• Title/Summary/Keyword: transgenic suspension culture

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Production and Secretion of Human Interleukin-18 in Transgenic Tobacco Cell Suspension Culture

  • Sharma, Niti;Kim, Tae-Geum;Yang, Moon-Sik
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.11 no.2
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    • pp.154-159
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    • 2006
  • Interleukin-18 (IL-18), otherwise known as interferon-gamma-inducing factor (IGIF), is one of several well characterized and important cytokines that contribute to host defenses. The complementary DNA (cDNA) of mature human interleukin-18 gene (hIL-18) was fused with the signal peptide of the rice amylase 1A gene (Ramy1A) and introduced into the plant expression vector under the control of a duplicated CaMV 35S promoter. The recombinant plasmid was transformed into tobacco (Nicotiana tabacum L. cv Havana) using the Agrobacterium-mediated transformation method. The integration of the hlL-18 gene into the genome of transgenic tobacco plants was confirmed by polymerase chain reaction (PCR) amplification and its expression was observed in the suspension cells that were derived from the transgenic plant callus by using Northern blot analysis. The hlL-18 protein was detected in the extracts of the transgenic callus and in the medium of the transgenic tobacco suspension culture by using immunoblot analysis. Based upon enzyme-linked immunosorbant assay (ELISA) results, the expression level of the hlL-18 protein approximated $166{\mu}g/L$ in the suspension culture medium. Bioassay results from the induction of $interferon-{\gamma}$ from a KG-1 cell line indicated that the hlL-18 secreted into the suspension culture medium was bioactive.

Production of Green Fluorescent Protein (GFP) from Transgenic Rice Cell Suspension Culture (형질전환된 벼세포배양에서 green fluorescent protein (GFP) 생산)

  • Lee, Jae-Hwa
    • Journal of Life Science
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    • v.17 no.2 s.82
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    • pp.293-297
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    • 2007
  • Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. In this study, GFP was produced and secreted from suspension cells derived from transgenic rice. The RAmy3E promoter placed before the GFP gene controlled by sugars such as sucrose. The effects of sucrose concentration on the secretion of GFP and total protein into the medium were investigated in batch suspension culture. It was possible, therefore, to induce the expression of the GFP by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. The dry cell weight (7.06 g/L) and GFP level were detected as highest at 12%, 3% sucrose after 20 day culture, respectively. However secreted GFP fluorescence at the other sucrose concentrations (6%, 12%, 18% and 24%) were a little amount in media.

Production of hGM-CSF from Cell Suspension Culture of Transformed Lettuce Using Agrobacterium-mediated Transformation System (Agrobacterium을 이용한 형질전환 상추의 세포 현탁배양으로부터 hGM-CSF의 생산)

  • Kim, Young-Sook;Kim, Mi-Young;Kwon, Tae-Ho;Yang, Moon-Sik
    • Journal of Plant Biotechnology
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    • v.30 no.1
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    • pp.97-102
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    • 2003
  • Lettuce (Lactuca sativa) was transformed with Agrobacterium tumefacience LBA4404 containing human granulocyte macrophage colony stimulating factor (hGM-CSF) gene to produce in cell suspension cultures. Cell suspension culture was established using callus from transgenic lettuce plant. Integration of hGM-CSF gene into plant chromosome was confirmed through genomic PCR and Southern blot analysis. In addition, Northern blot analysis indicated the expression of the introduced hGM-CSF gene in transformed lettuce. The recombinant hGM-CSF was expressed in transgenic cell cultures derived from transgenic plants as a yield of about 149.0 $\mu\textrm{g}$/L in culture filtrate, which was determined by ELISA. These results demonstrated that transformed lettuce cell suspension cultures could be used as a production system of therapeutic proteins such as hGM-CSF.

