• Journal of Plant Biotechnology
• /
• 제47권3호
• /
• pp.227-234
• /
• 2020

벼(Oryza sativa L.)에서 세포 현탁 배양을 통한 miraculin 단백질의 생산을 위해 miraculin 유전자(AB512278)가 도입된 Agrobacterium tumefacience EHA105를 매개로 벼 캘러스에 형질전환하였다. 현탁배양세포주는 형질전환 캘러스를 이용하여 몇번의 선발과정 및 계대배양을 통해 선발하였고, 게놈 PCR 분석을 통해 miraculin 유전자가 벼 염색체에 안정적으로 도입된 것을 확인하였다. 또한, RT-PCR 분석을 통해 형질전환 세포주에서 도입된 miraculin 유전자가 과발현 되었다. 재조합 miraculin은 형질전환 현탁배양 HK-2 세포주에서 가장 높게 발현되어 total soluble protein (TSP) 대비 2.0%를 보였다. 이러한 결과는 형질전환 현탁세포배양이 miraculin과 같은 미각 수식 단백질의 대량생산 시스템을 구축하는데 이용 가능 할 것으로 사료된다.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제9권1호
• /
• pp.12-16
• /
• 2004

Partitioning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) was achieved in the aqueous two-phase systems (ATPSs) using a crude extract of transgenic tobacco cell suspension culture. This study examined the effects of polyethylene glycol (PEG) molecular weight and concentration and the effects of sodium phosphate concentration in different PEG/sodium phosphate systems on the partition coefficient, K. The best ATPS system was 5% PEG 8,000/1.6 M sodium phosphate after 2 h of incubation at room temperature. In this system, hGM-CSF was partitioned in the PEG-rich phase with a yield of 57.99% and K$\_$hGM-CSF/ of 8.12. In another system, 3% PEG 10,000/1.6 M sodium phosphate, hGM-CSF was also partitioned primarily in the top phase with a yield of 45.66% and K$\_$hGM-CSF/ of 7.64 after 2 h of incubation at room temperature.

• Plant Biotechnology Reports
• /
• 제5권3호
• /
• pp.245-254
• /
• 2011

This study describes an efficient protocol for Agrobacterium tumefaciens-mediated transformation of two subgroups of genotype AAA bananas (Musa acuminata cv. Pei Chiao and Musa acuminata cv. Gros Michel). Instead of using suspension cells, cauliflower-like bud clumps, also known as multiple bud clumps (MBC), were induced from sucker buds on MS medium containing $N^6$-Benzylaminopurine (BA), Thidiazuron (TDZ), and Paclobutrazol (PP333). Bud slices were co-cultivated with A. tumefaciens C58C1 or EHA105 that carry a plasmid containing Arabidopsis root-type ferredoxin gene (Atfd3) and a plant ferredoxin-like protein (pflp) gene, respectively. These two strains showed differences in transformation efficiency. The EHA105 strain was more sensitive in Pei Chiao, 51.3% bud slices were pflp-transformed, and 12.6% slices were Atfd3-transformed. Gros Michel was susceptible to C58C1 and the transformation efficiency is 4.4% for pflp and 13.1% for Atfd3. Additionally, gene integration of the putative pflp was confirmed by Southern blot. Resulting from the pathogen inoculation assay, we found that the pflp transgenic banana exhibited resistance to Fusarium oxysporum f. sp. cubense tropical race 4. This protocol is highly advantageous to banana cultivars that have difficulties in setting up suspension cultures for the purpose of quality improvement through genetic transformation. In addition, this protocol would save at least 6 months in obtaining explants for transformation and reduce labor for weekly subculture in embryogenic cell suspension culture systems.

