• Title/Summary/Keyword: CHO cell

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Real-Time PCR for Validation of Minute Virus of Mice Safety during the Manufacture of Mammalian Cell Culture-Derived Biopharmaceuticals (세포배양 유래 생물의약품 생산 공정에서 Minute Virus of Mice 안전성 검증을 위한 Real-Time PCR)

  • Lee, Dong-Hyuck;Cho, Hang-Mee;Kim, Hyun-Mi;Lee, Jung-Suk;Kim, In-Seop
    • Microbiology and Biotechnology Letters
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    • v.36 no.1
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    • pp.12-20
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    • 2008
  • Validation of viral safety is essential in ensuring the safety of mammalian cell culture-derived biopharmaceuticals, because numerous adventitious viruses have been contaminated during the manufacture of the products. Mammalian cells are highly susceptible to minute virus of mice(MVM), and there are several reports of MVM contamination during the manufacture of biopharmaceuticals. In order to establish the validation system for the MVM safety, a real-time PCR method was developed for quantitative detection of MVM in cell lines, raw materials, manufacturing processes, and final products as well as MVM clearance validation. Specific primers for amplification of MVM DNA was selected, and MVM DNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be $6{\times}10^{-2}TCID_{50}/mL$. The real-time PCR method was proven to be reproducible and very specific to MVM. The established real-time PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with MVM. MVM DNA could be Quantified in CHO cell as well as culture supernatant. When the real-time PCR assay was applied to the validation of virus removal during a virus filtration process, the result was similar to that of virus infectivity assay. Therefore, it was concluded that this rapid, specific, sensitive, and robust assay could replace infectivity assay for detection and clearance validation of MVM.

Zinc and Selenium Requirements for Glutathione Peroxidase Activity and Cell Survival in Chinese Hamster Ovary Cells Overexpressing Metallothionein

  • Kwun, In-Sook;John R. Arthur;John H. Beattie
    • Preventive Nutrition and Food Science
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    • v.8 no.1
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    • pp.36-39
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    • 2003
  • Many defined cell culture media were formulated over 3() years ago and may be deficient in certain micronutrients whose essentiality has only subsequently been recognised. The objective of this study was to evaluate whether alpha-minimal essential medium (MEM) supplemented with 10% foetal bovine serum contained sufficient selenium for optimal activity of the selenium containing enzymes cytosolic glutathione peroxidase (cGPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in cultured Chinese hamster ovary (CHO) cells. Additionally, the effect of zinc deficiency and metallothionein (MT) overexpression on cGPx and PHGPx activity was studied. The addition of 100 nM of selenous acid to the culture medium increased cGPx expression by 10-fold and PHGPx by about 2-fold in both wild-type CHO-K1 cells and CHO-K1 cells overexpressing mouse MT-1. Zinc deficiency had no significant effect on enzyme activity, but cells overexpressing mouse MT-1 had higher levels of cGPx activity. Zinc deficiency decreased cell survival but overexpression of MT-1 was partially protective, probably because its presence in quantity favoured the uptake, sequestration and cellular retention of any remaining zinc. This study demonstrates that selenium in complete alpha-MEM is insufficient for optimal cGPx and PHGPx activity and may compromise the cellular response to oxidative stress.

Btg-1 Induction by Oxidative Stress

  • Cho, Il-Je;Lee, Song-Jin;Kim, Sang-Geon
    • Proceedings of the PSK Conference
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    • 2003.04a
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    • pp.151.1-151.1
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    • 2003
  • B cell translocation gene-1 (Btg-1), originally discovered from chromosomal translocation in chronic B-cell lymphocytic leukemia. belongs to the APRO family. Btg-1 exhibit antiproliferative function. being expressed during the $G_{0}/G_{1}$ transition phase of cell cycle. Btg-1 is fully expressed in quiescent and differentiated cells. while the protein expression decreases as the cell progresses through the cell cycle. (omitted)

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Expression and Purification of Recombinant Human Angiopoictin-2 and Its Analog in Chinese Hamster Ovary Cells

