• The Korean Journal of Nuclear Medicine
• /
• v.38 no.1
• /
• pp.62-73
• /
• 2004

Purpose: The herpes simplex virus type 1 thymidine kinase gene(HSV1-tk) is an attractive candidate as a reporter gene in noninvasive reporter gene monitoring system. The HSV1-tk gene was chosen as a reporter gene, because it has been extensively studied, and there are appropriate reporter probes, substrates of HSV1-tk gene product, to apply for HSV1-tk gene imaging. We used radiolabeled 5-iodovinyl-2'-deoxyuridine (IVDU) and 5-iodovinyl-2'-fluoro-2'-deoxyuridine (IVFRU) as reporter probes for HSV1-tk gene monitoring system. Materials and Methods: We prepared HSV1-tk gene transduced Morris hepatoma cell line using retroviral vector, MOLTEN containing HSV1-tk gene. And we confirmed the HSV1-tk gene expression by Northern blotting and Western blotting. We compared in vitro uptakes of radioiodinated IVDU and IVFRU to monitor HSV1-tk gene expression in Morris hepatoma cell line (MCA) and HSV1-tk gene tranduced MCA (MCA-tk) cells until 480 minutes. We also peformed correlation analysis between percentage of HSV1-tk gene tranduced MCA cell % (MCA-tk%) and uptakes of radiolabeled IVDU or IVFRU. Results: MCA-tk cell expressed HSV1-tk mRNA and HSV1-TK protein. Two compounds showed minimal uptake in MCA, but increased uptake was observed in MCA-tk. IVDU showed 4-fold higher accumulation than IVFRU at 480 min in MCA-tk (p<0.01). Both IVDU and IVFRU uptake were linearly correlated ($R^2>0.96$) with increasing MCA-tk%. Conclusion: The radiolabeld IVDU and IVFRU showed higher specific accumulation in retrovirally HSV1-tk gene transfected Morris hepatoma cell line. Both IVDU and IVFRU could be used as good substrates for evaluation of HSV1-tk gene expression.

• Nuclear Medicine and Molecular Imaging
• /
• v.42 no.5
• /
• pp.394-400
• /
• 2008

Purpose: Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirus-mediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Materials and Methods: Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was $19.1{\pm}4.7%$, $54.0{\pm}6.4%$, $85.7{\pm}8.7%$, and $98.4{\pm}1.3%$ at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and 1-125 uptake. Results: Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro 1-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC ($29,704{\pm}6,659\; picomole/10^6\;cells$) was greater than that in adeno-hNIS-rMSC at MOI 100 ($6,168{\pm}2,134\;picomole/10^6\;cells$). Conclusion: Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.

• Nuclear Medicine and Molecular Imaging
• /
• v.43 no.5
• /
• pp.487-494
• /
• 2009

Purpose: Small size of recombinant scFv antibody has many advantages such as rapid blood clearances and improved targeting antibodies to tumor region. On the other hand owing to small size, number of amino group is insufficient in conjugation with chelator and fluorescence labeling. This study is to introduce poly lysine tag to the C-terminal end of scFv lym-1 sequence for fluorescence chelator conjugation. Materials and Methods: Poly lysine scFv lym-1 gene, cloned into pET-22b (+) vector, was expressed in E. coli BL21 (DE3) strain. Antibody purification was performed with Ni-NTA column and then size exclusion column chromatography. Expression and purification levels of poly lysine tagged scFv lym-1 antibody were confirmed by western blot analysis. I-124, I-125, I-131 and Tc-99m were used for radiolabeling of purified poly lysine scFv lym-1. Flow cytometry analysis of FIT( conjugated poly lysine scFv lym-1 was performed for confirmation of immunoreactivity of human Burkitt's lymphoma cells. Results: Poly lysine scFv lym-1 antibody was purified through two steps and identified as molecular weight of 48 KDa. Radiolabeling yields of I-124, I-125, I-131 and Tc-99m into poly lysine scFv lym-1 were >99%, >99%, >95% and >99%, respectively. Flow cytometry analysis of poly lysine scFv and scFv lym-1 was showed similar immunoreactivity to human Burkitt's lymphoma cells. Conclusion: Poly lysine tag was useful for the sufficient number of amino groups to scFv lym-1 antibody for chelator conjugation with minimizing loss of immunoreactivity.

