• Title/Summary/Keyword: Gene modified

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Investigation of Possible Gene Transfer to Soil Microorganisms for Environmental Risk Assessment of Genetically Modified Organisms

  • Kim, Young-Tae;Park, Byoung-Keun;Hwang, Eui-Il;Yim, Nam-Hui;Kim, Na-Rae;Kang, Tae-Hoon;Lee, Sang-Han;Kim, Sung-Uk
    • Journal of Microbiology and Biotechnology
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    • v.14 no.3
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    • pp.498-502
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    • 2004
  • The current study was conducted to monitor the possibility of the gene transfer among soil bacteria, including the effect of drift due to rain and surface water, in relation to the release of genetically modified organisms into the environment. Four types of bacteria, each with a distinct antibiotic marker, kanamycin-resistant P. fluorescens, rifampicin-resistant P. putida, chloramphenicol-resistant B. subtilis, and spectinomycin-resistant B. subtilis, were plated using a small-scale soil-core device designed to track drifting microorganisms. After three weeks of culture in the device, no Pseudomonas colonies resistant to both kanamycin and rifampicin were found. Likewise, no Bacillus colonies resistant to both chloramphenicol and spectinomycin were found. The gene transfer from glyphosate-tolerant soybeans to soil bacteria, including Rhizobium spp. as a symbiotic bacteria, was examined by hybridization using the DNA extracted from soil taken from pots, in which glyphosate-tolerant soybeans had been growing for 6 months. The results showed that 35S, T-nos, and EPSPS were observed in the positive control, but not in the DNA extracted from the soilborne microorganisms. In addition, no transgenes, such as the 35S promoter, T-nos, and EPSPS introduced into the GMO soybeans were detected in soilborne bacteria, Rhizobium leguminosarum, thereby strongly rejecting the possibility of gene transfer from the GMO soybeans to the bacterium.

Production of Cloned Pigs Derived from Double Gene Knockout Cells Using CRISPR/Cas9 System and MACS-based Enrichment System

  • Cho, Bumrae;Kim, Su Jin;Lee, Eun-Jin;Ahn, Sun Mi;Lee, Jin Seok;Ji, Dal-young;Lee, Sang Hoon;Kang, Jung-Taek
    • Journal of Embryo Transfer
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    • v.33 no.4
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    • pp.245-254
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    • 2018
  • Pigs are considered as optimal donor animal for the successful xenotransplantation. To increase the possibility of clinical application, genetic modification to increase compatibility with human is an important and essential process. Genetic modification technique has been developed and improved to produce genetically modified pigs rapidly. CRISPR/Cas9 system is widely used in various fields including the production of transgenic animals and also can be enable multiple gene modifications. In this study, we developed new gene targeting vector and enrichment system for the rapid and efficient selection of genetically modified cells. We conducted co-transfection with two targeting vectors for simultaneous inactivation of two genes and enrichment of the genetically modified cells using MACS. After this efficient enrichment, genotypic analysis of each colony showed that colonies which have genetic modifications on both genes were confirmed with high efficiency. Somatic cell nuclear transfer was conducted with established donor cells and genetically modified pigs were successfully produced. Genotypic and phenotypic analysis of generated pigs showed identical genotypes with donor cells and no surface expression of ${\alpha}$-Gal and HD antigens. Furthermore, functional analysis using pooled human serum revealed dramatically reduction of human natural antibody (IgG and IgM) binding level and natural antibody-mediated cytotoxicity. In conclusion, the constructed vector and enrichment system using MACS used in this study is efficient and useful to generate genetically modified donor cells with multiple genetic alterations and lead to an efficient production of genetically modified pigs.

Application of a Promoter Isolated from Chlorella Virus in Chlorella Transformation System