  • Hwang, Su-Jeong;Kim, In-Jun;Go, Gyu-Yeong;Lee, Gyun-Min
    • 한국생물공학회:학술대회논문집
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    • 2001.11a
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    • pp.459-462
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    • 2001
  • Angiopoietin-2 (Ang2) is a naturally occurring antagonist for angiopoietin-l (Angl) and its Tie2 receptor during vasculogenesis, Although angiopoietins have been expressed in several mammalian cell lines, their expression levels are low. Recombinant Chinese hamster ovary (CHO) cell lines expressing a high level of human Ang2 or its analog, human $Ang2_{443}$, with an amino-terminal FLAG-tag were constructed by transfecting the expression vectors into dhfr-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate level. Secreted Ang2 or human $Ang2_{443}$ were purified from the cultured medium using an anti-FLAG- agarose affinity chromatography, The purified Ang2 and $Ang2_{443}$ migrated on SOS-PAGE as a broad band, characteristic of glycosylated protein. Their biological activity in vitro was demonstrated in a serum deprivation-induced apoptosis assay. Ang2 at high concentration, like AngI, can be an apoptosis survival factor for endothelial cells through the activation of the Tie2 receptor.

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Effect of Carcinogenic Chromium(VI) on Cell Death and Cell Cycle in Chinese Hamster Ovary Cells

  • Lee, San-Han;Nam, Hae-Seon;Kim, Sung-Ho
    • Environmental Mutagens and Carcinogens
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    • v.24 no.3
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    • pp.113-120
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    • 2004
  • Chromium compounds are known human and animal carcinogens. In this study, the effects of sodium chromate on apoptosis and cell cycle were investigated in order to unveil the elements of early cellular responses to the metal. Using Chinese hamster ovary cells(CHO-K1-BH4), we found taht chromium (VI) treatment induced apoptosis in these cells, as signified by nuclear fragmentation, DNA laddering on agarose gel electrophoresis, and an increased proportionof cells with hypodiploid DNA. Preceding these changes, chromium (VI) treatment increased caspase 3 pritease activity and also increased expression of p53 protein, while the level of bcl2 protein was not changed. Coincubation with caspase inhibitor, Z-DEVD-FMK, inhibited chromium-induced apoptosis. In the flow cytometric analysis using propidium iodide fluorescence, an increase of cell population in G2/M phase was shown in cells exposed to at least 160 $\mu\textrm{m}$ of sodium chromate for 72h, form 9.8% for 0$\mu\textrm{m}$ chromium (VI) to 26.4% for 320$\mu\textrm{m}$ chromium(VI). Taken together, these findings suggest that chromium(VI)-induced apoptosis is accompanied by G2/M cell cycle arrest, and that p53-mediated pathway may be involved in positive regulation of G2/M arrest and a concurred apoptosis in CHO cells.

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The Effect of Barrier Layer on Thin-film Silicon Solar Cell Using Graphite Substrates (탄소 기판을 이용한 박막 실리콘 태양전지의 배리어 층 효과)

  • Cho, Young Joon;Lee, Dong Won;Cho, Jun Sik;Chang, Hyo Sik
    • Journal of the Korean Institute of Electrical and Electronic Material Engineers
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    • v.29 no.8
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    • pp.505-509
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    • 2016
  • We have investigated the characteristics of amorphous silicon (a-Si) thin-film solar cell by inserting barrier layer. The conversion efficiency of a-Si thin-film solar cells on graphite substrate shows nearly zero because of the surface roughness of the graphite substrate. To enhance the performance of solar cells, the surface morphology of the back side were modified by changing the barrier layer on graphite. The surface roughness of graphite substrate with the barrier layer grown by plasma enhanced chemical vapor deposition (PECVD) reduced from ~2 um to ~75 nm. In this study, the combination of the barrier layer on graphite substrate is important to increase solar cell efficiency. We achieved ~ 7.8% cell efficiency for an a-Si thin-film solar cell on graphite substrate with SiNx/SiOx stack barrier layer.