• Journal of Microbiology and Biotechnology
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• v.23 no.8
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• pp.1176-1184
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• 2013

Anti-hepatocyte growth factor (anti-HGF) monoclonal antibodies (mAbs) are potential therapeutics against various cancers. Screening for high-producer clones is a time-consuming and complex process and is a major hurdle in the development of therapeutic mAbs. Here, we describe an efficient approach that allows the selection of high-producer Chinese hamster ovary (CHO) cell lines producing the novel anti-HGF mAb SFN68, which was generated previously by immunizing HGF bound to its receptor c-Met. We selected an SFN68-producing parental cell line via transfection of the dihydrofolate reductase-deficient CHO cell line DG44, which was preadapted to serum-free suspension culture, with an SFN68-expression vector. Subsequent gene amplification via multiple passages of the parental cell line in a methotrexate-containing medium over 4 weeks, followed by clonal isolation, enabled us to isolate two cell lines, 2F7 and 2H4, with 3-fold higher specific productivity. We also screened 72 different media formulated with diverse feed and basal media to develop a suboptimized medium. In the established suboptimized medium, the highest anti-HGF mAb yields of the 2F7 and 2H4 clones were 842 and 861 mg/l, respectively, which were about 10.5-fold higher than that of the parental cell line in a non-optimized basal medium. The selected CHO cell lines secreting high titers of SFN68 would be useful for the production of sufficient amounts of antibodies for efficacy evaluation in preclinical and early clinical studies.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.6
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• pp.479-499
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• 2022

The lack of a clonal renin-secreting cell line has greatly hindered the investigation of the regulatory mechanisms of renin secretion at the cellular, biochemical, and molecular levels. In the present study, we investigated whether it was possible to induce phenotypic switching of the renin-expressing clonal cell line As4.1 from constitutive inactive renin secretion to regulated active renin secretion. When grown to postconfluence for at least two days in media containing fetal bovine serum or insulin-like growth factor-1, the formation of cell-cell contacts via N-cadherin triggered downstream cellular signaling cascades and activated smooth muscle-specific genes, culminating in phenotypic switching to a regulated active renin secretion phenotype, including responding to the key stimuli of active renin secretion. With the use of phenotype-switched As4.1 cells, we provide the first evidence that active renin secretion via exocytosis is regulated by phosphorylation/dephosphorylation of the 20 kDa myosin light chain. The molecular mechanism of phenotypic switching in As4.1 cells described here could serve as a working model for full phenotypic modulation of other secretory cell lines with incomplete phenotypes.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.347-352
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• 1999

This cDNAs were cloned with the aid of the polymerase chain reaction (PCR) by sequences based on cloned rat LH/CG and FSH receptor cDNAs. A cDNAs of LHR and FSHR were transfected into the 293 cells. Several clonal cell lines were obtained expressing different numbers of cell surface receptors. One cell lines for each LHR and FSHR were chosen, and a corresponding cell lines expressing the wild type LHR and FSHR were selected based on the number of cell surface receptor for the particular LHR and FSHR. The abilities of the LHR and FSHR to transduce the hCG and FSH signals were measured by quantitating cAMP accumulation in cells incubated with increasing concentrations of hCG and FSH. The cAMP accumulation effects for these receptors were increased by the increasing concentrations of hCG and FSH. Thus, most of the receptors expressed in cells transfected with LHR and FSHR could be detected by measuring hormone binding and cAMP response, and can utilize to study the structure function and signal transduction of the choriogonadotropins and glycoprotein hormones.

• The Korean Journal of Zoology
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• v.17 no.1
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• pp.37-42
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• 1974

Comparative LDH isozyme studies on normal, malignant and virus-transformed derivatives of Chinese and Armenian hamsters were illustrated. With respect to LDH isozyme patterns, there were good indications that epithelial-like cells have varying levels of LDH-1-2-3 and -4, in addition to the "standard" LDH-5 band, which was exhibited by the majority of fibroblast-like cell lines.ell lines.

