• Asian Pacific Journal of Cancer Prevention
• /
• 제13권3호
• /
• pp.781-784
• /
• 2012

The aim of this study was to evaluate the effect of the adenovirus-mediated double suicide gene (CD/TK) for selective killing of gastric cancer cells. Gastric cancer cells SCG7901 and normal gastric epithelial cell lines were infected by adenoviruses Ad-survivin/GFP and Ad-survivin/CD/TK. GFP expression and CD-TK were detected by fluorescence microscopy and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. After treatment of the infected cells with the pro-drugs ganciclovir (GCV) and/or 5-FC, the cell growth status was evaluated by methyl thiazolyl tetrazolium assay. Cell cycle changes were detected using flow cytometry. In nude mice bearing human gastric cancer, the recombinant adenovirus vector was injected directly into the tumor followed by an intraperitoneal injection of GCV and/or 5-FC. The subsequent tumor growth was then observed. The GFP gene driven by survivin could be expressed within the gastric cancer line SCG7901, but not in normal gastric epithelial cells. RT-PCR demonstrated the presence of the CD/TK gene product in the infected SCG7901 cells, but not in the infected normal gastric epithelial cells. The infected gastric cancer SCG7901, but not the gastric cells, was highly sensitive to the pro-drugs. The CD/TK fusion gene system showed significantly greater efficiency than either of the single suicide genes in killing the target cells (P<0.01). Treatment of the infected cells with the pro-drugs resulted in increased cell percentage in G0-Gl phase and decreased percentage in S phase. In nude mice bearing SCG7901 cells, treatment with the double suicide gene system significantly inhibited tumor growth, showing much stronger effects than either of the single suicide genes (P<0.01). The adenovirus-mediated CD/TK double suicide gene driven by survivin promoter combined with GCV an 5-FC treatment could be an effective therapy against experimental gastric cancer with much greater efficacy than the single suicide gene CD/TK combined with GCV or 5-FC.

• 생명과학회지
• /
• 제23권2호
• /
• pp.290-298
• /
• 2013

Mycobacterium 속은 Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis와 같은 동물과 인체에 병원성을 나타내는 세균 종을 다수 포함하고 있다. 이들의 숙주에서의 생존과 병원성에 관한 유전학적 정보를 확보하는 것은 매우 중요하지만, 효과적인 유전학적 도구가 부족하였기 때문에 이들에 관한 연구가 미비하였다. 따라서 mycobacteria의 연구를 위한 분자생물학적 실험 도구로서 다양한 기능성 vector들이 고안되었고, 이러한 기능성 vector의 개발은 실질적으로 mycobacteria에서의 연구 효과를 증진시켰다. 본 연구에서는 Mycobacterium smegmatis에 적용 가능하고 기존에 제시되었던 mycobacteria 연구에 있어서의 한계점을 극복하기 위한 노력의 일환으로, 기능성 vector인 temperature-sensitive replication origin (TSRO)과 counterselectable marker로 levansucrase를 암호화하는 sacB 유전자를 포함하는 suicide vector pKOTs, chromosomal DNA로 site-specific recombination을 통해 삽입되는 lacZ transcriptional fusion vector pMV306lacZ, 그리고 TSRO를 가지는 minitransposon vector pTnMod-OKmTs를 개발하였다. 이 vector들은 실질적으로 M. smegmatis에서 효과적으로 작동하는 것이 확인되었으며 목적으로 하는 실험 결과 도출 가능성 또한 보여주었다. 따라서 이들 vector는 앞으로의 mycobacteria에 대한 효과적인 연구 기반이 될 것으로 기대된다.

• Journal of Microbiology and Biotechnology
• /
• 제13권2호
• /
• pp.305-308
• /
• 2003

We earlier reported the identification of bif-k, t-3, and a third ORF from an enterotoxigenic strain of Bacteroides fragilis 419, which was isolated from the blood of a Korean patient suffering from systemic infections. In the present study, the deleted fragments of the t-3 and the bft-k genes from B. fragilis 419 were cloned into suicide vector PJST55 and used to create a mutant with chromosomal disruption of the t-3 and bft-k genes. Structures of the selected mutants, DMP-2 and DBT-4, were found to be intermediate forms that integrated the suicide vector into the chromosome. t-3 disrupted Dmp-2 and Bft-k disrupted DBT-4 did not react with polyclonal antibodies against T-3 or BFT-K, and had no biological activity in $HT29/C_1$, cells.

