• Title/Summary/Keyword: Knock-out

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Development of a toxA Gene Knock-out Mutant of Pasteurella multocida and Evaluation of its Protective Effects

  • Kim Tae-Jung;Lee Jae-Il;Lee Bong-Joo
    • Journal of Microbiology
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    • v.44 no.3
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    • pp.320-326
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    • 2006
  • Pasteurella multocida is an important veterinary and opportunistic human pathogen. In particular, strains of P. multocida serogroup D cause progressive atrophic rhinitis, and produce a potent, intracellular, mitogenic toxin known as P. multocida toxin (PMT), which is encoded by the toxA gene. To further investigate the toxigenic and pathogenic effects of PMT, a toxA-deleted mutant was developed by homologous gene recombination. When administrated to mice, the toxigenicity of the toxA mutant P. multocida was drastically reduced, suggesting that the PMT constributes the major part of the toxigenicity of P, multocida. Similar results were obtained in a subsequent experiment, while high mortalities were observed when toxA(+) P. multocida bacterial culture or culture Iysate were administrated. Mice immunized with toxA(-) P. multocida were not protected (none survived) following challenge with toxA(+) P. multocida or bacterial culture Iysate (toxin). These results suggest that the toxigenicity of P. multocida is mainly derived from PMT.

Use of Tumor Necrosis Factor Receptor (TNFR)-Knockout Mice to Probe the Mechanism of Chemically-Induced Asthma

  • Karol, Meryl H.;Matheson, Joanna M.;Lange, Robert W.;Lemus, Ranulfo;Luster, Michael I.
    • Toxicological Research
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    • v.17
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    • pp.305-307
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    • 2001
  • Toluene diisocyanate (TDI) is widely used in the manufacture of polyurethanes and is a recognized cause of occupational asthma. Although extensive investigations have been undertaken, the molecular mechanism(s) of the disease is still unclear. We hypothesized that inflammatory cytokines are required during both the sensitization and elicitation phases of the disease and have utilized TNF-R knock-out (KO) mice to address the hypothesis. Black C57 TNFR knock-out mice were exposed to TDI by sc injection and challenged by inhalation of 100 ppb TDI vapor. Control animals included: wild type C57 animals, sham-exposed animals that were challenged with TDI, and animals that were injected with anti-TNF antibodies prior to sensitization and again prior to challenge. Total IgE was increased in the knock-out animals compared with the wild type sensitized and challenged animals whereas TDI-specific IgG antibodies did not differ significantly in KO and wild type animals. There was less inflammation in the nares and trachea in KO animals compared with the wild type animals exposed to TD1 as well as less goblet cell hyperplasia and epithelial damage. Airway reactivity was assessed in animals treated with anti-TNF$\alpha$ antibody and found to be substantially reduced compared with that in sensitized and challenged animals. These results indicate that TNF$\alpha$ plays a role in the immunologic and physiologic responses and in airways inflammation in this animal model and suggests a role for TNF in occupational asthma due to TDI.

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A Case Study on Battle of Forms in International Commercial Contracts (국제상사계약에서 서식분쟁 사례에 관한 연구)

  • Han, Na-Hee;Ha, Choong-Lyoung
    • Korea Trade Review
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    • v.42 no.5
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    • pp.19-42
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    • 2017
  • Nowaday typically international commercial transaction, buyer and seller communicate with each other using standard forms. So called "the battle of forms" results from the exchanges of these forms. There are several problems that have to be solved in the battle of the forms. For example, Do the exchanges of the different terms form a contract? What are the contract terms? Which party's terms could apply? etc. Around the world, two basic types of rules are applied to solve the problem as to the battle of forms : last-shot rule and knock-out rule. In 2015, Hague Conference in Private International Law finally approved Hague Principles. The principles deal with the battle of the forms. Also in 2013, CISG Advisory Council adopted the "Black letter rules" to provide an effective way of resolving regarding the inclusion of standard terms under the CISG. This study would try to comprehensively review the battle of forms concerning Hague Principles and CISG. The aim of this study is to propose the most appropriate way to resolve the problem of the battle of forms both parties.

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Analysis of Frozen-Thawed Sperm Characteristic in Alpha 1,3-Galactosyltransferase(GalT) Knock-out Cloned Miniature Pig (Alpha 1,3-Galactosyltransferase (GalT) Knock-out 복제 미니돼지 정액의 동결-융해 후 정액 성상 분석)

  • Woo, Jea-Seok;Lee, Yong-Seung;Yoo, Han-Jun;Hwang, Seong-Soo;Oh, Keon-Bong;Cheong, Hee-Tae;Yang, Boo-Keun;Park, Soo-Bong;Park, Choon-Keun
    • Reproductive and Developmental Biology
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    • v.35 no.3
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    • pp.385-390
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    • 2011
  • The purpose of this study was undertaken to evaluate of cryopreservation efficiency in ${\alpha}$ 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen ($LN_2$) vapours for 10 min before placing them into $LN_2$ for cryopreservation. After thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.

