• Title/Summary/Keyword: Transgene

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Characterization, detection and identification of transgenic chili pepper harboring coat protein gene that enhances resistance to cucumber mosaic virus

  • Seo, Sang-Gyu;Kim, Ji-Seong;Jeon, Seo-Bum;Shin, Mi-Rae;Kang, Seung-Won;Lee, Gung-Pyo;Hong, Jin-Sung;Harn, Chee-Hark;Ryu, Ki-Hyun;Park, Tae-Sung;Kim, Sun-Hyung
    • Journal of Plant Biotechnology
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    • v.36 no.4
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    • pp.384-391
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    • 2009
  • Previously, two events (H15 and B20) of transgenic pepper (Capsicum annuum L.) that enhanced resistance to Cucumber mosaic virus (CMV) by the introduction of CMV coat protein (CP) gene were constructed. Presently, a single copy number of the CP gene was revealed in H15 and B20 by Southern blot. To predict possible unintended effects due to transgene insertion in an endogenous gene, we carried out sequencing of the 5'-flanking region of the CP gene and a Blastbased search. The results revealed that insertion of the transgene into genes encoding putative proteins may occur in the H15 and B20 transgenic event. Mutiplex polymerase chain reaction (PCR) for simultaneous detection and identification of transgenic pepper was conducted with a set of nine primers. Both transgenic event were differentiated from non-transgenic event by the presence of 267 bp and 430 bp PCR products indicative of CP gene specific primer pairs and primer pairs targeting the CP gene and 35S promoter. H15 and B20 uniquely possessed a 390 bp and 596 bp PCR product, respectively. The presence of a 1115 bp product corresponding to intrinsic pepper actin gene confirmed the use of pepper DNA as the PCR template. The primer set and PCR conditions used presently may allow the accurate and simple identification of CMV resistant transgenic pepper.

Environmental risk assessment of genetically modified Herbicide-Tolerant zoysiagrass (Event: Jeju Green21) (제초제저항성 들잔디(Zoysia japonica Steud.) 이벤트 Jeju Green21의 환경위해성평가)

  • Bae, Tae-Woong;Kang, Hong-Gyu;Song, In-Ja;Sun, Hyeon-Jin;Ko, Suk-Min;Song, Pill-Soon;Lee, Hyo-Yeon
    • Journal of Plant Biotechnology
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    • v.38 no.2
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    • pp.105-116
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    • 2011
  • Transgenic zoysiagrass (Zoysia japonica Steud.) expressing the bar gene inserted in the plant genome has been generated previously through Agrobacterium tumefaciens-mediated transformation. The GM zoysiagrass (event: JG21) permits efficient management of weed control of widely cultivated zoysiagrass fields, reducing the frequency and cost of using various herbicides for weed control. Now we have carried out the environmental risk assessment of JG21 prior to applying to the governmental regulatory agency for the commercial release of the GM turf grass outside of test plots. The morphological phenotypes, molecular analysis, weediness and gene flow from each test plot of JG21 and wild-type zoysiagrasses have been evaluated by selectively analyzing environmental effects. There were no marked differences in morphological phenotypes between JG21 and wild-type grasses. The JG21 retained its stable integration in the host plant in T1 generation, exhibiting a 3:1 segregation ratio according to the Mendelian genetics. We confirmed the copy number (1) of JG21 by using Southern blot analysis, as the transgenic plants were tolerant to ammonium glufosinate throughout the culture period. From cross-fertilization and gene flow studies, we found a 9% cross-pollination rate at the center of JG21 field and 0% at distances over 3 m from the field. The JG21 and wild-type zoysiagrass plants are not considered "weed" because zoysiagrasses generally are not dominant and do not spread into weedy areas easily. We assessed the horizontal gene transfer (HGT) of the transgene DNA to soil microorganisms from JG21 and wild-type plants. The bar gene was not detected from the total genomic DNA extracted from each rhizosphere soil of GM and non-GM Zoysia grass fields. Through the monitoring of JG21 transgene's unintentional release into the environment, we found no evidence for either pollen mediated gene flow of zoysiagrass or seed dispersal from the test field within a 3 km radius of the natural habitat.

