http://chineseinput.net/에서 pinyin(병음)방식으로 중국어를 변환할 수 있습니다.
변환된 중국어를 복사하여 사용하시면 됩니다.
Characterization of the KG1a Cell Line for Use in a Cell Migration Based Screening Assay
KarlFrancis,이균민,BernhardO.Palsson 한국생물공학회 2002 Biotechnology and Bioprocess Engineering Vol.7 No.3
for potentially therapeutic compounds. Further screening of these lead compounds is typicaly done with secondary assays which may utilize living, functioning cels as screening tools. A prob-lem (or benefit) with these cell-based assays is that living cells are very sensitive to their environ-ment. We have been interested in the process of stem cel migration and how it relates to the cel-lular therapy of bone marrow transplantation. In this study we describe a secondary, cel-based as-say for screening the effects of various in-vitro conditions on Imature Hematopoietic Cell (IHC) or the effect of a cultures growth phase, that need to be accounted for in a screning protocol. Fi-nally, we show that exponentially growing KG1a cells (a human IHC cell line) were 10 times more motile than those in the lag or stationary phases. These data strongly sugest that KG1a cels secrete a chemokinetic factor during the exponential growth phase of a culture.
김민수,김원희,이균민 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.4
The establishment of erythropoietin (EPO) producing Chinese hamster ovary (CHO) cell lines was conducted using Cre-mediated cassette exchange. The characterization of site-specific recombination mediated by Cre-recombinase during the cell line development was also performed. A total of six parental clones, which had various green fluorescence levels ranging from high to low and containing a single copy of insertion vector (pEGFP-m2), were screened. The EPO targeting vector (pIC-m2-EPO) was targeted into the 6 parental clones by Cre-mediated cassette exchange. Correctly targeted clones were obtained from 4 out of 6 parental clones with 0~15% of targeting efficiencies. Moreover, there was a positive relationship (R² = 0.87) between fluorescence levels of the parental clones before Cre-mediated cassette exchange and specific EPO productivities (qEPO) of the correctly targeted clones after Cre-mediated cassette exchange. Therefore, it was verified that the chromosomal loci’s characteristic gene expression level was not modified even after cassette exchange mediated by Cre recombinase during the development of EPO producing CHO cell lines. This finding implies that the reproducible development of CHO cell lines largely producing a desired protein is expected to be achieved by Cre-mediated cassette exchange.
A Role of GADD153 in ER Stress-induced Apoptosis in Recombinant Chinese Hamster Ovary Cells
Chaya Mohan,Madhavi Sathyamurthy,이균민 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.3
The imbalance between the folding capacity and the folding demand imposed on the endoplasmic reticulum (ER) of therapeutic protein-producing host cells results in a stressed ER. This initiates a series of cellular signaling events termed the unfolded protein response (UPR) aimed at restoring homeostasis. In order to alleviate ER stress and ER stress-induced apoptosis in recombinant Chinese hamster ovary (rCHO) cells, silencing of the growth arrest and DNA damage 153 gene (GADD153), the main pro-apoptotic factor of UPR, was attempted. The rCHO cells were cultured under four ER stress inducing conditions, including thapsigargin, brefeldin A, glucose deprivation, glucose and glutamine deprivation. In these conditions, the functions of stably GADD153-silenced clones were investigated. It was found that under exclusive ER stress-inducing conditions of thapsigargin and brefeldin A treatments, the GADD153-silenced clones showed a less incidence of apoptosis (about 38%) and less cell viability (about 58% non-viable cells) than the control cells. However, under nutrient deprivation, the beneficial effect of GADD153 silencing was not pronounced because nutrient deprivation led to a cascade of various events including GADD153-induced cell death. GADD153-overexpressing pool cells also substantiated the findings of GADD153 downregulation. Investigation of the underlying mechanism revealed that increased GADD153 expression results in an exaggerated production of reactive oxygen species (ROS) and that GADD153 silencing promotes translational attenuation facilitating cell