Efficient Gene Targeting using Nuclear Localization Signal (NLS) and Negative Selection Marker Gene in Porcine Somatic Cells

  • Kim, Hye Min;Lee, Sang Mi;Park, Hyo Young;Kang, Man-Jong
    • Reproductive and Developmental Biology
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    • v.38 no.2
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    • pp.71-77
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    • 2014
  • The specific genetic modification in porcine somatic cells by gene targeting has been very difficult because of low efficiency of homologous recombination. To improve gene targeting, we designed three kinds of knock-out vectors with ${\alpha}1,3$-galactosyltransferase gene (${\alpha}1,3$-GT gene), DT-A/pGT5'/neo/pGT3', DT-A/NLS/pGT5'/neo/pGT3' and pGT5'/neo/ pGT3'/NLS. The knock-out vectors consisted of a 4.8-kb fragment as the 5' recombination arm (pGT5') and a 1.9-kb fragment as the 3' recombination arm (pGT3'). We used the neomycin resistance gene (neo) as a positive selectable marker and the diphtheria toxin A (DT-A) gene as a negative selectable marker. These vectors have a neo gene insertion in exon 9 for inactivation of ${\alpha}1,3$-GT locus. DT-A/pGT5'/neo/pGT3' vector contain only positive-negative selection marker with conventional targeting vector. DT-A/NLS/pGT5'/neo/pGT3' vector contain positive-negative selection marker and NLS sequences in upstream of 5' recombination arm which enhances nuclear transport of foreign DNA into bovine somatic cells. pGT5'/neo/pGT3'/NLS vector contain only positive selection marker and NLS sequence in downstream of 3' recombination arm, not contain negative selectable marker. For transfection, linearzed vectors were introduced into porcine ear fibroblasts by electroporation. After 48 hours, the transfected cells were selected with $300{\mu}g/ml$ G418 during 12 day. The G418-resistant colonies were picked, of which 5 colonies were positive for ${\alpha}1,3$-GT gene disruption in 3' PCR and southern blot screening. Three knock-out somatic cells were obtained from DT-A/NLS/ pGT5'/neo/pGT3' knock-out vector. Thus, these data indicate that gene targeting vector using nuclear localization signal and negative selection marker improve targeting efficiency in porcine somatic cells.

Production of ${\alpha}$1,3-Galactosyltransferase (GalT) Double Knock-out (-/-) Transgenic Pigs for Xenotransplantation (${\alpha}$1,3-Galactosyltransferase(GalT) 유전자가 완전 Knock-out(-/-)된 바이오장기용 형질 전환 돼지 생산)

  • Hwang, Seong-Soo;Oh, Keun-Bong;Kim, Dong-Hoon;Woo, Jea-Seok;Shim, Ho-Sup;Yun, Ik-Jin;Park, Jin-Ki;Im, Gi-Sun
    • Journal of Embryo Transfer
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    • v.27 no.1
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    • pp.9-14
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    • 2012
  • This study was conducted to analyze the transgenic efficiency and sex ratio in ${\alpha}$-1,3-galactosyltransferase (GalT) knock-out (KO) transgenic pigs according to generation. GalT KO piglets were produced by artificial insemination or natural mating. The transgenic confirmation of GalT KO was evaluated by PCR amplification using specific primers. After electrophoresis, three types of bands were detected such as 2.3 kb single band (Wild), 2.3 and 3.6kb double bands (GalT KO -/+; heterozygote), and 3.6kb single band (GalT KO -/-; homozygote). Transgenic efficiency in F1 generation was 64.5% (23/35) of GalT KO (-/+). In F2 generation, GalT KO transgenic efficiency was 36.4% (21/57, Wild), 47.5% (28/57, GalT KO -/+), and 16.1% (8/57, GalT KO -/-), respectively. Interestingly, no homozygote piglets were born in 6 deliveries among total 11 deliveries, although they were pregnant between male (M) and female (F) $F_1$ heterozygote. In the 5 litters including at least one GalT KO -/- piglet, the transgenic efficiency was 13.3% (2/24, Wild), 51.3% (14/24, GalT KO -/+), and 35.3% (8/24, GalT KO -/-), respectively. The sex ratio of M and F was 40:60 in $F_1$ and 49:51 in $F_2$ generation, respectively. Based on these results, GalT KO transgenic pigs have had a reproductive ability with a normal range of transgenic efficiency and sex ratio.

Constructing Gene Regulatory Networks using Knock-out Data (Knock-out 데이터를 이용한 유전자 조절망의 구성)

  • Hong, Sung-Ryong;Sohn, Ki-Rack
    • Journal of the Korea Society of Computer and Information
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    • v.12 no.6
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    • pp.105-113
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    • 2007
  • A gene regulatory network is a network of genes representing how genes influence the activities of other genes. Nowadays from microarray experiments, a large number of measurements on the expression levels of genes are available. One of typical data is the so-called "steady-state model" data measuring the expression levels of other genes after knocking out a particular gene. This paper shows how to reverse engineer a parsimonious gene regulatory network, using these measurement data. Our model considers auto-regulation, which forms a cycle in a genetic network. We also model repressor and enhancer roles of genes. which are not considered in previous known methods.

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A Study on the Knocking Characteristics with Various Excess Air Ratio in a HCNG Engine (HCNG 엔진의 공기과잉율 변화에 따른 노킹 특성에 관한 연구)

  • Lim, Gihun;Park, Cheolwoong;Lee, Sungwon;Choi, Young;Kim, Changgi;Lee, Janghee
    • Journal of the Korean Institute of Gas
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    • v.17 no.1
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    • pp.7-12
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    • 2013
  • As emission regulation for vehicle has been reinforced, many researches carried out for HCNG(hydrogen-natural gas blends) fuel to the conventional compressed natural gas (CNG) engine. However, abnormal combustion such as backfire, pre-ignition or knocking can be caused due to high combustion speed of hydrogen and it can result in over heating of engine or reduction of thermal efficiency and power output. In the present study, improvement of combustion performance was observed with HCNG fuel since it can extend a flammability limit. Knocking characteristics for CNG and HCNG fuel were investigated. Feasibility of HCNG fuel was evaluated by checking the knock margin according to excess air ratio. The operation of engine with HCNG was stable at minimum advance for best torque(MBT) spark timing and knock phenomena were not detected. However, it is necessary to prepare higher knock tendency since possibility of knock is higher with HCNG fuel.