• 한국환경성돌연변이발암원학회지
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• 제16권2호
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• pp.83-87
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• 1996

This study was performed by the sister chromatid exchanges (SCEs) to investigate the effects of Aphidicolin (APC) or 2, 4-dinitrophenoi (DNP) on cross-adaptive response to ultraviolet radiation (UV) or ethyl methanesulfonate (EMS) in Chinese hamster ovary (CHO) cells. The pretreatment with 1 J/m$^2$ UV decreased the yield of SCEs induced by subsequent treatment with 8 mM EMS in CHO cells. And the treatment with 10 $\mu$g/ml APC or 50 $\mu$M DNP during incubation after pretreatment with 1 J/m$^2$ UV increased the yield of SCEs induced by 8 mM EMS. The pretreatment with 2 mM EMS decreased the yield of SCEs induced by subsequent treatment with 5 J/m$^2$ UV. The treatment with 10 $\mu$g/ml APC during incubation after 2 mM EMS increased the yield of SCEs induced by 5 J/m$^2$ UV. These results suggest that APC and DNP inhibit cross-adaptive response to pretreatment with UV and subsequent treatment with EMS, and also cross-adaptive response to pretreatment with EMS and subsequent treatment with UV is inhibited by APC in CHO cells.

• Journal of Microbiology and Biotechnology
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• 제21권12호
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• pp.1299-1305
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• 2011

An important modification of thrombolytic agents is resistance to plasminogen activator inhibitor-1 (PAI-1). In previous studies, a new truncated PAI-1-resistant variant was developed based on deletion of the first three domains in t-PA and the substitution of KHRR 128-131 amino acids with AAAA in the truncated t-PA. The novel variant expressed in a static culture system of Chinese Hamster Ovary (CHO) DG44 cells exhibited a higher resistance to PAI-1 when compared with the full-length commercial drug; Actylase. In the present study, the truncated-mutant protein was expressed in CHO DG44 cells in 50 ml orbital shaking bioreactors. The final yield of the truncated-mutant in the culture was 752 IU/ml, representing a 63% increase compared with the static culture system. Therefore, these results suggest that using the combined features of a transient and stable expression system is feasible for the production of novel recombinant proteins in the quantities needed for preclinical studies.

• 한국환경성돌연변이발암원학회지
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• 제15권2호
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• pp.94-99
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• 1995

The adaptive response and cross-adaptive response to sister chromatid exchanges (SCEs) and DNA single-strand breaks (SSBs) in Chinese hamster ovary (CHO)-K$_1$ cells treated with ultraviolet radiation (UV), ethyl methanesuffonate (EMS), or bleomycin (BLM) were investigated. Two assays were used in this study; SCEs and alkaline elution. The pretreatment with low conditioning dose of 2 mM EMS or 1 J/m$^2$ UV decreased the yield of SCEs induced by subsequent treatment with 8 mM EMS, 5 J/m$^2$ UV or 5 $\mu$g/ml BLM. And the pretreatment with low conditioning dose of 1 $\mu$g/ml BLM decreased the yield of SCEs induced by subsequent treatment with 5 $\mu$g/ml BLM or 5 J/m$^2$ UV. The rejoining of DNA SSBs in cells subsequently treated with 2 J/m$^2$ UV, 50 mM EMS or 400 $\mu$g/ml BLM is higher than that only treated with 2 J/m$^2$ UV, 50 mM EMS or 400 $\mu$g/ml BLM. These results suggest that there are the adaptive response and cross-adaptive response to SCEs, and is the adaptive response to the rejoining of DNA SSBs in CHO cells.