• Korean Journal of Microbiology
• /
• v.44 no.3
• /
• pp.178-185
• /
• 2008

Avian influenza virus (AIV) is recognized as key to the emergence of pandemic influenza for humans; there are growing concerns that AIV H9N2 may become more efficient to transmit to humans in the near future, since the infection of poultry with AIV H9N2 has been common in recent years. In this study, we aimed to produce antisera recognizing the HA and NA proteins of AIV H9N2. Initially, coding sequences corresponding to the N-terminal regions of the HA and NA proteins of the Korean AIV H9N2 (A/Ck/Kr/MS96/96) isolated from a domestic chicken were amplified from the genomic RNA. Following cloning of the amplified cDNA fragments into pGEX4T-1 vector, two GST-fusion proteins (GST-HAln and GST-NAn) were expressed in E. coli BL21 and purified with glutathione sepharose columns; the recombinant GST-HAln and GST-NAn proteins were both used as immunogens in rabbits. The antigenicity of the rabbit antisera was analyzed by immunoblotting of the cell lysates prepared from AIV H9N2-infected MDCK cells. Overall, the recombinant HAln and NAn proteins fused to the C-terminus of GST and the rabbit antisera raised against the corresponding recombinant proteins would provide a valuable reagent for AIV diagnosis and basic research.

• Journal of fish pathology
• /
• v.18 no.1
• /
• pp.67-80
• /
• 2005

Phosphoinositide-specific phospholipase $C\delta$ $PLC\delta$) plays an important role in many cellular responses and is involved in the production of second messenger. The present study was conducted to obtain the biochemical characteristics of the expressed recombinant $PLC\delta$ in E. coli cloned from Misgurnus mizolepis and partially purified $PLC\delta$ enzymes from liver tissues of M. mizolepis (wild ML-$PLC\delta$). The ML $PLC\delta$ gene was cloned and expressed under the previous report (Kim et al., 2004), and purified the recombinant protein by successive chromatography using $Ni^{2+}$-NTA affinity column and gel iltration FPLC column. The wild ML-$PLC\delta$ protein was solublized with 2 M KCI and purified by successive chromatography on open heparin-Sephagel and analytical TSKgel heparin-5PW. Both the recombinant and wild ML-$PLC\delta$ form of protein showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP$_2$) or phosphatidylinositol (PI). Its activity was absolutely $Ca^{2+}$- dependant, which was similar to mammalian $PLC\delta$ isozymes. Maximal PI-hydrolytic activations of recombinant and wild ML- TEX>$PLC\delta$ was at pH 7.0 and pH 7.5, respectively. In addition, the enzymatic activities of recombinant and wild ML-$PLC\delta$ were increased in concentration-dependent manner by detergent, such as sodium deoxycholate SDC), phosphatidylethanolamine (PE) and phosphatidylcholine (PC). The activities decreased in contrast by a polyamine, such as spermine. Western blotting showed that several types of $PLC\delta$ isozymes exist in various organs. Taken together our results, it suggested that the biochemical characteristics of ML-$PLC\delta$ are similar with those of mammalian $PLC\delta1$ and ${\delta}3$ isozymes.

• 한국공간정보시스템학회:학술대회논문집
• /
• 2004.12a
• /
• pp.169-181
• /
• 2004

SK Telecom has already constructed GIMS system as the base common framework of LBS/GIS service system based on OGC(OpenGIS Consortium)'s international standard for the first mobile vector map service in 2002, But as service content appears more complex, renovation has been needed to satisfy multi-purpose, multi-function and maximum efficiency as requirements have been increased. This research is for preparation ion of GML3-based platform to upgrade service from GML2 based GIMS system. And with this, it will be possible for variety of application services to provide location and geographic data easily and freely. In GML 3.0, it has been selected animation, event handling, resource for style mapping, topology specification for 3D and telematics services for mobile LBS multimedia service. And the schema and transfer protocol has been developed and organized to optimize data transfer to MS(Mobile Stat ion) Upgrade to GML 3.0-based GIMS system has provided innovative framework in the view of not only construction but also service which has been implemented and applied to previous research and system. Also GIMS channel interface has been implemented to simplify access to GIMS system, and service component of GIMS internals, WFS and WMS, has gotten enhanded and expanded function.

• Microbiology and Biotechnology Letters
• /
• v.33 no.2
• /
• pp.96-105
• /
• 2005

For screening of autoregulator receptor gene from Streptomyces longwoodensis, PCR was performed with primers of receptor gene designed on the basis of amino acid sequences of autoregulator receptor proteins with known function. PCR products were subcloned into the BamHIsite of pUC19 and transformed into the E. coli $DH5{\alpha}$. The isolated plasmid from transformant contained the fragment of 100 bp, which was detected on $2\%$ gel after BamHI treatment. The insert, 100 bp PCR product, was confirmed as the expected internal segment of gene encoding autoregulator receptor protein by sequencing. Southern and colony hybridizations with the 100 bp fragment as a probe allowed to select a genomic clone of S. longwoodensis, pSLT harboring a 4.4 kb SphI fragment. Nucleotide sequencing analyses revealed a 651 bp open reading frame(ORF) were isolated protein showing moderate homology ($35{\sim}46\%$) with the ${\Gamma}$-butyrolactone autoregulator receptors from Streptomyces sp., and this ORF was named sltR The sltR/pET-17b plasmid was constructed to overexpress the recombinant SltR protein (rSltR) in E. coli BL21 (DE3)/pLysS, and the rSltR protein was purified to homogeneity by DEAE-Sephacel column chromatography, and DEAE-5PW chromatography (HPLC). The molecular mass of the purified rSltR protein was 55 kDa by HPLC gel-filtration chromatography and 28 kDa by SDS-PAGE, indicating that the rSltR protein is present as a dimer. A binding assay with tritium-labeled autoregulators revealed that the rSltR has clear binding activity with a A-factor type autoregulator as the most effective ligand.