  • Park, Hyoun-Hyang;Park, Tae-Jin
    • The Plant Pathology Journal
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    • v.20 no.2
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    • pp.158-163
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    • 2004
  • Chlorella is a eukaryotic microalgae which shares metabolic pathways with higher plants. These charac-teristics make chlorella a potential candidate for eukaryotic overexpression systems. Recently, a foreign flounder growth hormone gene was stably introduced and expressed in transformed Chlorella ellipsoidea by using a modified plant transformation vector that contains cauliflower mosaic virus (CaMV) 35S pro-moter and the phleomycin resistant Sh ble gene as a selection marker. In this study, this same vector was modified by incorporating a promoter and a 3' UTR region of the 33kDa peptide gene from a chlorella virus that was isolated in our laboratory. The 33kDa gene promoter was used to replace the 35S promoter and the 3' UTR was introduced to separate the target gene and downstream Sh ble gene. Three different chlorella transformation vectors containing human erythropoietin (EPO) gene were constructed. The mp335EPO vector consists of a promoter from the 33kDa peptide gene, whereas the mp3353EPO vector contains the same promoter from the 33kDa peptide gene and its 3' UTR. The mp35S33pEPO vector contains the 35S promoter and the 3' UTR from the 33 kDa peptide gene. There was no significant difference in the expression levels of EPO protein in chlorella cells transformed with either of three of the transformation vectors. These data indicate that the promoters from the chlorella virus are comparable to the most common CaMV 35S promoter. Furthermore, these data suggest that other promoters from this virus can be used in future construction of chlorella transformation system for higher expression of target proteins.

Safety evaluation of gene therapy - a case study of naked DNA product

  • Ahn, Byung-Ok
    • Proceedings of the Korean Society of Toxicology Conference
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    • 2003.10b
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    • pp.86-86
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    • 2003
  • Gene therapy is a medical intervention based on modification of the genetic material of living cells. Gene transfer usually conducted using bacterial plasmid DNA and/or virus vector to express a specific protein. Gene transfer medicinal products classified as naked nucleic acid, complexed nucleic acid or non-viral vectors, viral vector, and genetically modified cells according to biological origin.(omitted)

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Gene Flow from GM Cabbage to Non-GM Control (유전자변형 양배추로부터 비유전자변형 모본으로의 유전자 이동성)

  • Kim, Young-Joong;Nam, Kyong Hee;Pack, In Soon;Park, Jung-Ho;Jeong, Soon-Chun;Harn, Chee Hark;Kim, Chang-Gi
    • Korean Journal of Agricultural Science
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    • v.41 no.3
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    • pp.157-161
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    • 2014
  • Understanding the gene flow from genetically modified (GM) crops to conventional crops is important to prevent and mitigate seed contamination caused by pollen-mediated gene flow. We conducted a field test to investigate the gene flow from diamondback moth resistant GM cabbage (Brassica oleracea var. capitata) containing cry1Ac1 gene, to a non-GM control line AD126. GM and non-GM cabbage plants were cultivated in the field and pollinated using Bombus terrestris under the nets during the flowering periods. After seeds were collected from non-GM plants, hybrids between them and the GM cabbages were screened by multiplex PCR targeting cry1Ac1 gene. Out of 878 germinated seedlings, 168 hybrids were found and the average gene flow frequency was 19.7%. Because cabbage is mainly pollinated by insect pollinators, large-scale field tests are needed to study gene flow of GM cabbage.

Applications of Genetically Modified Tools to Safety Assessment in Drug Development

  • Kay, Hee-Yeon;Wu, Hong-Min;Lee, Seo-In;Kim, Sang-Geon
    • Toxicological Research
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    • v.26 no.1
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    • pp.1-8
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    • 2010
  • The process of new drug development consists of several stages; after identifying potential candidate compounds, preclinical studies using animal models link the laboratory and human clinical trials. Among many steps in preclinical studies, toxicology and safety assessments contribute to identify potential adverse events and provide rationale for setting the initial doses in clinical trials. Gene modulation is one of the important tools of modern biology, and is commonly employed to examine the function of genes of interest. Advances in new drug development have been achieved by exploding information on target selection and validation using genetically modified animal models as well as those of cells. In this review, a recent trend of genetically modified methods is discussed with reference to safety assessments, and the exemplary applications of gene-modulating tools to the tests in new drug development were summarized.