recovery from stress. Taken together, this study suggests that GADD153 sensitizes cells to ER stress through mechanisms that involve enhanced oxidative injury and by manipulating the ER client protein load in rCHO cells. The imbalance between the folding capacity and the folding demand imposed on the endoplasmic reticulum (ER) of therapeutic protein-producing host cells results in a stressed ER. This initiates a series of cellular signaling events termed the unfolded protein response (UPR) aimed at restoring homeostasis. In order to alleviate ER stress and ER stress-induced apoptosis in recombinant Chinese hamster ovary (rCHO) cells, silencing of the growth arrest and DNA damage 153 gene (GADD153), the main pro-apoptotic factor of UPR, was attempted. The rCHO cells were cultured under four ER stress inducing conditions, including thapsigargin, brefeldin A, glucose deprivation, glucose and glutamine deprivation. In these conditions, the functions of stably GADD153-silenced clones were investigated. It was found that under exclusive ER stress-inducing conditions of thapsigargin and brefeldin A treatments, the GADD153-silenced clones showed a less incidence of apoptosis (about 38%) and less cell viability (about 58% non-viable cells) than the control cells. However, under nutrient deprivation, the beneficial effect of GADD153 silencing was not pronounced because nutrient deprivation led to a cascade of various events including GADD153-induced cell death. GADD153-overexpressing pool cells also substantiated the findings of GADD153 downregulation. Investigation of the underlying mechanism revealed that increased GADD153 expression results in an exaggerated production of reactive oxygen species (ROS) and that GADD153 silencing promotes translational attenuation facilitating cell recovery from stress. Taken together, this study suggests that GADD153 sensitizes cells to ER stress through mechanisms that involve enhanced oxidative injury and by manipulating the ER client protein load in rCHO cells.
Heparan Sulfate Proteoglycan Synthesis in CHO DG44 and HEK293 Cells
이소정,김미겸,김누리,허원도,이균민 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.3
Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells are the most popular host cells for transient gene expression (TGE) of therapeutic proteins. These host cells require high transfection efficiency in order to enhance TGE. Heparan sulfate proteoglycan (HSPG) at the cell surface is known to regulate endocytosis for gene delivery. The HSPG expression in CHO DG44 and HEK293E cells was investigated in an effort to enhance the TGE. Immunostaining of HSPGs followed by confocal microscopy and flow cytometry analyses revealed that CHO DG44 cells possessed a higher amount of cellsurface and intracellular HSPGs than HEK293E cells. The mRNA levels of the representative enzymes involved in the HSPG biosynthesis in CHO DG44, which were determined by quantitative real time PCR, were quite different from those in HEK293E cells. Taken together, the results obtained here would be useful in improving TGE in CHO DG44 and HEK293E cells through genetic engineering of HSPG synthesis.
Won Hee Kim,Yeon Jung Kim,이균민 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.3
Gadd45 is a p53-regulated protein and isinvolved in cell cycle arrest in the G2/M phase. In an effortto improve transient gene expression (TGE) in Chinesehamster ovary (CHO) cells, the effect of Gadd45-inducedcell cycle arrest on TGE in CHO cells was investigatedusing the two different expression vectors encoding Fcfusionprotein and recombinant antibody. To regulate theexpression of Gadd45 in CHO cells, the CHO-TRExgadd45cell line was established using the T-REx systemcontrolled by doxycycline. During the cultures for TGE,Gadd45 overexpression severely inhibited cell growth, butsignificantly enhanced TGE. Compared with the culturewithout Gadd45 overexpression, the TGE of Fc fusionprotein and humanized antibody were increased by 111 and93%, respectively. The enhanced TGE, despite the cellgrowth arrest induced by Gadd45 overexpression, was dueto the significantly increased specific productivity, resultingfrom enhanced transfection efficiency, increased cell size,and active DNA demethylation. Taken together, the dataobtained here demonstrate that Gadd45-induced cell cyclearrest in G2/M phase can significantly enhance TGE inCHO cells.