• Journal of Biosystems Engineering
• /
• v.4 no.1
• /
• pp.1-23
• /
• 1979

The use of barley seeder attached to rotary tiller in the rural area has a significant meaning not only for the solution of labor peak season, but also for the increase of land utilization efficiency. The facts that presently being used barley seeders are all based on the mechanical principles of the reverse rotation, center drive and are all using forward rotating tine, which is used to be easily and heavily worn out when it rotates reversely, raise problem of recommending them to rural area in Korea. Therefore, the main objective of the study was to develop new type of rotary tine attachable to barley seeders. To attain the objective the following approaches were applied. (1) The kinematic analysis of reverse rotating barley seeders. (2) The studies on the soil bin and artificial soil. (3) The comparative experiment on the power requirement of prototype tine. The results obtained from the studies are summarized as follow: 1. The kinematic analysis of barley seeder attached to rotary tiller: The following results were obtained from the kinematic analysis for deriving general formulae of the motion and velocity characterizing the rotary tine of barley seeders presently being used by farmers. a) The position vector (P) of edge point (P) in the rotary tine of reverse rotating, center drive was obtained by the following formula. $$P=(vt+Rcos wt)i+Rsin wt j+ \{ Rcos \theta r sin \alpha cos (wt- \beta +\theta r) +Rsin \theta r sin \alpha sin (wt-\beta + \theta r) \} lk $$ b) The velocity of edge point $(P^')$ of reverse rotating, center drive rotary tine was obtained by the following formula. $$(P^')=(V-wR sin wt)i+(w\cdot Rcoswt)j + \{ -w\cdot Rcos \theta r\cdot sin \alpha \cdot sin (wt-\beta +\theta r) + w\cdot Rsin \theta r\cdot sin \alpha \cdot cos (wt- \beta + \theta r \} k $$ c) In order to reduce the power requirement of rotary tine, the angle between holder and edge point was desired to be reduced. d) In order to reduce the power requirement, the edge point of rotary tine should be moved from the angle at the begining of cutting to center line of machine, and the additional cutting width should be also reduced. 2. The studies on the soil bin and artificial soil: In order to measure the power requirement of various cutting tines under the same physical condition of soil, the indoor experiments Viere conducted by filling soil bin with artificially made soil similar to the common paddy soil and the results were as follows: a) When the rolling frequencies$(x)$ of the artificial soil were increased, the densIty$(Y)$ was also increased as follows: $$y=1.073200 +0.070780x - 0.002263x^2 (g/cm^3)$$ b) The absolute hardness $(Y)$ of soil had following relationship with the rolling frequencies$(x)$ and were increased as the rolling frequencies were increased. $$Y=37.74 - \frac {0.64 + 0.17x-0. 0054x^2} {(3.36-0.17x + 0.0054x^2)^3} (kg/cm^3)$$ c) The density of soil had significant effect on the cohesion and angle of internal friction of soil. For instance, the soil with density of 1.6 to 1.75 had equivalent density of sandy loam soil with 29.5% of natural soil moisture content. d) The coefficient of kinetiic friction of iron plate on artificial soil was 0.31 to 0.41 and was comparable with that of the natural soil. e) When the pulling speed of soil bin was the 2nd forward speed of power tiller, the rpm of driving shaft of rotary was similar to that of power tiller, soil bin apparatus is indicating the good indoor tester. 3. The comparative experiment on the power requirement of prototype tine of reverse rotating rotary: According to the preliminary test of rotary tine developed with various degrees of angle between holder and edge pcint due to the kinematic analysis, comparative test between prototype rotary tine with $30 ^\circ $ and $10 ^\circ$ of it and presently being used rotary tine was carried out 2nd the results were as follows: a) The total cutting torque was low when the angle between holder and edge point was reduced. b) $\theta r$ (angle between holder and edge point) of rotary tine seemed to be one: of the factors maximizing the increase of torque. c) As the angle between holder and edge point ($\theta r$) of rotary tine was $30 ^\circ $ rather than $45 ^\circ $, the angle of rotation during cutting soil was reduced and the total cutting torque was accordingly reduced about 10%, and the reduction efficiency of total cutting torque was low when the angle between holder and edge point ($\theta r$) of rotary tine was $10 ^\circ $, which indicates that the proper angle between holder and edge point of rotary tine should be larger than $10 ^\circ $ and smaller than $30 ^\circ $ . From above results, it could be concluded that the use of the prototype rotary tine which reduced the angle between holder and edge point to $30 ^\circ $, insted of $45 ^\circ $, is disirable not only decreasing the power requirements, but also increasing the durabie hour of it. Also forward researches are needed, WIlich determine the optimum tilted angle of rotary brocket, and rearrangement of the rotary tine on the rotary boss.

