• 한국생물공학회:학술대회논문집
• /
• 2001.11a
• /
• 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.

• Proceedings of the Korea Society of Poultry Science Conference
• /
• 2001.11a
• /
• pp.40-46
• /
• 2001

The use of pluripotent stem cells has tremendous advantages for various purposes but these cell lines with proven germ-line transmission have been completely established only in the mouse. Embryonic germ (EG) cell lines are also pluripotent and undifferentiated stem cells established from primordial germ cells (PGCs). This study was conducted to establish and characterize the chicken EG cells derived from gonadal primordial germ cells. We isolated gonadal PGCs from 5.5-day-old (stage 28) White leghorn (WL) embryos and established chicken EG cells lines with EG culture medium supplemented with human stem cell factor (hSCF), murine leukemia inhibitory factor (mLIF), bovine basic fibroblast growth factor (bFGF), human interleukin-11 (hIL-11), and human insulin-like growth factor-I (hIGF-I). These cells grew continuously for 4 months (10 passages) on a feeder layer of mitotically active chicken embryonic fibroblasts. These cells were characterized by screening with the Periodic acid-Shiff＇s reaction, anti-SSEA-1 antibody, and a proliferation assay after several passages. As the results, the chicken EG cells maintained characteristics of undifferentiated stem cells as well as that of gonadal PGCs. When cultured in suspension, the chicken EG cells successfully formed an embryoid body and differentiated into a variety of cell types when re-seeded onto culture dish. The chicken EG cells were injected into blastodermal layer at stage X and dorsal aorta of recipient embryo at stage 14 (incubation of 53hrs) and produced chimeric chickens with various differentiated tissues derived from the EG cells. The germline chimeras were also successfully induced by using EG cells. Thus, Chicken EG cells will be useful for the production of transgenic chickena and for studies of germ cell differentiation and genomic imprinting.

• Journal of Life Science
• /
• v.14 no.6 s.67
• /
• pp.1018-1022
• /
• 2004

Optimized conditions for Agrobacterium-mediated lisianthus pollen transformation were adjusted using various factors such as temperature, pH and sucrose concentration. Pollen tube growth was successfully achieved in a medium (pollen germination medium; PGM) containing $7-15\%$ sucrose with pH in the range of 5.5-7.0 at temperature of $20-27^{\circ}C$. Lisianthus pollen was vacuum-infiltrated with Agrobacterium cell suspension for 20 min, and transformed pollen was confirmed by GUS histochemistry and Southern hybridization following RT-PCR. Transgenic pollen system may be utilized for establishing an area of plant transient expression systems based on the convenient pollen transformation procedure presented in here.

• KSBB Journal
• /
• v.24 no.3
• /
• pp.246-252
• /
• 2009

Rice cells were transformed to express human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) using RAmy3D promoter. hCTLA4Ig was produced and secreted into culture media inducibly when sugar was depleted. The obstacles of this system are the cell death and release of proteases by sugar starvation. These problems resulted in the losses of stability and productivity of hCTLA4Ig. Therefore, the effects of proline as an inhibitor of cell death were investigated. When 4 mM proline was added in sugar-free media, the cell death and release of proteases were reduced. As a consequence, the production of hCTLA4Ig was enhanced. In addition, the effects of protein stabilizers such as gelling agents were studied. It was found that the application of 0.01 g/L gelatin led to an increase in hCTLA4Ig production. This increase might be originated from the stabilization of hCTLA4Ig. In conclusion, the production of hCTLA4Ig could be enhanced by the additions of proline and gelatin in transgenic rice cell cultures.

• Korean Journal of Animal Reproduction
• /
• v.18 no.4
• /
• pp.299-307
• /
• 1995

The goal of cell stem cell technology is to produce a viable and genetically normal animal. To achieve this goal various laboratories have followed 2 different pathways beginning with either the culture of 1) single or pooled ICMs grown with or without a feeder layer or 2) single or pooled 16-20 cell stage embryos grown with a feeder layer. Also, thus far embryonic cell cultures or lines have been established by several methods including loose suspension culture for short-term cultures and more commonly murine or bovine fibroblast feeder layers for long-term culture. Pluripotent lines have been derived from 16-cell through blastocyst inner cell mass stages. The efficiency of establishing cell lines and cell proliferation apper to be affected by the number of cells or embryos starting the line. Most attempts to produce offspring from long term STO cell feeder layer cultured ICM or morulae derived ES cells have resulted in pregnancy failure in the first trimester when ES cells were used in cuclear transfer or have failed to retain ES cells in the progeny produced by chimerization. The exception is 1 chimeric fetus from use of morula ES cells in the chimerization with early embryonic cells. There is much to be learned yet about ES cell culture requirements for maintenance of totipotency. If bovine ES cell lines loose imprinting pattern and totipotency with long-term culture and passage as suggested for mouse ES cells, we may be limited to the use of short-term cultures for multiplication of embryos and efficient production of transgenic animals. No bovine ES cell system has yet met all of the criteria indicated for a totipotent ES cell line.