The expression of Foxp3 protein by retroviral vector-mediated gene transfer of Foxp3 in C57BL/6 mice (C57BL/6 마우스에서 Retroviral 벡터를 이용한 Foxp3 유전자의 도입에 의한 Foxp3 단백의 발현 양상)

  • Hwang, Insun;Ha, Danbee;Bing, So Jin;Jeon, Kyong-Leek;Ahn, Ginnae;Kim, Dae Seung;Cho, Jinhee;Lim, Jaehak;Im, Sin-Hyeog;Hwang, Kyu-Kye;Jee, Youngheun
    • Korean Journal of Veterinary Research
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    • v.52 no.3
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    • pp.183-191
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    • 2012
  • The maintenance of peripheral immune tolerance and prevention of chronic inflammation and autoimmune disease require $CD4^{+}CD25^{+}$ T cells (regulatory T cells). The transcription factor Foxp3 is essential for the development of functional, regulatory T cells, which plays a prominent role in self-tolerance. Retroviral vectors can confer high level of gene transfer and transgene expression in a variety of cell types. Here we observed that following retroviral vector-mediated gene transfer of Foxp3, transductional Foxp3 expression was increased in the liver, lung, brain, heart, muscle, spinal cord, kidney and spleen. One day after vector administration, high levels of transgene and gene expression were observed in liver and lung. At 2 days after injection, transductional Foxp3 expression level was increased in brain, heart, muscle and spinal cord, but kidney and spleen exhibited a consistent low level. This finding was inconsistent with the increase in both $CD4^{+}CD25^{+}$ T cell and $CD4^{+}Foxp3^{+}$ T cell frequencies observed in peripheral immune cells by fluorescence-activated cell-sorting (FACS) analysis. Retroviral vector-mediated gene transfer of Foxp3 did not lead to increased numbers of $CD4^{+}CD25^{+}$ T cell and $CD4^{+}Foxp3^{+}$ T cell. These results demonstrate the level and duration of transductional Foxp3 gene expression in various tissues. A better understanding of Foxp3 regulation can be useful in dissecting the cause of regulatory T cells dysfunction in several autoimmune diseases and raise the possibility of enhancing suppressive functions of regulatory T cells for therapeutic purposes.

Confirmation of Drought Tolerance of Ectopically Expressed AtABF3 Gene in Soybean

  • Kim, Hye Jeong;Cho, Hyun Suk;Pak, Jung Hun;Kwon, Tackmin;Lee, Jai-Heon;Kim, Doh-Hoon;Lee, Dong Hee;Kim, Chang-Gi;Chung, Young-Soo
    • Molecules and Cells
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    • v.41 no.5
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    • pp.413-422
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    • 2018
  • Soybean transgenic plants with ectopically expressed AtABF3 were produced by Agrobacterium-mediated transformation and investigated the effects of AtABF3 expression on drought and salt tolerance. Stable Agrobacterium-mediated soybean transformation was carried based on the half-seed method (Paz et al. 2006). The integration of the transgene was confirmed from the genomic DNA of transformed soybean plants using PCR and the copy number of transgene was determined by Southern blotting using leaf samples from $T_2$ seedlings. In addition to genomic integration, the expression of the transgenes was analyzed by RT-PCR and most of the transgenic lines expressed the transgenes introduced. The chosen two transgenic lines (line #2 and #9) for further experiment showed the substantial drought stress tolerance by surviving even at the end of the 20-day of drought treatment. And the positive relationship between the levels of AtABF3 gene expression and drought-tolerance was confirmed by qRT-PCR and drought tolerance test. The stronger drought tolerance of transgenic lines seemed to be resulted from physiological changes. Transgenic lines #2 and #9 showed ion leakage at a significantly lower level (P < 0.01) than ${\underline{n}}on-{\underline{t}}ransgenic$ (NT) control. In addition, the chlorophyll contents of the leaves of transgenic lines were significantly higher (P < 0.01). The results indicated that their enhanced drought tolerance was due to the prevention of cell membrane damage and maintenance of chlorophyll content. Water loss by transpiration also slowly proceeded in transgenic plants. In microscopic observation, higher stomata closure was confirmed in transgenic lines. Especially, line #9 had 56% of completely closed stomata whereas only 16% were completely open. In subsequent salt tolerance test, the apparently enhanced salt tolerance of transgenic lines was measured in ion leakage rate and chlorophyll contents. Finally, the agronomic characteristics of ectopically expressed AtABF3 transgenic plants ($T_2$) compared to NT plants under regular watering (every 4 days) or low rate of watering condition (every 10 days) was investigated. When watered regularly, the plant height of drought-tolerant line (#9) was shorter than NT plants. However, under the drought condition, total seed weight of line #9 was significantly higher than in NT plants (P < 0.01). Moreover, the pods of NT plants showed severe withering, and most of the pods failed to set normal seeds. All the evidences in the study clearly suggested that overexpression of the AtABF3 gene conferred drought and salt tolerance in major crop soybean, especially under the growth condition of low watering.