Secretory Production of hGM-CSF with a High Specific Biological Activity by Transgenic Plant Cell Suspension Culture

  • Kwon, Tae-Ho;Shin, Young-Mi;Kim, Young-Sook;Jang, Yong-Suk;Yang, Moon-Sik
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.8 no.2
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    • pp.135-141
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    • 2003
  • The human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene was introduced into tobacco plants. The cell suspension culture was established from leaf-derived calli of the transgenic tobacco plants in order to express and secrete a biologically active hGM -CSF. The recombinant hGM-CSF from the transgenic plant cell culture (prhGM-CSF) was identified as a yield of about 180 ${\mu}$g/L in the culture filtrate, as determined by ELISA. The addition of 0.5 g/L polyvinylpyrrolidone (PVP) to the plant cell culture medium both stabilized the secreted prhGM-CSF and increased the level of production approximately 1.5-fold to 270 ${\mu}$g/L. The biological activity of the prhGM-CSF was confirmed by measuring the proliferation of the hGM-CSF-dependent cell line, TF-1. Interestingly, the specific activity of the prhGM-CSF was estimated to be approximately 2.7 times higher than that of a commercially available preparation from E. coli.

북한산 국립공원의 식생군집형에 대하여

  • 송호경;이근복
    • Proceedings of the Botanical Society of Korea Conference
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    • 1985.08b
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    • pp.23-33
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    • 1985
  • Plant cell culture is emerging to express bioactive foreign proteins because it has several advantages in that it is safe, economical, genetically stable and eukaryotic expression system comparing with other expression systems. However several limitations such as slow growth rate, low expression level and lack of well established down stream process need to be answered. As a preliminary approach to produce the immunologically interested molecules through the plant cell culture, we tested if granulocyte-macrophage colony stimulating factors (GM-CSFs) from both murine (mGM-CSF) and human (hGM-CSF) are produced as a biologically active form through plant cell culture. The murine and human GM-CSF genes were cloned into the plant expression vector, pBI121, and Ti-plasmid mediated transformation of tobacco leaves was conducted using Agrobacterium tumefaciens harboring both recombinant GM-CSF (rGM-CSF) genes. Cell suspension culture was established from the leaf-derived calli of transgenic tobacco plant. Northern blot analysis indicated the expression of the introduced mGM-CSF gene in both transgenic plant and cell suspension cultures. In addition, the biological activities of both murine and human GM-CSF from plant cell culture were confirmed by measuring the proliferation of the GM-CSF dependent FDC-PI and TF-1 cells, respectively.

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The Growth of Transgenic Tobacco′s Suspension Culture and the Production of β-Glucuronidase in Bubble Column Bioreactor (Bubble column bioreactor에서 형질전환된 담배세포의 성장양상 및 β-Glucuronidase의 생산)

  • 김석우;이동근;현진원;이상현;하종명;하배진;이재화
    • Journal of Life Science
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    • v.12 no.5
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    • pp.577-583
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    • 2002
  • The growth kinetics and the production of $\beta$-glucuronidase from transgenic tobacco's suspension culture was investigated in the flask culture and a 2.5 L bubble column reactor. The growth of bubble column reactor was similar to that of flask culture. However, in the bubble column reactor, the production of $\beta$ -glucuronidase reached 2850 U/mg (85-fold higher than that of flask culture). In both case, the production level of $\beta$ -glucuronidase was fluctuated, which was resulted from periodical degradation of the protein. Sucrose is important component in plant culture medium. Twice addition of sucrose in bubble column reactor could not improve cell growth, since other components in a medium were already depleted. However, the addition of sugar decreased cell size, which facilitated the operation of bioreactor. The production of $\beta$ -glucuronidase was continuously increased, however final concentration of $\beta$ -glucuronidase was similar to that without sucrose addition.

Enhanced production of hGM-CSF by temperature shifting in transgenic Nicotiana tabacum cell suspension cultures

  • Kim, Yong-Hoon;Lee, Sang-Yoon;Kim, Dong-Il
    • 한국생물공학회:학술대회논문집
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    • 2003.10a
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    • pp.329-333
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    • 2003
  • Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is a glycoprotein that stimulates the production of granulocytes, macrophages and white blood cells. hGM-CSF secreted by transgenic Nicotiana tabacum suspension cells was unstable in the culture medium and rapidly degraded by extracellular preteases. In order to reduce extracellular pretense activity, culture temperature was lowered. Then, the production of hGM-CSF by transgenic plant suspension cell cultures could be enhanced by reduced degradation of hGM-CSF at low temperature.