• Journal of Korean Neurosurgical Society
• /
• 제30권sup1호
• /
• pp.13-19
• /
• 2001

Objective : We investigated the feasibility of a double suicide gene/prodrug therapy, involving direct introduction of the herpes simplex virus Type 1 thymidine kinase(TK) gene and the Escherichia coli cytosine deaminase(CD) gene, via a recombinant adenoviral vector and ganciclovir(GCV) and/or 5-fluorocytosine(5-FC) treatment, in C6 glioma cells. Methods : Efficient gene transfer and transduction of C6 glioma cells via a recombinant adenovirus were evaluated by infecting cells with adenovirus bearing the ${\beta}$-galactosidase gene and then staining cells with 5-bromo-4-chloro-3-indolyl-13-D-galactoside. CD/TK expression in cells infected with adenovirus bearing the CD/TK gene(ad-CD/TK) was examined by immunoblotting analysis. For in vitro cytotoxicity experiments, the cells were infected with ad-CD/TK or ad-${\Delta}E1$(as a control). After addition of a variety of concentrations of GCV and 5-FU, either separately or in combination, cell viability was determined by staining the cells with crystal violet solution 6 days after infection. Result : C6 glioma cells were efficiently transduced with recombinant adenoviral vector at multiplicities of infection of 200 or more. In vitro cytotoxicity of GCV and/or 5-FC, either alone or in combination, was exclusively observed in the cells transduced with ad-CD/TK. Obvious cytotoxicity(>50% inhibition) was observed in the presence of 5-FC at concentrations greater than 30ug/ml or GCV at concentrations greater than 0.3ug/ml at a multiplicity of infection of 100. Additionally, cytotoxicity in the presence of both GCV and 5-FC was greater than that after sinlge-prodrug treatments, indicating additive effects of the prodrug treatments. Conclusion : The administration of a double-suicide gene/prodrug therapy might have great potential in the treatment of brain tumors.

• 생명과학회지
• /
• 제14권2호
• /
• pp.362-367
• /
• 2004

장염비브리오균의 숙주 내 감염을 일으키는 기작을 이해하기 위하여 세포외 효소 중의 하나인 콜라겐분해효소의 유전자가 불활성화된 돌연변이체를 제작하였다. 콜라겐분해효소의 유전자인 vppC 유전자에 항생제 내성 유전자인 nptII를 삽입하여 제작된 재조합 DNA를 suicide vector인 pDMS197에 클로닝하여 pVCM03이라 명명하였다. 재조합 suicide 플라스미드 pVCM03을 E. coli 7213에 형질전환하여 접합을 통하여 원 균주인 V. varahaemolyticus 04에 전달하였다. 전달된 pVCM03 유래의 재조합 vvpC::npfII DNA는 homologous recombination에 의해 wild-type allele와 교환되어 돌연변이체를 형성하게 되고, 돌연변이체는 10% sucrose가 함유된 TCBS 배지에서 선별되었다. Allele exchange는 PCR에 의한 증폭된 DNA의 크기 비교로 확인하였다. 돌연변이체인 V. parahaemolyticus CM은 원 균주와 비교하였을 때 약 4배정도 낮은 콜라겐 분해 활성을 나타내었고, vero cell을 이용한 MTT assay에서도 원 균주에 비하여 낮은 세포독성을 보였다.

• 대한수의학회지
• /
• 제39권2호
• /
• pp.327-337
• /
• 1999

The transposon TnphoA was used to generate avirulent mutants from a type A Pasteurella multocida. A suicide vector plasmid pRT733 carrying TnphoA, having the kanamycin resistant gene and harbored in Escherichia coli K-12 strain SM10(${\lambda}pir$), was mated with streptomycin resistant P. multocida P-1059 strain as recipient. This resulted in the generation of two TnphoA insertion mutants (transconjugants, tc95-a and tc95-b) which were resistant both to kanamycin ($Km^{R}$) and streptomycin ($Sm^{R}$), secreted alkaline phosphatase, and were avirulent to turkeys. Southern blot hybridization using two probes derived from internal fragments of TnphoA, confirmed the insertion of TnphoA into 12.9kb or 13.7kb DNA fragment from the EcoRV digested genomic fragments of transconjugants. The two transconjugants, tc95-a and tc95-b, were distinguishable from their parent strains by differences in ribotypes, and outer membrane protein profiles. TnphoA insertion in both transconjugants also resulted in constitutive expression of a 33Kd iron regulated outer membrane protein (IROMP). The gene encoding $Sm^{R}$ was also located within the same 12.9kb EcoRV genomic fragment from both transconjugants. Furthermore, our finding that the recipient P. multocida P-1059 $Sm^{R}$ strain and both transconjugants were avirulent to turkeys suggest that the either 12.9kb or 13.7kb genomic DNA contains the virulence gene and speculate that the presence of $Sm^{R}$ gene or TnphoA insertion may be responsible for regulating and inactivating the gene(s) encoding virulence in P. multocida.