• Korean Journal of Animal Reproduction
• /
• v.25 no.4
• /
• pp.339-347
• /
• 2001

Sperm-mediated DNA transfer has a potential to markedly simplify techniques for the generation of transgenic animals. The exogenous DNA transfer by intracytoplasmic sperm injection (ICSI) procedure has been recently introduced in the production of transgenic animals. In this study, the developmental competence and tile expression rates of transgene were investigated after injection of spermatozoon or sperm head with enhanced green fluorescent protein (EGFP) gene into the mature porcine oocytes. The porcine oocytes were injected with intact sperm, membrane-disrupted sperm or sperm head. After injection. embryos were cultured in NCSU23 medium up to the blastocyst stage, and the developmental competence and expression rates were studied. The developmental rate (67.0%) of sperm injection group was higher than that (59.7%) of sperm head injection group, and the rates of EGFP expression were also significantly different between sperm injection and sperm head injection groups (42.1 vs 20.0%) (F＜0.05). In the porcine oocytes injected with sperm treated with different methods of membrane disruption, the removal of sperm membrane did not alter the developmental competence of embryos. The rate of blastocysts at 7 days after injection with intact and membrane disrupted sperm were 15.0 and 14.2%, respectively. The EGFP expression rates, 38.4% in embryos injected with frozen-thawed sperm was higher than that, 22.4% of embryos injected with the Triton X-100 treated sperm. Prior to injection, sperm were cultured in different EGFP gene concentrations from 0.Ol to 1ng/u${mu}ell$. However, no significant difference in developmental rates of embryos among different concentrations of EGFP gene were observed. The highest expression rate of EGFP gene, 37.4% was obtained from the embryos injected with spermatozoa treated with 0.1 ng/${mu}ell$ EGFP gene. These results suggested that exogenous DNA could be attached to the membrane disrupted sperm, and that these sperm could be used as a vector carrying foreign DNA into embryos.

• Tuberculosis and Respiratory Diseases
• /
• v.55 no.5
• /
• pp.488-498
• /
• 2003

Background : Activation of the transcription factor NF-${\kappa}B$ has been shown to protect cells from tumor necrosis factor-alpha, chemotherapy, and radiation-induced apoptosis. NF-${\kappa}B$-dependent cIAP expression is a major antiapoptotic mechanism for that. NF-${\kappa}B$ activation and cIAP expression in A549 lung cancer cells which is relatively resistant to radiation-induced cell death were investigated for the mechanism of radioresistance. Materials and methods : We used A549 lung cancer cells and Clinac 1800C linear accelerator for radiation. Cell viability test was done by MTT assay. NF-${\kappa}B$ activation was tested by luciferase reporter gene assay, Western blot for $I{\kappa}B{\alpha}$ degradation, and electromobility shift assay. For blocking ${\kappa}B$, MG132 and transfection of $I{\kappa}B{\alpha}$-superrepressor plasmid construct were used. cIAP expression was analyzed by RT-PCR and cIAP2 promoter activity was performed using luciferase assay system. Results : MTT assay showed that cytotoxicity even 48 hr after radiation in A549 cells were less than 20%. Luciferas assay demonstrated weak NF-${\kappa}B$ activation of $1.6{\pm}0.2$ fold compared to PMA-induced $3.4{\pm}0.9$ fold. Radiation-induced $I{\kappa}B{\alpha}$ degradation was observed in Western blot and NF-${\kappa}B$ DNA binding was confirmed by EMSA. However, blocking NF-${\kappa}B$ using MG132 and $I{\kappa}B{\alpha}$-superrepressor transfection did not show any sensitizing effect for radiation-induced cell death. The result of RT-PCR for cIAP1 & 2 expression was negative induction while TNF-${\alpha}$ showed strong expression for cIAP1 & 2. The cIAP2 promoter activity also did not show any change compared to positive control with TNF-${\alpha}$. Conclusion : We conclude that activation of NF-${\kappa}B$ does not determine the intrinsic radiosensitivity of cancer cells, at least for the cell lines tested in this study.