• 식물조직배양학회지
• /
• 제25권2호
• /
• pp.89-93
• /
• 1998

Tomato phenylalanine ammonia-lyase 5 (tPAL5) was identified that alternate initiation sites were utilized differentially in response to environmental stimuli (Lee et al, 1992b). In this study, we tried to look into tissue -or cell- specific expression pattern of tPAL5 gene by fusing with ${\beta}-glucuronidase$ (GUS) gene in transgenic tobacco plants. In transgenic plants, root and stem extracts contained 8~12 fold higher levels of GUS activity than petiole or leaf tissue while the highest levels of induction was observed from leaf tissue by mechanical wounding (5~11 fold). In trans-sections of stems and petioles, GUS activity was restricted to phloem cells(outer region) of developing vascular bundle and mainly at apical tip region in the root tissues. The levels of GUS activity was drastically reduced (10~12 fold reduction) when the 5'-upstream region of tPAL5 gene (-1151bp from ATG codon) was deleted up to -665. The levels of GUS expression, however, raised up by 6~8 fold when deleted up to -455. Therefore, we conclude that there are positive cis-elements at the region -1151 to -1008 and at -455 to -195 while the negative cis-element is at -1008 to -455.

• 한국식물학회:학술대회논문집
• /
• 한국식물학회 1995년도 식물학심포지움 식물로부터 유용 2차대사산물의 생산 PRODUCTION OF USEFUL SECONDARY METABOLITES FROM PLANTS
• /
• pp.67-78
• /
• 1995

Monoclonal antibodies (MoAbs) are a highly diversified class of proteins with major research and commercial applications such as diagnostics and therapeutics. Currently, the dominant method for producing MoAbs is through the hybridoma technique. However, this technique is slow, tedious, labor intensive, and expensive. The production of MoAbs in cultured transgenic plant cells can offer some advantages over that in the over that in the mammalian systems. The media to cultivate plant cells are well defined and inexpensive. Contamination by bacteria or fungi is easily monitored in plant tissue cultures. Furthermore, these contaminants are usually not potent pathogens to human beings. In our interdisciplinary research efforts, heavy chain monoclonal antibody (HC MAb) was inserted into Ti plasmid vector and transferred into A. tumefaciens for the transformation in tobacco cells. It was found that 76% of the transformants produced HC MAb. The presence of HC MAb in the cell membrane fraction indicated that the signal peptide was functional and efficient. The change of the HC MAb concentration during a batch culture followed a similar trend as dry cell concentration, indicating that the production of HC MAb was growth related. The long-term repeated subcultures of 11 cell lines showed that there was no obvious trend of neither the decrease nor the increase of the productivity with the repeated subcultures.

• 한국생명과학회:학술대회논문집
• /
• 한국생명과학회 2002년도 제38회 학술심포지움
• /
• pp.48-60
• /
• 2002

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that stimulates the production of granulocytes, macrophages, and white blood cells. The effects of osmotic pressure on secretion of human GM-CSF into the culture medium were investigated in suspension cultures of transgenic tobacco cells. An increase in osmotic pressure caused by the addition of mannitol decreased the cell size index, with the effect being more pronounced when cells were measured wet rather than dry. Increased osmotic pressure enhanced the secretion of hGM-CSF. At 90 g/L mannitol, the maximum concentration tested, hGM-CSF was present in the culture medium at 980 ug/L. As the concentration of mannitol increased, the total amount of protein secreted also increased, but was disproportionately enriched in GM-CSF NaCl, another osmoticum, had very similar effects on cell growth and hGM-CSF production, but did not cause enrichment for hGM-CSF Additionally, protein-stabilizing polymer was added to culture broth to enhance stability of secreted recombinant protein. Finally, above two method were applied together to maximize the productivity.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2001년도 추계학술발표대회
• /
• pp.193-196
• /
• 2001

IL-18. formerly known as IGIF(interferon -gamma inducing factor), is structurally IL-l related but functionally IL-12 related pro-inflammatory cytokine. The human IL -18(hIL-lS), like IL-$1{\beta}$, is synthesized as a biologically inactive precursor of 24kDa lacking a signal peptide, and then cleaved into an active mature form by cystein protease IL-$1{\beta}$ converting enzyme (ICE: caspase- 1), We tested if the mature hIL -18 can be expressed and secreted into culture medium by transforming the forming gene construct consisting of a mature hIL-18 gene fused to signal peptide of rice amylase lA. Secondly, we were tested if the pro- IL-18 could be processed into a biologically active form by caspase-l like protease in plant. Cell suspension culture was established from the leaf-derived calli of transgenic tobacco plant. Southern and Northern blot analysis indicated the expression of both pro-hIL-18 and mature hIL-18 plant cells. Western blot analysis introduced the protein products of pro- hIL -18 and mhIL -18 were observed in transigenic cell lines. In addition, the molecular size of recombinant pro-hILl-18 and mhIL-18 were estimated to be 24kDa and 18kDa, respectively. ELISA revealed that the amount of pro- hIL -18 was 1.3ug per gram of fresh weight calli. Moreover, the presence of mhIL-18 was detected in the culture medium and it appeared to be 25ug/L.