Delivery of growth factor-associated genes to mesenchymal stem cells for cartilage and bone tissue regeneration

  • Ahn, Jongchan;Park, Seah;Cha, Byung-Hyun;Kim, Jae Hwan;Park, Hansoo;Joung, Yoon Ki;Han, Inbo;Lee, Soo-Hong
    • Biomaterials and Biomechanics in Bioengineering
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    • v.1 no.3
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    • pp.151-162
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    • 2014
  • Genetically-modified mesenchymal stem cells (GM-MSCs) have emerged as promising therapeutic tools for orthopedic degenerative diseases. GM-MSCs have been widely reported that they are able to increase bone and cartilage tissue regeneration not only by secreting transgene products such as growth factors in a long-term manner, also by inducing MSCs into tissue-specific cells. For example, MSCs modified with BMP-2 gene increased secretion of BMP-2 protein resulting in enhancement of bone regeneration, while MSCs with TGF-b gene did cartilage regeneration. In this review, we introduce several growth factors for gene delivery to MSCs and strategies for bone and cartilage tissue regeneration using GM-MSCs. Furthermore, we describe strategies for strengthening GM-MSCs to more intensively induce tissue regeneration by co-delivery system of multiple genes.

Quantitative Analysis of Two Genetically Modified Maize Lines by Real-Time PCR

  • Lee Seong-Hun;Kang Sang-Ho;Park Yong-Hwan;Min Dong-Myung;Kim Young-Mi
    • Journal of Microbiology and Biotechnology
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    • v.16 no.2
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    • pp.205-211
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    • 2006
  • A quantitative analytical method to detect new lines of genetically modified (GM) maize, NK603 and TC1507, has been developed by using a real-time polymerase chain reaction (PCR). To detect these GM lines, two specific primer pairs and probes were designed. A plasmid as a reference molecule was constructed from an endogenous DNA sequence of maize, a universal sequence of a cauliflower mosaic virus (CaMV) 35S promoter used in most GMOs, and each DNA sequence specific to the NK603 and TC1507 lines. For the validation of this method, the test samples of 0, 0.1, 0.5, 1.0, 3.0, 5.0, and 10.0% each of the NK603 and TC1507 GM maize were quantitated. At the 3.0% level, the biases (mean vs. true value) for the NK603 and TC1507 lines were 3.3% and 15.7%, respectively, and their relative standard deviations were 7.2% and 5.5%, respectively. These results indicate that the PCR method developed in this study can be used to quantitatively detect the NK603 and TC1507 lines of GM maize.

Comparison of Expression Pattern of Housekeeping Genes in Mice fed Genetically Modified Rice (유전자 이입에 따른 GM쌀 섭취 마우스의 Housekeeping Gene 발현 패턴 비교)

  • Lee, Dong-Yeob;Heo, Jin-Chul;Lee, Kyu-Hyun;Kim, Dong-Ho;U, Sang-Uk;Cho, Hyun-Suk;Lee, Sang-Han
    • Food Science and Preservation
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    • v.14 no.6
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    • pp.688-694
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    • 2007
  • To evaluate the human risk of long-term intake of genetically modified (GM) rice, we carried out RT-PCR of housekeeping genes. Housekeeping genes, which show highly uniform expression in living organisms during various stages of development and under different environmental conditions, were normalized by RT-PCR. We assessed the expression of 10 common housekeeping genes (18s rRNA, 25S rRNA, UBC, UBQ5, UBQ10, ACT11, GAPDH, eEF-$1{\alpha}$, ${\beta}$-TUB, GAPDH, ${\beta}$-actin, B2m, G6pd2, Gyk, Gus, Hprt, Cyclophlin A, Tfrc, ${\alpha}$-tubulin and RPL13A) in the liver, stomach, small intestine, large intestine, kidney and spleen of mice fed GM or non-GM rice. We found no significant differences in the expression of housekeeping genes between the two groups of mice.

Mtatioal Analysis of the Role of vir-box in the Expression of the virE Gene

  • Han, Seong-Su;Sim, Woong-Seop
    • Journal of Microbiology
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    • v.37 no.3
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    • pp.175-179
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    • 1999
  • To elucidate the role of vir-box in the expression of the virE gene, the vir-box was modified by site-directed mutagenesis and tested for ${\beta}$-galactosidase activities. A, C, T T, A, C substitutions at -62, -63, and -65 positions, destroying the 5'-region of the vir-box and A T at position -55, destroying the 3'-region of the vir-box respectively, showed only 17% promoter activity. When the vir-box was modified to contain perfect dyad symmetry structure (DSR) by the substitutions T, G A, T at -60 an d-61 positions, ${\beta}$-glactosidase activity increased 302%. These results indicate that the 5' and 3'-region of vir-box as well as the imperfect DSR of the vir-box itself may play a very important role in the regulation of virE gene expression.

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