• Korean Journal of Plant Tissue Culture
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• v.24 no.4
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• pp.239-256
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• 1997

Plants belonging to the category of 2n apomixis or agamospermy form embryos and seeds without the processes of normal meiosis and syngamy. Seeds produced in this way have identical genotype of their maternal parent. Three different types of agamospermy are recognized: diplospory, apospory, and adventitious (adventive) embryony. $F_1$ hybrid cultivars cannot be used as seed sources in the next ($F_2$) generation because this generation would be extremely variable as a result of genetic segregation. Hybrid vigor is also reduced in the $F_2$ generation. Therefore, parental stocks for hybrid seed production need to be maintained and cross must be continuously repeated. Agamospermic 2n apomixis would make it possible to fix the genotype of a superior variety so that clonal seeds faithfully representing that genotype could be continuously and cheaply produced independent of pollination. That is, $F_1$ hybrid seeds could be produced for many generations without loss of vigor or genotype alteration. Production of apomictic $F_1$ hybrid seed would be simplified because line isolation would not be necessary to produce seed or to maintain parental lines, and the use of male-sterile lines could be avoided. Overall, apomixis would enable a significant reduction in hybrid seed production costs. Additionally, the production of clonal seed is not only important for seed propagated crops, but also for the propagation of heterozygous fruit trees and timbers. Clonal seed would help avoid costly and time-consuming vegetative propagating methods that are currently used to ensure the large-scale production of these plants. Apomixis is scattered throughout the plant kingdom, but few important agricultural crops possess this trait Therefore, most research to date has centered on introgressing the trait of apomixis into agricultural crops such as wheat, maize, and some forage grasses from wild distant relatives by traditional cross breeding. The classical breeding approach, however is slow and often impeded by many breeding barriers. These problems could be surmounted by taking mutagenesis or molecular approach. Arabidopsis thaliana is a tiny sexually reproducing plant and is convenient in constructing and screening in molecular researches. Male-sterile mutants of Arabidopsis are particularly suitable genetic background for mutagenesis and screening for apomictic mutants. Molecular approaches towards isolating the genes controlling the apomictic process are feasible. Direct isolation of genes conferring apomixis development would greatly facilitate the transfer of this trait to wide variety of crops. Such studies are now in progress.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.759-766
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• 2003

Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level of human thrombopoietin (hTPO) or its analog, TPO33r, were obtained by transfecting expression vectors into dihydrofolate reductase-deficient (dhfr) CHO cells and subsequent gene amplification in media containing stepwise increments in methotrexate (MTX) level such as 20, 80, and 320 nM. The parental clones with a hTPO expression level $>0.40\;{\mu}g/ml$ (27 out of 1,200 clones) and the parental clones with a TPO33r expression level $>0.20\;{\mu}g/ml$ (36 out of 400 clones) were subjected to 20 nM MTX. The clones that displayed an increased expression level at 20 nM MTX were subjected to stepwise increasing levels of MTX such as 80 and 320 nM. When subjected to 320 nM MTX, most clones did not display an increased expression level, since the detrimental effect of gene amplification on growth reduction outweighed its beneficial effect of specific TPO productivity ($q_{TPO}$) enhancement at 320 nM MTX. Accordingly, the highest producer subclones ($1-434-80^{*}$ for hTPO and $2-3-80^{*}$ for TPO33r), whose $q_{TPO}$ was 2- to 3-fold higher than that of their parental clones selected at 80 nM MTX, were isolated by limiting dilution method and were established as rCHO cel1 lines. The $q_{TPO}$ of $1-434-80^{*}\;and\;2-3-80^{*}\;was\;5.89{\pm}074\;and\;1.02{\pm}0.23\;{\mu}g/10^6$ cells/day, respectively. Southern and Northern blot analyses showed that the enhanced $q_{TPO}$ of established rCHO cell lines resulted mainly from the increased TPO gene copy number and subsequent increased TPO mRNA level. The hTPO and TPO33r produced from the established rCHO cell lines were biologically active in vivo, as demonstrated by their ability to elevate platelet counts in treated mice.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.5
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• pp.332-336
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• 2001