• 한국자원식물학회지
• /
• 제24권2호
• /
• pp.121-129
• /
• 2011

분자육종의 발전에 따라 좀 더 유용한 형질전환 식물체를 더 많이 얻어내기 위한 많은 형질전환 기술들이 개발되어오고 있으나 새로운 수요를 충족시키기 위한 다양한 종류의 새로운 품종들이 실시간으로 새롭게 개발되고 있으며 개발된 품종에 적합한 새로운 형질전환 기술에 대한 개발 필요성 또한 지속적으로 요구되고 있다. 카네이션(Dianthus caryophyllus L.)은 전 세계적인 주요 화훼작물 중 하나로 경제적 파급효과가 매우 큰 작물이다. 따라서 기술개발의 파급효과가 큰 카네이션을 대상으로 조직배양 및 형질전환의 효율성을 향상시키기 위하여 시장에서 주요하게 거래되고 있는 주요 품종 18종을 수집하고 이 중 조직배양을 통한 신초 분화 능력이 우수한 4개 품종 Yellow dotcom, Jakarta, Belmonte, Polar tessino 등을 선발하였다. 선발된 4개 품종에 공통으로 적용시킬 수 있는 가장 효율적인 생장조절제는 MS+Sucrose 3%+NAA 1.0 mg/L+TDZ 1.0 mg/L.이었으며 MS+Sucrose 3%+NAA 3.0 mg/L+BA 1.0 mg/L 처리는 Belmonte.에 적용 가능한 생장조절제 조합이었다. 캘러스 형성과 신초 분화에 가장 효율적인 기본 배지는 MS이었으며 탄소원으로는 Sucrose를 3%로, Phytagel 0.3%를 배지 경화제로 처리하는 것이 조직배양을 통한 재분화 식물체 획득에 가장 효과적이었다. 가장 효율적 조직배양 체계를 구축하기 위한 조직배양 부위는 줄기 부위 이었으며 이 부위를 배양재료로 사용할 경우 신초 생성율을 80.2%까지 올릴 수 있었고 배양소요 기간도 6일 정도 단축할 수 있었다.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2000년도 춘계학술발표대회
• /
• pp.317-320
• /
• 2000

유전자 재조합된 Nicotiana tabacum을 이용하여 동결 보존 후의 세포 생장을 관찰하였다. 형질 전환된 Nicotiana tabacum의 동결 보존에서 회복배지에 계면 활성제와 산소 전달 물질을 첨가한 경우 첨가하지 않은 경우에 비해 세포 생장을 증대 시켰고 산소 전달 물질 보다 계면활성제의 영향이 크다는 결과를 관찰하였다.

• KSBB Journal
• /
• 제26권5호
• /
• pp.386-392
• /
• 2011

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), an immunosuppressive agent, was expressed in transgenic rice cells using RAmy3D promoter and RAmy1A signal peptide for the inducible production and secretion into culture media by sugar depletion. In this study, sodium butyrate was used as a small molecular enhancer (SME) to enhance the production of hCTLA4Ig in transgenic rice cell suspension cultures. When 1 mM sodium butyrate was added in sugar-free media, relative viability was not reduced, while the productivity was improved 1.3-fold. In addition, by supplementing 87 mM sodium pyruvate as an alternative energy source during the production phase, death rate of the cells was decreased. When sodium pyruvate was not added, most cells became dead at day 6. However, by adding sodium pyruvate, 18% of viability can be maintained until day 10 and the production of hCTLA4Ig was enhanced 1.4-fold. When the combination of sodium pyruvate and sodium butyrate at optimum concentrations was added, the highest viability and hCTLA4Ig production could be obtained. The highest level of hCTLA4Ig reached up to 35 mg/L at day 10.