• Journal of Life Science
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• v.20 no.11
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• pp.1577-1581
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• 2010

Granulocyte colony stimulating factor (G-CSF) is a hematopoietic cytokine that stimulates bone marrow cells to proliferate and differentiate into granulocytes. G-CSF is approved and used for therapeutic purposes. The endoplasmic reticulum (ER) signal peptide of hG-CSF was replaced with silkworm-specific signal peptides to express and efficiently secrete recombinant hG-CSF by silkworm cells. Plasmids that contain cDNAs for hG-CSF and hG-CSF fused with silkworm- specific signal peptides of prophenoloxidase activating enzyme (PPAE), protein disulfide isomerase (PDI), and bombyxin (BX) were constructed. The G-CSF protein was expressed in insect cell line BM5 and was detected by western blot analysis. The cells transfected with plasmids containing rhG-CSF genes with silkworm-specific signal sequences released mature rhG-CSF protein more efficiently than the cells transfected with pG-CSF, the plasmid containing human G-CSF gene, including its own signal sequence. The production of hG-CSF reached maximal level at four days post-transfection and remained at a high level until 7 days post-transfection. These data demonstrate that the modification of the human G-CSF mimic to insect proteins synthesized in ER greatly improves the production of the protein.

• Development and Reproduction
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• v.13 no.3
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• pp.191-198
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• 2009

In an attempt to simultaneously produce two human proteins, hGH and hG-CSF, in the milk of transgenic mice, we constructed goat $\beta$-casein-directed hGH and hG-CSF expression cassettes individually and generated transgenic mice by co-injecting them into mouse zygotes. Out of 33 transgenic mice, 29 were identified as double transgenic harboring both transgenes on their genome. All analyzed double transgenic females secreted both hGH and hG-CSF in their milks. Concentrations ranged from 2.1 to $12.4\;mg/m{\ell}$ for hGH and from 0.04 to $0.13\;mg/m{\ell}$ for hG-CSF. hG-CSF level was much lower than hGH level but very similar to that of single hG-CSF mice, which were introduced with hG-CSF cassette alone. In order to address the causes of concentration difference between hGH and hG-CSF in milk, we examined mRNA level of hGH and hG-CSF in the mammary glands of double transgenic mice and tissue specificity of hG-CSF mRNA expression in both double and single transgenic mice. Likewise protein levels in milk, hGH mRNA level was much higher than hG-CSF mRNA, and hG-CSF mRNA expression was definitely specific to the mammary glands of both double and single transgenic mice. These results demonstrated that two transgenes have distinct transcriptional potentials without interaction each other in double transgenic mice although two transgenes co-integrated into same genomic sites and their expressions were directed by the same goat $\beta$-casein promoter. Therefore goat $\beta$-casein promoter is very useful for the multiple production of human proteins in the milk of transgenic animals.

• KSBB Journal
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• v.16 no.4
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• pp.376-380
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• 2001

The human granulocyte-macrophage colony-stimulating factor (hGM-CSF) was produced from cell suspension culture of transgenic tobacco which was transformed by using Agrobacterium harboring the hGM-CSF gene. To improve the production of hGM-CSF in batch culture system, the effects of initial sucrose concentration and inoculum size were investigated. The results show that the hGM-CSF production was not affected by small inoculum size in medium containing either low or high concentration of sucrose. However, the production of hGM-CSF was increased under increasing of the inoculum sizes and sucrose concentration. Under the combination of inoculum and sucrose concentration, the maximum hGM-CSF production of 720 $\mu$g/L was obtained at 90 g/L of initial sucrose concentration and 110 g/L of inoculum size.

• Journal of Life Science
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• v.18 no.7
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• pp.912-917
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• 2008

To investigate the biological activity of recombinant human granulocyte colony-stimulating factor (rec-hG-CSF) in mammalian cells, hG-CSF gene was cloned using the eDNA extracted from the human squamous carcinoma cell lines and rec-hG-CSF was produced in CHO cell lines. To analyze the biological activity in vivo, the rec-hG-CSF protein was injected into mice subcutaneously on days 0 and 2. Blood was withdrawn for white blood cell (WBC) determination 5 days after the first injection. WBC values were found to have increased significantly. A pEGFP-mUII-hG-CSF vector was transfected into somatic cell lines isolated from bovine fetal cells. The colony expressing EGFP signals was observed with a confocal microscope. These data suggest that the rec-hG-CSF produced in this study has potent activity in vivo. Thus, the results of this biological activity show that rec-hG-CSF can be enhanced considerably by genetic engineering that affects potential activity, including mutations, which add the oligosaccharide chain and construct double-fusion proteins. A pEGFP-mUII-hG-CSF vector can be utilized for the production of cloned transgenic livestock.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.153-159
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• 2007

In this study, using FIV-based lentivirus vector system, we tried to express hG-CSF in tetracycline-controllable manner. hG-CSF influences the proliferation, differentiation, and survival of cells in the neutrophil lineage. To enhance stability and translation of hG-CSF transcript, WPRE sequence was also introduced into FIV-Tet-On vector at downstream region of either the hG-CSF gene or the sequence encoding rtTA. Primary culture cells (CEF, chicken embryonic fibroblast; PFF, procine fetal fibroblast) infected with the recombinant FIV were cultured in the medium supplemented with or without doxycycline for 48 hours, and induction efficiency was measured by comparing the hG-CSF gene expression level using quantitative real-time PCR, Western blot and ELISA. Higher hG-CSF expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hG-CSF (in CEF) or rtTA (in PEE) gene. This FIV-Tet-On vector system may be helpful in solving serious physiological disturbance problems which has continuously hampered successful production of transgenic animals and gene therapy.