• Journal of Korea Artificial Intelligence Association
• /
• 제1권1호
• /
• pp.1-6
• /
• 2023

This study developed models using decision forest, support vector machine, and logistic regression methods to predict and prevent suicidal ideation among Korean adolescents. The study sample consisted of 51,407 individuals after removing missing data from the raw data of the 18th (2022) Youth Health Behavior Survey conducted by the Korea Centers for Disease Control and Prevention. Analysis was performed using the MS Azure program with Two-Class Decision Forest, Two-Class Support Vector Machine, and Two-Class Logistic Regression. The results of the study showed that the decision forest model achieved an accuracy of 84.8% and an F1-score of 36.7%. The support vector machine model achieved an accuracy of 86.3% and an F1-score of 24.5%. The logistic regression model achieved an accuracy of 87.2% and an F1-score of 40.1%. Applying the logistic regression model with SMOTE to address data imbalance resulted in an accuracy of 81.7% and an F1-score of 57.7%. Although the accuracy slightly decreased, the recall, precision, and F1-score improved, demonstrating excellent performance. These findings have significant implications for the development of prediction models for suicidal ideation among Korean adolescents and can contribute to the prevention and improvement of youth suicide.

• Journal of Computing Science and Engineering
• /
• 제6권2호
• /
• pp.143-150
• /
• 2012

Computational techniques for topic classification can support qualitative research by automatically applying labels in preparation for qualitative analyses. This paper presents an evaluation of supervised learning techniques applied to one such use case, namely, that of labeling emotions, instructions and information in suicide notes. We train a collection of one-versus-all binary support vector machine classifiers, using cost-sensitive learning to deal with class imbalance. The features investigated range from a simple bag-of-words and n-grams over stems, to information drawn from syntactic dependency analysis and WordNet synonym sets. The experimental results are complemented by an analysis of systematic errors in both the output of our system and the gold-standard annotations.

• IMMUNE NETWORK
• /
• 제1권1호
• /
• pp.45-52
• /
• 2001

Background: Many types of cancer become resistant to current chemotherapeutic and radiotherapeutic intervention. To overcome this situation application of gene therapy by the introduction of suicide genes followed by their prodrugs may be promising. A viral enzyme, Herpes simplex thymidine kinase (HSV-tk), which converts ganciclovir from an inactive prodrug to a cytotoxic agent by phosphorylation, are being actively investigated for use in gene therapy for cancer. The purpose of this study was to determine whether combining prodrug-activating gene therapy and irradiation might result in enhanced antitumor effects. Methods: The HSV-tk gene was cloned into the retroviral vector, pLXSN and established the clones producing retroviruses carrying the HSV-tk gene. The carcinoma cell line, HCT116 and Huh-7 were transduced with high-titer recombinant retroviruses. These cell lines were treated with ganciclovir before or after irradiation for the defining combinational effect of suicide gene therapy and radiotherapy. Results: The titers of cloned PA3 17 amphotropic retroviruses ranged from 4 to 6 X $10^6CFU/ml4$. After selectional periods, the expression of HSV-tk was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). The growth of cells expressing HSV-tk was inhibited as increase of GCV dose after 48 hr and the growth inhibitory effect of GCV was much higher after 72 hr. When the cells transduced with HSV-tk gene were exposed to radiation, the growth inhibitory effect of GCV was significantly increased, as compared with non-transduced parental cells. Conclusions: The results suggest that the addition of HSV-tk gene therapy to standard radiation therapy may improve the effectiveness of treatment for solid tumors.

• 생명과학회지
• /
• 제16권7호
• /
• pp.1112-1118
• /
• 2006

돼지 위축성 비염과 개의 kennel cough의 원인균인 B. bronchiseptica는 각 숙주의 상부 호흡기관의 점막에 집락을 형성하는 병원균으로서 철이 부족한 환경에서 hydroxamate type의 alcaligin이 라는 siderophore를 생산한다. Alcaligin의 생합성에 관련하는 구조유전자 중 alcA 유전자의 기능을 밝히고자 alcA 결손돌연변이주 구축을 통하여 확인하였다. alcA 유전자 결손 돌연변이를 위해 0.6 kb alcA 5' flanking DNA와 0.7 kb alcA 3' flanking DNA fragment들을 pCP1.11을 주형으로 하여 PCR법으로 증폭한 후, 5' flanking과 3' flanking DNA가 연결된 재조합 suicide vector pDMl을 구축하여 세포 접합을 통해 B. bronchiseptica로 도입시켰다. 도입된 pDM1으로부터 allelic exchange법에 의해 alcA 유전자가 결손된 돌연변이주 B. bronchiseptica H1을 얻을 수 있었다. B. bronchiseptica H1은 야생형인 B. bronchiseptica에 비하여 alcaligin siderophore를 거의 생성하지 못하였다. alc 오페론 중 promoter와 alcA 유전자만을 가지는 재조합 플라스미드를 B. bronchiseptica Hl에 도입하였을 때 alcaligin siderophore의 생산이 회복됨을 확인할 수 있었다. 이상의 결과로부터 alcA 유전자가 alcaligin 생합성에서 매우 중요한 역할을 수행하는 것을 알 수 있었다.