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( Kyong Leek Jeon ),황규계 ( Kyu Kye Hwang ) 한국예방수의학회 2014 예방수의학회지 Vol.38 No.1
A cell line of bovine origin was immortalized to isolate foot-and-mouth disease virus (FMDV). The immortalization was performed by infection of bovine primary epithelial cells with a recombinant retrovirus that over expressed the human telomerase (hTERT), after primary culture of fetal bovine kidney tissue and removal of fibroblasts. After cloning the immortalized cell line into single cells, the cloned cell lines were named JNUBK-1, JNUBK-2, JNUBK-3, and JNUBK-4, according to their characteristics. To confirm the epithelial phenotype of the cell lines JNUBK-3 and JNUBK-4, which showed stable proliferation capability over 35 generations after immortalization, the expression of cytokeratin and fibronectin was measured. Finally, the FMDV titer in the JNUBK-3 and JNUBK-4 cell lines was measured and was 800∼2,000 times higher than that of the currently used cell line IRBS-2. In conclusion, more sensitive isolation and production of FMDV became possible through the use of the immortalized JNUBK-3 and JNUBK-4 cell lines.
Original articles : Investigation of national live stock breeding facilities workers in Korea
( Kyong Leek Jeon ),( Seong Geun Yun ),( Kyu Kye Hwang ) 한국예방수의학회(구 한국수의공중보건학회) 2015 예방수의학회지 Vol.39 No.2
Several national control plans have been formulated and carried out to prevent domestic animals from various infectious diseases in Korea. Typical plans include the prevention of diseases by vaccination and the quarantine activities to reduce or cut off the occurrence and transmission. Although such great efforts to prevent diseases every year, difficulties have been experienced in controling transmittable diseases like foot-and-mouth disease and bird influenza. The present study was conducted to investigate the awareness, knowledge, difficulties and routine activities in controling infectious diseases on persons-in-charge of preventive measures in national live stock breeding facilities. A questionnaire survey composed of 81 questions in three categories was carried out on 30 subjects (12 veterinarians and 18 ordinary workers) serving in nine national breeding stock farms. According to the results of the survey analysis, the difficulties and concerns in carrying out preventive measures experienced by the veterinarians and the ordinary workers were similar. Shortage of budgets and manpower was the top ranked concern in performing preventive measures for both the veterinarians and the ordinary workers. Manual or guideline on controling infectious diseases for routine or emergency case were established in all of the facilities, however those were not executed practically in most cases. Instructional training on those manual and controling wild animal to prevent contacting breeding stock were not performed in most facilities surveyed.
College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Korea
Kyong-Leek Jeon,Kyu-Kye Hwang 충북대학교 동물의학연구소 2015 Journal of Biomedical and Translational Research Vol.16 No.1
Kidney cells of canine embryos were separated into single cells using collagenase and dispase. Primary culture was conducted using these cells. To remove fibroblasts, these cells were treated with edetate disodium dihydrate (Na2EDDA), and pure epithelial cells were separated. Recombinant retrovirus particles that manifest teromerase were produced and inoculated into primary culture cells to produce immortalized canine cell strains (JNUCK-1 and JNUCK-2). To examine the characteristics of the produced cell strains, the growth curve, maximum cultured households, and expressed proteins (keratin) were identified. The JNUCK-1 and JNUCK-2 cell lines showed division ability until the 30th generation without growth retardation. JNUCK-1 and JNUCK-2 cell lines clearly expressed telomerase until the 25th generation. The canine distemper virus (CDV) was inoculated into the JNUCK-1 and JNUCK-2 cell lines, as well as in the Madin–Darby canine kidney (MDCK) cell line. The maximum titer of CDV from the JNUCK-1 cell strain was about 200 times higher than that from the MDCK cell strain. However, the JNUCK-2 cell strain produced a lower titer than the MDCK cell strain. We established a new canine kidney epithelial cell line (JNUCK-1) that could produce CDV with high titer.