• Journal of Plant Biotechnology
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• v.31 no.2
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• pp.163-167
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• 2004

The effects of sucrose concentration on the secretion of hGM-CSF, total protein and protease into the medium were investigated in transgenic tobacco cells. The dry cell weight (11.22 g/L), hGM-CSF (181.53 $\mu\textrm{g}$/L) and total protein (66.8 mg/L) were detected as highest at 30 g/L sucrose and protease activity (2660 U/L) was highest at 120 g/L sucrose after 5-day culture. However after 10-day culture, the maximum dry cell weight (28.36 g/L) was found at 60 g/L sucrose while the maximum hGM-CSF (95 $\mu\textrm{g}$/L) was appeared at 150 g/L sucrose. The total protein and protease activity was secreted as 52.28mg/L and 3430 U/L, respectively in the same culture.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.321-326
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• 2000

To examine the feasibility of the long-term production of the human granulocyte colony stimulating factor (hG-CSF) using the $_{L}-arabinose$ promoter system of Escherichia coli, flask relay culture and cyclic fed-batch culture were performed. In the flask relay culture, it was found that the pismid was maintained stably up to about 170 generations in an uninduced condition, whereby the cells could also maintain the capability of expressing hG-CSF expression were maintained stably up to at least 100 generations. In contrast, in the cyclid fed-batch culture, segregational plasmid instability was observed within about 4 generations after induction, even though the cell growth and hG-CSF production reached their maximum balues, 78.0 g/l of dry cell weight and 7.0 g/l of hG-CSF, respectively. It would appear that, when compared to the flask relay culture, the high-cell density and high-level expression of hG-CSF in the cyclic fed-batch cultrure led to the segregational plasmid instability; in other words, a severe metabolic burden existe on the cells due to the high-level expression of hG-CSF. Accordingly, based on these long-term cultures, the segregational and structural plasmid instability was observed and a strategy to overcome such problems could be designed.

• Microbiology and Biotechnology Letters
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• v.32 no.2
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• pp.135-141
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• 2004

Human granulocyte colony-stimulating factor (hG-CSF) is a hematopoiesis agent that principally affects the differentiation of neutrophils in the bone marrow. At present, recombinant hG-CSF is used successfully in the treatment of chemotheraphy-induced neutropenia and its indication has been expanded to bone marrow transplantation and aplastic anemia. In this study, we have constructed rhG-CSF secretion plasmid pYRC1 in which OmpA signal sequence/hG-CSF gene was expressed under the control of the T7 promoter. rhG-CSF produced in E. coli BL21 (pYRC1) grown at $37{\circ}C$ was found in aggregates. However, 15% of the periplasmic protein was soluble rhG-CSF when the E. coli BL21 (pYRC1) was cultured at $25^{\circ}C$ for 7 h in the modified MBL medium containing 10 g/$\ell$ glucose with 10 $\mu$M IPTG induction. The production of soluble rhG-CSF in E. coli BL21 (pYRC1) using fed batch culture was also studied. In the fed batch culture system, the final yield of rhG-CSF produced from E. coli BL21 (pYRC1) was increased from 4.4 mg/$\ell$to 24 mg/$\ell$by controlling the specific growth rate from $0.43 h^{-1}$ to $0.14 h^{-1}$, and optimizing the time of induction.

• Development and Reproduction
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• v.14 no.1
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• pp.13-19
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• 2010

We developed a novel dicistronic system for the expression of target cDNA sequences in the milk of transgenic animals using goat beta-casein/hGH fusion construct, pGbc5.5hGH (Lee, 2006) and internal ribosome entry site (IRES) sequences of encephalomyocarditis virus (EMCV). Granulocyte colony-stimulating factor (hG-CSF) cDNA was linked to 3' untranslated region of hGH gene in the pGbc5.5hGH via EMCV IRES sequences. Transgenic mice were generated by microinjection and transgene expression was examined in the milk and mammary gland of transgenic mice at 10 days of lactation. Northern blot analysis showed that hGH gene and hG-CSF cDNA were transcribed as a single dicistronic mRNA. The hG-CSF and hGH proteins were independently translated from the dicistronic mRNA and secreted into the milk of transgenic mice. The highest concentration of hG-CSF and hGH in the milk of transgenic mice were $237{\mu}g/m{\ell}$ and $8,990{\mu}g/m{\ell}$, respectively. In contrast, another hG-CSF expression cassette, in which hG-CSF genomic sequences were inserted into a commercial milk-specific expression vector (pBC1), generated a lower level ($91{\mu}g/m{\ell}$) of hG-CSF expression in the milk of transgenic mice. These results demonstrated that the novel pGbc5.5hGH-based dicistronic construct could be useful for an efficient cDNA expression in the milk of transgenic animals.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.341-345
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• 2003

Effect of immobilization on the production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) by Nicotiana tabacum cells was investigated using polyurethane foam as immobilization matrices. The cell activity and the hGM-CSF production were maintained for 16 days in spite of 3 times of media exchange. Under the same conditions of temperature and agitation rate, maximum concentrations of hGM-CSF in a 500-mL spinner flask and 100-mL Erleuneyer flasks were 17.3 ${\mu}g/L$ and 9.8 ${\mu}g/L$, respectively. Consequently high hGM-CSF production could be possible in spinner flask when the rate and amount of media exchange were optimized.