• Toxicological Research
• /
• v.23 no.4
• /
• pp.383-389
• /
• 2007

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) regulates proliferation and differentiation of hematopoietic progenitor cells and modulates function of the mature hematopoietic cells. In the previous study, we reported that hGM-CSF could be produced in transgenic rice cell suspension culture, termed rhGM-CSF. In the present study we examined the single and repeated dose toxicity of rice cells-derived hGM-CSF in SD rats. During single dose toxicity study for 7 days, there were no any toxic effects at any dose of from 10 to $1000{\mu}g/kg$. The lethal dose ($LD_{50}$) was not found in this range. Moreover, repeated dose toxicity study of 14-days period and at the doses of 50 and $200{\mu}g/kg$ (i. v.) of rhGM-CSF did not show any changes in food and water intake. There were also no significant changes in both body and organ weights between the control and the test groups. The hematological and blood biochemical parameters were statistically not different in all the groups. These results suggest that rhGM-CSF has no toxicity in SD rats.

• Toxicological Research
• /
• v.24 no.4
• /
• pp.315-320
• /
• 2008

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a glycoprotein and hematopoietic growth factors that regulates the proliferation of myeloid precursor cells and activates mature granulocytes and macrophages. In a previous study, we reported that hGM-CSF could be produced in transgenic rice cell suspension culture, termed rhGM-CSF. In the present study, we examined the repeated dose toxicity of rhGM-CSF in SD rats. The repeated dose toxicity study was performed at each dose of 50 and 200 ${\mu}g/kg$ subcutaneous administration of rhGM-CSF everyday for 28-days period. The results did not show any changes in food and water intake. There were also no significant changes in both body and organ weights between the control and the tested groups. The hematological and blood biochemical parameters were statistically not different in all groups. These results suggest that rhGM-CSF may show no repeated dose toxicity in SD rats under the conditions.

• Korean Journal of Animal Reproduction
• /
• v.21 no.2
• /
• pp.167-176
• /
• 1997

This study was carried out to evaluate the potential for preselection of transgenic embryos prior to transfer into recipient animals. In these experiments, I used a 3.2 kb transgene which contained the neomycin resistance gene (neo) and lac Z gene driven by the $\beta$ actin promoter (iacZ Ineo). Oocytes were aspirated from abattoir ovaries, matured in TCM-199 supplemented with 10% fetal bovine serum (FBS), 5 ${\mu}\textrm{g}$/ml LH, 0.5 ${\mu}\textrm{g}$/ml FSH, 100 unit/ml penicillin, and 100 ${\mu}\textrm{g}$/ml streptomycin for 22 to 24 hrs then inseminated with a sperm suspension of 1 X 10$^6$ sperm/ml containing 5 ${\mu}\textrm{g}$/ml of heparin. At 18-20 hrs after insemination, cumulus cells were removed by vortexing and pronuclei of centrifuged zygotes microinjected with the lacZ/neo construct (3 ng/$\mu$l). All cultures were carried out in CR1aa with transfected BRL monolayers containing 3 mg/ml BSA, 20 $\mu$/ml NEM amino acids and 40 $\mu$I/ml BME amino acids. To identify the appropriate concentration of G418 for selection, non-microinjected zygotes were cultured in the presence of 0, 50, 100 and 200 $\mu$g/ml of G418. After 8 days of culture in these treatments, 44/145 (30.3%), 13/150 (8. 7%), 1/151 (0.7%) and 0/134 (0.0%) devel-oped to the blastocyst stage in 0, 50, 100 and 200 $\mu$g/ml of G418, respectively. A total of 1,127 zygotes were microinjected and placed into culture (without G418) and subsequently 710 (63.0%) cleaved. At 48 hrs post-injection, embryos ($\geq$2-cell) were randomly assigned to control (medium alone) or G418 (100 ${\mu}\textrm{g}$/ml) treatments. A control culture of 740 non-microinjected embryos from the same replicates of embryos were also placed into control medium. After 8 days in culture, 54/343 (15.7%) and 22/367 (6.0 %) of the microinjected embryos developed to the blastocyst stage in control and G418 media, respectively. A total of 151/740 (27.2%) of the non-microinjected embryos placed in the control medium developed to the blastocyst stage. The blastocysts in the control treatment had a mean of 70.7 ${\pm}$ 4.7 cells of which 23.1 ${\pm}$ 2.6 (32.7%) stained for $\beta$-Gal activity. B1astocysts in the G418 treatment had a mean of 48.8${\pm}$7.5 cells of which 40.3 ${\pm}$ 4.1 (82.6%) stained for $\beta$-Gal ac tivity (P<0.05). In the control treatment 26 of 30 (87.0%) blastocysts had some cells with $\beta$-Gal activity while all of the blastocysts in the G418 treatment had some cell with $\beta$-Gal ac tivity (P<0.05). However, ICM colonies in either control or G418 treatments were not expressed any epiblast cell except of trophetoderm celIs. The $\beta$-actin promoter/lacZ gene may not be e expression or silence expression in epiblast cells These results clearly show an enrichment of blastocysts expressing the transgene in the majority of their cells after culture in the presence of G418. The exogeneous gene was not express a and silence in ICM colonies especiallly epiblast cells except of trophectederm cells. Even though the higher rate cell number expressed of exogeneous gene in the G418 treatments, a total cell number was decrease significantly (P<0.05) of which might be also drop of the establishment of ES like-cell colonies and production of transgenic animals. However, futher studies need to determine the viability of these selected embryos and the avability of production of transgenic offspring.