When parental Chinese hamster ovary (CHO) cell clones that are capable of producing thrombopoietin (TPO) were subjected to high methotrexate (MTX) concentrations, clonal variations in cell growth were apparent. In the clones that had no significant enhancement in specific TPO productivity (q$\_$Tpo/)when a higher level of MTX was administered, their growth was not depressed significantly nor their cell size changed significantly. On the other hand, those clones that showed a significant-enhancement in q$\_$Tpo/ at higher a MTX dosage, cell growth was depressed initially but recovered during successive sub-cultures. Furthermore, their cell size increased, which suggested that changes in cell size may be indicative of an enhanced q$\_$Tpo/. When the enhancement of the q$\_$Tpo/ of 9 clones after a high MTX dosage was plotted against the extent of the increase of their size, there was a linear correlation (γ$^2$=0.80, p<0.001, ANOVA), which suggested that an enhancement of q$\_$Tpo/ after high MTX administration can be measured by the increase in their cell size. Taken together, our data demonstrate that the selection of amplified CHO cell clones with enhanced q$\_$Tpo/ can be done upon their increased size and growth pattern. This facilitates the development of highly productive recombinant CHO cell lines.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.6
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• pp.507-513
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• 2000

ErbB3/HER3 is a cell surface receptor which belongs to the ErbB/HER subfamily of receptor protein tyrosine kinases. When expressed in NIH/3T3 cells, ErbB3 can form heterodimeric coreceptor with endogenous ErbB2. Among known intracellular effectors of the ErbB2/ErbB3 are mitogen-activated protein kinase (MAPK) and phosphoinositide (PI) 3-kinase. In the present study, we studied relative contributions of above two distinct signaling pathways to the heregulin-induced mitogenic response via activated ErbB3. For this, clonal NIH-3T3 cell lines expressing wild-type ErbB3 and ErbB3 mutants were stimulated with $heregulin{\beta}_1$. While cyclin D1 level was markedly high and further increased by treatment of heregulin in cells expressing wild-type ErbB3, the elimination of either Shc binding or PI 3-kinase binding lowered both levels. This result was supported by the reduction of cyclin $D_1$ expression by preteatment with MAPK kinase inhibitor or PI 3-kinase inhibitor before stimulation with heregulin. In accordance with the cyclin $D_1$ expression, elimination of either Shc binding or PI 3-kinase binding reduced the heregulin-induced DNA synthesis and cell growth rate. Our results obtained by the comparison of wild-type and ErbB3 mutants indicate that the full induction of the cell cycle progression through $G_1/S$ phase by ErbB3 activation is dependent on both Shc/MAPK and PI 3-kinase signal transduction pathways.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.4
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• pp.499-509
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• 2008

Foxo1 plays an important role in the integration of hormone-activated signaling pathways with the complex transcriptional cascade that promotes preadipocyte differentiation of clonal cell lines from rodents. We isolated the full-length cDNA of porcine FoxO1 gene using RACE, confirmed by visual Northern blotting. The deduced amino acids indicated 94% and 90% identities with the corresponding human and mice aa. Analysis of the aa sequence, showed that it included a Forkhead domain (aa 167-247), a transmembrane structure domain (aa 90-113), a LXXLL motif (aa 469-473), and 51 Ser, 8 Thr, and 4 Tyr phosphorylation sites, indicating a potential important role for FoxO1 transcriptional activity in vivo. Using the IMpRH panel, we mapped FoxO1 gene to chromosome 11p13. Our data provide basic molecular information useful for the further investigation on the function of FoxO1 gene. Time-course analysis of FoxO1 expressions indicated that levels of mRNA and protein gradually increased from day 0 to 3, and it reached almost maximal level at day 3, then decreased from day 5 to 7 in porcine primary preadipocyte differentiation. After induction by IGF-1, GPDH activity and accumulation of lipid increased, however, expressions of FoxO1 mRNA and protein were inhibited in a dose dependent manner. These results suggest that FoxO1 takes part in porcine preadipocyte differentiation and expressions of FoxO1 were regulated by IGF-1.