Production of Transgenic Porcine haboring the Human Erythropoietin(EPO) Gene (사람 조혈인자 유전자(Human Erythropoietin Gene)를 도입한 형질전환돼지 생산)

  • 이연근;박진기;민관식;이창현;성환후;전익수;임석기;양병철;임기순
    • Korean Journal of Animal Reproduction
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    • v.26 no.2
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    • pp.95-104
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    • 2002
  • This study was performed during the four seasons for the production of transgenic pigs containing the human erythropoietin(hEPO) transgene. Purebred Landrace gilts and sows approximately 8∼15 months of age (n=42) were used fur the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Synchronization and superovulation procedures were used that cyclic gilts were synchronized with 20mg altrenogest (ALT) per day for 9days after PG600 administration followed by superovulation with 1500IU pregnant mares serum gonadotropin (PMSG) and 500IU human chorionic gonadotrophin (hCG). Preparation of recombinant gene for microinjection is mice whey acidic protein promoter (mWAP) linked to human erythropoietin (hEPO) gene. After hormone treatment, 650 embryos were collected from 23 donors and 83.1% (540/650) embryos were in 1-cell stage which can be visualized the pronuclei for DNA microinjection. A total of 543 DNA microinjected embryos fiom donors were transferred to 19 synchronized recipients, seven of them maintained pregnancy and delivered 47 piglets. One of the 47 offsprings were determined to have transgene by PCR analysis. The overall rate of transgenic production was 2.13% (tansgenic/offspring). This study provides the success and useful information regarding production of transgenic pig for bioreactor research.

Effects of osmoticum treatments and shooting chances on the improvement of particle gun-mediated transformation in Phalaenopsis (유전자총을 이용한 팔레놉시스 형질전환 효율향상에 삼투압 조절제 및 발사횟수차이가 미치는 영향)

  • Roh, Hee Sun;Kim, Jong Bo
    • Journal of Plant Biotechnology
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    • v.41 no.4
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    • pp.216-222
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    • 2014
  • This study was carried out to develop an efficient transformation protocol via particle bombardment with PLBs (protocorm-like bodies) in Phalaenopsis. To achieve this aim, osmoticum treatment and an increasing shooting chances in particle bombardment process were applied for this study. In addition, pCAMBIA3301: ORE7 vector which contains a herbicide-resistance bar gene as a selectable marker and ORE7 gene as a gene of interests were employed. With regard to the increasing chances of shooting in particle bombardment, double shooting was the best results with 1.5 ~ 2.5 times higher than those of a single or triple shooting treatment in the productioon of PPT (D-L-phosphinothricin)-resistant PLBs. However, regeneration rate of shoots in double shooting was not high as a single shooting. Further, double shooting showed 35 ~ 40% higher than that of a single shooting in the frequency of browning. Regarding effects of different osmotic treatments, combination of 0.2 M sorbitol with 0.2 M mannitol showed the best results in transformation efficiency, regeneration of transformants and reduction of browning. Putative transgenic Phalaenopsis plants were analyzed by PCR analysis and confirmed the presence of bar and ORE 7 gene. Also, real-time PCR was conducted by using 21 transgenic plants and showed only 4 plants had one copy of transgene; whereas, the other 17 plants had more than 2 copies of transgene. Transgenic phalaenopsis plants produced in this study were transferred to pots and flowered normally without morphological variations in flower and leaf.

