Recently, several approaches have been used to measure the oxygen consumption rates of individual embryos, but relationship between oxygen consumption and pregnancy rates of Hanwoo following embryo transfer has not yet been reported. In this study, we investigated the correlation between oxygen consumption rate and pregnancy rates of Hanwoo embryo using a SECM. In addition to, the expression of apoptosis-related genes was determined using real-time PCR by extracting RNA according to the oxygen consumption of in vivo embryo. First, we found that the oxygen consumption significantly increased in blastocyst-stage embryos (blastocyst) compared to early blastocyst stage embryos, indicating that oxygen consumption reflects the embryo quality (Grade I). The oxygen consumption or GI blastocysts were significantly higher than those of GII blastocysts (10.2x1014/mol s-1 versus 6.4x1014/mol s-1, p＜0.05). Pregnant rate in recipient cow was 0, 60 and 80% in the transplantation of embryo with the oxygen consumption of below 10.0, 10.0~12.0 and over 12.0x1014/mol s-1, respectively. Apoptosis regulatory genes, Hsp-70.1 were significantly increased in over-10.0 group than below 10.0 group but in Caspase-3, Bax and P53 gene, there was no significant difference. In conclusion, These results suggest that measurement of oxygen consumption maybe help increase the pregnant rate of Hanwoo embryos.

This study investigated the developmental ability and gene expression of somatic cell nuclear transfer embryos using ear skin fibroblast cells derived from miniature pig. When miniature pig (m) and landrace pig (p) were used as donor cells, there were no differences in cleavage (79.2 vs. 78.2%) and blastocyst rates (27.4 vs. 29.7%). However, mNT blastocysts showed significantly higher apoptosis rate than that of pNT blastocysts (6.1 vs. 1.7%) (p<0.05). The number of nuclei in pNT blastosysts was significantly higher than that of mNT (35.8 vs. 29.3) (p<0.05). Blastocysts were analyzed using Realtime RT-PCR to determine the expression of Bax-, Bcl-xl, H19, IGF2, IGF2r and Xist. Bax- was higher in mNT blastocyst than pNT blastocyst (p<0.05). There was no difference in Bcl-xl between two NT groups. Bax-/Bcl-xl was, however, significantly higher in mNT blastocyst compared to pNT. The expression of imprinting genes were aberrant in blastocysts derived from NT compared to in vivo blastocysts. H19 and IGF2r were significantly lower in mNT blastocysts (p<0.05). The expression of IGF2 and Xist was similar in two NT groups. However, imprinting genes were expressed aberrantly in mNT compared to pNT blastocysts. The present results suggest that the NT between donor cells derived from miniature pig and recipient oocytes derived from crossbred pig might affect reprogramming of donor cell, resulting in high apoptosis and aberrant expression patterns of imprinting genes.

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.

Promoters for milk proteins have been used far producing transgenic animals due to their temporal and spatial expression patterns. -casein, a calcium-sensitive casein, is a major milk protein that corresponds ca. 30 per cent of total milk protein. Expression of -casein is controlled by lactogenic hormones such as prolactin (PRL), composite response elements (CoREs) and transcription factors. CoREs are clusters of transcription factor binding sites containing both positive and negative regulatory elements. -casein gene promoter contains various regions (CoREs) for gene transcription. We analyzed the promoter region by mutagenesis using exonuclease III and linker-scanning. Transcription control elements usually are positioned in 5'-flanking region of the gene. However, in some cases, these elements are located in other regions such as intron 1. The nucleotide sequences of -casein promote. region has been reported (E12614). However, the properties of the promoter is not yet clear. In this study, we plan to investigate the properties of cis-regulating elements of porcine -casein by mutation analysis and expression analysis using dual-luciferase repoter assay system.