• 한국환경성돌연변이발암원학회지
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• 제9권1호
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• pp.13-22
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• 1989

The effect of novobiocin (NOV), and inhibitor of topoisomerase II, on ethyl methanesulfonate (EMS)-or bleomycin (BLM)-induced DNA repair synthesis was examined during the cell cycle of Chinese hamster ovary (CHO)-K1 cells. Three assays were employed in this study: cell survival, alkaline elution and unscheduled DNA synthesis. EMS was effective at killing CHO cells in G1 phase, wheras BLM preferentially killed cells in G2 and S phases. EMS induced the much more amount of DNA damage in G1 phase, while BLM induced in G2 phase than the other phases. The both of pre- and post-treatment with BOV inhibitied EMS- or BLM-induced DNA repair synthesis in G1 and G2 phases, and pretreatment with NOV inhibited more effectively than the post-treated group. These results suggested that CHO cells exhibited a differential sensitivity to cell lethality and DNA damage in relation to cell cycle according to used chemical agents, and that DNA topoisomerase II participated in an initial stage of DNA repair.

• 한국환경성돌연변이발암원학회지
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• 제9권1호
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• pp.23-32
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• 1989

Chinese hamster ovary (CHO)-K1 cells echibited a differential sensitivity in the process of DNA repair synthesis induced by ethyl methanesulfonate (EMS) or bleomycin (BLM) in relation to cell cycle. Two assays were employed in this study: alkaline elution and unscheduled DNA synthesis. The post-treat-ment with aphidicolin (APC), an inhibitor of DNA polymerase alpha, inhibited DNA repair synthesis induced by EMS in G2 phase, while APC did not show any effect on BLM-induced DNA repair synthesis in all phases. On the other hands, the 2', 3'-dideoxythymidine (ddTTP), an inhibitor of DNA polymerase beta, inhibited DNA repair synthesis induced by EMS or BLM in both of G1 and G2 phases. These results suggested that the involvement of DNA polymerase alpha and beta in DNA repair was dependent on cell stage or used chemical agent.

• Journal of Microbiology and Biotechnology
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• 제14권4호
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• pp.844-851
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• 2004

Efficient mammalian erythropoietin (EPO)-expression systems are required for therapeutic applications. The accumulation of ammonia is a major problem in the production of recombinant proteins in cultured animal cells. To counter this problem we introduced the first two genes of the urea cycle, carbamoyl phosphate synthetase (CPSI) and ornithine transcarbamylase (OTC), into IBE Chinese Hamster Ovary (CHO) cells by stable transfection. The resulting cell line, CO5, had a higher growth rate and accumulated less ammonia per cell than the parental cell line, IBE. In addition, it produced 2 times more EPO than the parent, and the purified EPO contained a higher proportion of acidic isoforms with approximately 15% more sialic acid.

• Molecules and Cells
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• 제25권4호
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• pp.504-509
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• 2008

Three G-protein-linked acetylcholine receptors (GARs) exist in the nematode C. elegans. GAR-3 is pharmacologically most similar to mammalian muscarinic acetylcholine receptors (mAChRs). We observed that carbachol stimulated ERK1/2 activation in Chinese hamster ovary (CHO) cells stably expressing GAR-3b, the predominant alternatively spliced isoform of GAR-3. This effect was substantially reduced by the phospholipase C (PLC) inhibitor U73122 and the protein kinase C (PKC) inhibitor GF109203X, implying that PLC and PKC are involved in this process. On the other hand, GAR-3b-mediated ERK1/2 activation was inhibited by treatment with forskolin, an adenylate cyclase (AC) activator. This inhibitory effect was blocked by H89, an inhibitor of cAMP-dependent protein kinase A (PKA). These results suggest that GAR-3b-mediated ERK1/2 activation is negatively regulated by cAMP through PKA. Together our data show that GAR-3b mediates ERK1/2 activation in CHO cells and that GAR-3b can couple to both stimulatory and inhibitory pathways to modulate ERK1/2.