High titer production of canine distemper virus using cloned MDCK cell Lines
( Kyong Leek Jeon ),( Soon Beom Cho ),( Kyu Kye Hwang ) 한국수의공중보건학회 2012 예방수의학회지 Vol.36 No.1
Canine distemper virus (CDV) causes serious and often fatal disease in dogs. Currently, various cells or cell lines have been used to detect or produce CDV. In order to set up the conditions, we separated two different cell lines from Madin-Darby canine kidney (MDCK) and named as MDCK-F (fibroblast-like) and MDCK-E (epithelial-like) by Na2EDDA treatment. CDV seed virus was prepared using MDCK cells and inoculated into MDCK-F and MDCK-E including MDCK with various ranges of multiplicity of infection (MOI) to confirm the optimal amount of virus inoculation. The virus titer of TCID50/ml was calculated by inoculation of serially diluted virus into 96-well plate of MDCK cells. The titer and cytopathic effect (CPE) in MDCK-E were compared to those in MDCK-F. The titer of seed CDV was 1.24×106 TCID50/ml. Optimal MOI was about 0.1 for both MDCK-F and MDCK-E to obtain highest titers of 108 TCID50/ml and 5×108 TCID50/ml respectively. CPE in MDCK-E was shown 4 days after inoculation whether in MNCK-F 5~6 days after inoculation. We can obtain highest titer of 5×108 TCID50/ml with 0.1 MvvOI using MDCK-E. MDCK-E was more susceptible for CDV production than MDCK-F.
High Titer Production of Equine Herpes Virus-1 Using MDBK Cell Culture System
Kyong-Leek Jeon,Soon-Beom Cho,Okjin Kim,Yoon-Kyu Lim,Hong-Gu Ju,Young-Heun Jee,Young-Min Yun,Joo Myoung Lee,Kyu-Kye Hwang 한국실험동물학회 2009 Laboratory Animal Research Vol.25 No.3
Equine herpesvirus-1 (EHV) is one of the major causes of economic loss in the equine industry. High titer production of the viral antigen is essential for vaccine development and diagnostic applications. In order to produce high titer EHV-1, we investigated various culture conditions such as amount of virus inoculation, cell population, timing of virus harvesting, and the fetal bovine serum (FBS) content of culture media using Madin-Darby bovine kidney (MDBK) cell. We obtained highest titer of EHV when 80% mono-layered MDBK cells were infected with 1 multiplicity of infection (MOI) and the culture supernatant were harvested 36 hours post-infection. The virus titer was higher for media containing 2% FBS than for serum-free media. Conclusively, we could produce EHV up to 1.4×10¹¹ plaque forming unit (PFU)/mL in culture supernatant.
Primary Bovine Cell Cultures Using Various Digestion Conditions
Kyong-Leek Jeon,Kyu-Kye Hwang 한국실험동물학회 2009 Laboratory Animal Research Vol.25 No.3
We investigated various culture conditions to determine most appropriate method of culturing primary cells from bovine fetus. Organs were extracted from a fetus, minced and digested with several proteolytic enzymes (trypsin, collagenase and dispase). Removing of red blood cell (RBC) was carried out either by dilution method (dilution of minced tissues with culture medium) or by lysis method (treatment of minced tissues with a reagent containing NH₄Cl in HEPES). The dilution method was almost as efficient in removing RBC as the lysis method. But the lysis method damaged the cells greatly, resulting in very low survival rate for digested cells. The optimal digestive conditions differed slightly but were very similar for different organs. Trypsin digested tissues very quickly but greatly damaged cells. In the case of minced kidney tissues, treatment with 0.25% collagenase for 30 minutes followed by application of 0.01% dispase for 10 minutes resulted in most efficient digestion with minimal damage to digested cells.
Establishing Bovine Epithelial Cells by Removing Fibroblast During Primary Culture
Kyong-Leek Jeon,Kyu-Kye Hwang 한국실험동물학회 2009 Laboratory Animal Research Vol.25 No.3
Fibroblasts are usually mixed into primary cultures of epithelial cells during enzymatic digestion of animal tissues or organs. They proliferate quickly and grow over the population of epithelial cells. In order to establish epithelial cell culture, we investigated possible ways to eliminate fibroblasts using three epithelial-derived cultures of bovine fetus. After digestion of fetal tissues with collagenase and dispase, cells were cultured in the medium of Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) for 24 hours. The cultured cells were treated with 0.01% edetate disodium dihydrate (Na₂EDDA) and detached fibroblasts were removed by changing media. The epithelial cells with their own shape were confirmed under a microscope. If fibroblast like cells were still present after the first elimination, they were removed again with Na₂EDDA treatment after culturing cells in DMEM containing 2% FBS. This procedure was repeated until almost pure primary epithelial cultures were established. The optimal method of fibroblast elimination was treating cells with Na₂EDDA for 3 minutes, 24 hours after primary cultivation in 10% FBS media and followed by treating cells with Na₂EDDA for 2 minutes, 6 hours after cultivation in 2% FBS media. We were able to obtain almost pure epithelial cells by the additional treatment with Na₂EDDA for 3 minutes, 12-24 hours after cultivation in 2% FBS media.