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Effects of Antioxidants on Cell Viability and hGM-CSF Production by Transgenic Nicotiana tabacum Suspension Cultures (형질전환된 Nucotiana tabacum 현탁세포배양에서 항산화제가 세포생존도 및 hGM-CSF 생산에 미치는 영향)

  • Kim Yong Hoon;Lee Sang Yoon;Kim Dong Il
    • KSBB Journal
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    • v.19 no.5
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    • pp.374-380
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    • 2004
  • Production of therapeutic proteins by transgenic plant cell suspension cultures is an attractive system alternative to the other expression system. However, plant cell cultures have shown low expression level of foreign proteins and decreased cell viability by the changes of culture conditions. Therefore, it is necessary to enhance cell viability during the culture period. In this study, a quantitative analysis technique was designed to measure relative cell viability for plant suspension cells which have cell wall and aggregates. It was found that the programmed cell death of plant cells by apoptosis was essentially linked with the apoptotic pathway of animal cells. Therefore, effects of nicotinamide, 3-aminobenzamide and antioxidants on cell viability and apoptosis were examined in transgenic Nicotiana tabacum cells producing hGM-CSF. With those additives, cell viability could be maintained and apoptosis could be redued. In the result, the extracellular production of hGM-CSF could be enhanced 2.5 fold. It was also found that the supplementation of glutathione and ascorbic acid suppressed both the cold stress-induced decrease in cell viability and the increase of total genomic DNA fragmentation.

Effects of Silkworm Hemolymph on Cell Viability and hCTLA4Ig Production in Transgenic Rice Cell Suspension Cultures

  • Cheon, Su-Hwan;Lee, Kyoung-Hoon;Kwon, Jun-Young;Ryu, Hyun-Nam;Yu, Da-Hyun;Choi, Yong-Soo;Kim, Dong-Il
    • Journal of Microbiology and Biotechnology
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    • v.17 no.12
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    • pp.1944-1948
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    • 2007
  • Silkworm hemolymph (SH), prepared from fifth-instar larvae of Bombyx mori and heat-treated at $60^{\circ}C$ for 30 min, was used to improve cell viability and the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) in transgenic Oryza sativa L. cell suspension cultures. Even though SH could not elevate cell viability at the concentrations up to 3% (v/v), addition of 0.3% (v/v) SH to a culture medium enhanced the production of hCTLA4Ig by 36.8% over an SH-free medium. Moreover, the production period of hCTLA4Ig could be shortened in a 0.3% (v/v) SH-added medium compared with that in an SH-free culture. As a result, addition of 0.3% (v/v) SH improved the productivity of hCTLA4Ig significantly in transgenic rice cell cultures.

Production of Useful Proteins by Plant Cell Culture

  • Kwon, Tae-Ho;Kim, Dae-Hyun;Jang, Yong-Suk;Yang, Moon-Sik
    • Proceedings of the Botanical Society of Korea Conference
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    • 1999.07a
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    • pp.45-49
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    • 1999
  • Plant cell culture is emerging to express bioactive foreign proteins because it has several advantages in that it is safe, economical, genetically stable and eukaryotic expression system comparing with other expression systems. However several limitations such as slow growth rate, low expression level and lack of well established down stream process need to be answered. As a preliminary approach to produce the immunologically interested molecules through the plant cell culture, we tested if granulocyte-macrophage colony stimulating factors (GM-CSFs) from both murine (mGM-CSF) and human (hGM-CSF) are produced as a biologically active form through plant cell culture. The murine and human GM-CSF genes were cloned into the plant expression vector, pBI121, and Ti-plasmid mediated transformation of tobacco leaves was conducted using Agrobacterium tumefaciens harboring both recombinant GM-CSF (rGM-CSF) genes. Cell suspension culture was established from the leaf-derived calli of transgenic tobacco plant. Northern blot analysis indicated the expression of the introduced mGM-CSF gene in both transgenic plant and cell suspension cultures. In addition, the biological activities of both murine and human GM-CSF from plant cell culture were confirmed by measuring the proliferation of the GM-CSF dependent FDC-PI and TF-1 cells, respectively.

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