.33 vs 0.14 g) compared with the placenta weight of IVF and SCNT clones. (Key words : parthenogenetic oocytes, cytochalasin B, cloned mice)

To investigate the biochemical nature of changes in vaginal physiology during estrus and pregnancy, we examined the cytology and viscosity, and monitored the protein expression profile in vaginal mucus during estrus and pregnancy. The viscosity progressively decreased from estrus to pregnancy. Cell type analysis revealed that white blood cells progressively increased from estrus to pregnancy, while red blood cells progressively decreased during pregnancy. The cornification index (CI) was higher in estrus than in pregnancy. Protein mass spectrumetry identified the presence of ribosome-binding protein 1, GRIP 1 (Glutamate receptor-interacting protein 1)-associated protein 1, DUF729 (Domain of unknown function729) domain-containing protein 1, prolactin precursor, dihydrofolatereductase, and MMP (Matrix metalloprotease)-9 in vaginal mucus. MMP-2 and MMP-9 proteins in the vaginal mucus were active throughout estrus and gestation, as measured by a gelatinase assay, but most abundant in the vaginal mucus on day 0 of estrus. Results from ELISA of MMP-2 and MMP-9 were in accordance with the gelatinase assay. In light of the crucial role of metalloproteinases in extracellular matrix remodeling, the level of MMP-9 in vaginal mucus might be useful as an indicator of estrus and pregnancy to increase the efficiency of reproduction.

Human eryhropoietin (EPO) is acidic glycoprotein hormone that plays key role in hematopoiesis by facilitating differentiation of erythrocyte and formation of hemoglobin (Hb) and is used for the treatment of anemia. Human EPO is consist of 166 amino acids which is modified by three N-glycosylations (24, 38, 83) and single O-glycosylation (126). N-glycosylation is reported to be related to the cellular secretion and activity of EPO. In this study, we examined effects of mutagenesis in glycosylation site of recombinat hEPO for the cellular secretion during production from cultured CHO cell. We produced rhEpo which was cloned by PCR from human liver cDNA (TaKaRa) in cultured CHO cell. Using supernatant of the culture, ELISA assay and western analysis were performed. To estimate biological activity, 20IU of rhuEpo was subcutaneously injected into four ICR mice. After 8 days, HCT level was increased average 13 per cent, RBC was increased ca. 2106//. In disease model Rat (anemia c-kit, WSRC-WS/WS), HCT was increased ca. 12%, RBC was increased ca. 1.6106//. These results suggests that rhEpo we produced has biological activity. To remove glycosylation site by substituting 24, 38, 83, and 126th asparagine (or serine) with glutamic acid, overlapping -extension site-directed mutagenesis was performed. To add novel glycosylation sites, 69, 105th leucine was mutated to asparagine. Mutant EPO construct was transfected into CHO cell. Supernatant of the cell culture was analyzed using ELISA assay with monoclonal anti-EPO antibody (Medac, Germany). Since, several reports for mutagenesis of glycosylation sites showed case-by-case results, we examined both transient expression and stable expression. Addition of novel glycosylation sites resulted no secretion while deletion mutants had little effect except some double deletion mutants (24/83 and 38/83) and triple mutant. We suggest that not single but combination of glycosyl group affect secretion of EPO.