The Chinese Hamster Ovarian cells CHOK1 are one of the most extensively used cells for the evaluation of gene expression and toxicology. However, these cells are frequently used for biomedical research without consideration of their cytogenetic characteristics. Therefore, we carried out to investigate the karyologic profiles, the frequency and type of chromosome aberration, and the distribution of telomeric DNA on chromosomes of the CHOK1 cells. The GTGbanding and fluorescence in situ hybridization on CHOK1 cells were performed to characterize the karyotype and the distribution of telomeric DNA. The present study revealed that the chromosome modal number of CHOK1 cells was 2n=20; eight chromosomes appeared to be identical with those of the normal Chinese hamster, whereas the remaining 12 chromosomes were shown to be translocated, deleted, inversed, or rearranged from Chinese hamster chromosomes. The telomeric DNA on CHOK1 chromosomes was intensively distributed at the centromeres rather than the ends of chromosomes. In addition, three chromosomes had interstitial telomeres and one marker chromosome entirely consisted of telomeric DNAs. The frequency and type of chromosome aberrations in CHOK1 cells were examined. Of the 822 metaphase spreads, 68 (8.3%) cells resulted in chromosome aberrations of which the chromosome breakage was the most frequently occurred.

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.