• Archives of Pharmacal Research
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• 제28권2호
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• pp.220-226
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• 2005

Temperature and medium composition were changed with the aim of increasing growth and erythropoietin (EPO) production in EPO-producing Chinese hamster ovary (CHO) cells. We used the CHO cell line, IBE, and its derivative, CO5, which over-expresses the first two enzymes of the urea cycle, carbamoyl phosphate synthetase I (CPS I) and ornithine transcar-bamoylase (OTC). When supplements were added to the medium at $33\;^{\circ}C$, the growth of IBE and CO5 cells increased by $27\%\;and;26\%$, respectively and the maximum yield of EPO was increased by $40\%$ in both cell lines. The absolute EPO concentration in the CO5 cells was always $55{\sim}60\%$ higher than in the IBE cells. In addition, when the two cell lines were continuously cultured with supplements at $33\;^{\circ}C$ until their growth rates approached those at $37\;^{\circ}C$, the growth rates of both IBE and CO5 cells increased by $54\%$ and their maximum EPO levels increased by up to $73\%\;and\;56\%$, respectively. Therefore, the growth and EPO expression levels of CO5 cells increased 2.2-fold and 2.6-fold, respectively, compared to those of the IBE cells. These results indicate that adaptation to lower temperature as well as medium supplementation could be important for improving cell growth and EPO production.

• Journal of Microbiology and Biotechnology
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• 제29권1호
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• pp.55-58
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• 2019

Development of stable rCHO cell lines is still time consuming and labor intensive, although it is a critical step in the commercial development of recombinant antibodies. The current work demonstrates, for the first time, that electroporation of CHO cells with DMSO can enhance stable expression of recombinant antibodies in rCHO cells. Electroporation with DMSO resulted in an average 3.7-fold and 2.8-fold increases in expression levels of aflibercept and pembrolizumab, respectively, in pools of stable rCHO cells. It also resulted in an average of 2.2-fold and 2.6-fold increases in the expression of aflibercept and pembrolizumab, respectively, in single-cell derived rCHO clones. Simple batch cultures of rCHO cell clones with the highest expression produced 1.0 g/l for aflibercept and 1.4 g/l for pembrolizumab without a time-consuming gene amplification process. Electroporation with DMSO also shortened the development of rCHO cell lines to 2-3 months, allowing rapid establishment of stable rCHO cell lines with a desirable expression level antibodies.

• 한국가축번식학회지
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• 제27권3호
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• pp.259-268
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• 2003

모유에 존재하는 유지방구의 막을 구성하는 주된 당단백질인 하나인 lactadherin(과거에는 BA46로 일컬어짐)은 rotavirus에 의한 감염증상을 예방하는 것으로 보고되고 있다. 본 연구에서는 retrovirus vector system을 이용하여 Chinese Hamster Ovary (CHO) 세포에 tetracycline에 의해 발현이 제어되는 promoter 하의 lactadherin 유전자를 전이 시킨 후 lactadherin이 tetracycline에 의해 발현이 유도되는지의 여부를 실험하였다. 먼저 기초 실험으로 대장균의 LacZ 유전자를 이용하여 tetracycline에 의한 유도 여부를 시험하였다. RevTet-On과 RevTRE-LacZ retrovirus를 동시감염시킨 NIH3T3는 doxycycline (tetracycline 유도체)에 의해 투여량에 비례하여 반응정도가 증가하는 양상을 나타내었으며 최대의 반응은 doxycycline 농도가 1 $\mu\textrm{g}$/ml 이상에서부터 관찰되었다. 이 예비실험의 결과를 바탕으로 RevTet-On과 RevTRE-Ltd retrovirus vectyor를 이용하여 사람의 lactadherin 유전자의 유도적 발현을 검정하였는데 CHO 세포에서 lactadherin 유전자의 유도적 발현을 RT-PCR 기법을 이용하여 확인하였다. 표적세포 내에서 외부에서 도입된 유전자가 지속적으로 발현될 경우 심각한 생리적 부작용을 야기시킨다는 사실을 감안할 때 본 실험의 결과는 유전자 치료와 형질전환동물의 생산에 크게 도움이 될 것으로 예상된다.