Development of Salt-Tolerant Transgenic Rice Using Soybean PR10 Gene (콩의 Pathogenesis-Related 10 유전자를 이용한 내염성 벼 형질전환 계통 개발)

  • Kim, Hyo Jin;Baek, So Hyeon;Shin, Woon Chul;Seo, Chun Sun;Park, Myoung Ryoul;Ko, Jae Kwon;Yun, Song Joong
    • Korean Journal of Breeding Science
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    • v.42 no.5
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    • pp.540-546
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    • 2010
  • This study was conducted to understand the role of soybean pathogenesis-related 10 (GmPR10) gene in salt tolerance and to develop salt-tolerant rice using GmPR10 cDNA. GmPR10 transgene was expressed constitutively in the shoot and root of the $T_1$ transgenic rice plants. Interestingly, however, the levels of the transgene expression were increased temporally up to over four- to five-fold in the shoot and root by 125 mM NaCl treatment, peaking at six hours after the treatment and decreasing thereafter. Electrolyte leakage of leaf cells under 125 mM NaCl treatment was lower in all the transgenic lines than in the control variety, Dongjin-byeo. Ability of seedlings to recover from 125 mM NaCl treatment for two weeks was higher in the transgenic plants than in the control plants. These results demonstrated that GmPR10 had function to increase cell integrity and promote growth under the saline stress imposed by NaCl. The transgenic line GmPR10-3 which showed highest ability to recover from the saline stress could be used as a potential source for salt tolerance in rice breeding programs.

Bioloistic-mediated Transformation of Cotton (Gossypium hirsutum L.): Embryogenic Calli as Explant

  • Haq Ikram-ul;Asad Shaheen;Zafar Yusuf
    • Journal of Plant Biotechnology
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    • v.7 no.4
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    • pp.211-218
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    • 2005
  • Genetic transformation was carried out by using biolistic gun method. The hypocotyl derived embryogenic calli (explants) of cotton (Gossypium hirsutum L.) cv. Cocker-312 were transformed with a recombinant pGreen II plasmid, in which both, bar (selection marker) and GUS (${\beta}$-glucuronidase) reporter genes were incorporated. Explants were arranged on osmoticum-containing medium (0.5M mannitol) 4 hours prior to and 16 hours after bombardment that was resulted into an increase about >80% for GUS stable expression. 3 days after bombardment, GUS assay was performed, which exhibited, $18.36{\pm}1.00$ calli showed blue spots. The transformed embryogenic calli were cultured on selection medium (@ 6 mg/L basta) for 3 months. The putative transgenic plants were developed via selective somatic embryogenesis (@1.50 mg/L basta); maximum $27.58{\pm}1.25$ somatic embryos were obtained while $17.47{\pm}1.00$ embryos developed into plantlets (@ 0.75mg/L basta). In five independent experiments, up to 7.24% transformation efficiency was recorded. The presence of the transgenes was analyzed by using PCR and southern hybridization analysis. The transgenic plants were developed with in 6-7 months, but mostly transformants were abnormal in morphology.

Expression of Arabidiopsis CAX4 in tomato fruits increases calcium level with no accumulation of other metallic cations

  • Jeong, Se-Woon;Han, Jeung-Sul;Kim, Kyung-Min;Oh, Jung-Youl;Kim, Byung-Oh;Kim, Chang-Kil;Chung, Jae-Dong
    • Journal of Plant Biotechnology
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    • v.35 no.4
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    • pp.337-343
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    • 2008
  • We generated transgenic tomato plants with Arabidopsis thaliana $H^+$/cation exchanger gene (C4X4) by Agrobactrium-mediated transformation. We confirmed transgene copy number and transcription by Southern and Northern blot analyses. The intact CAX4-expressing tomato (Lycopersicon esculentum) fruits contained 63-71% more calcium ($Ca^{2+}$) than wild-type fruits. Moreover, ectopic expression of C4X4 in tomato fruits did not show any significant increase of the four kinds of metallic cations analyzed ($Mg^{2+}$, $Fe^{2+}$, $Mn^{2+}$, and $Cu^{2+})$. The C4X4-expressing tomato plants including their fruits did not show any morphological alternations during whole growth period. These results suggest the enhanced Ca-substrate specificity of CAX4 exchanger in tomato. Therefore, intact CAX4 exchanger can be a useful tool for $Ca^{2+}$ nutrient enrichment of tomato fruits with reduced accumulation of undesirable cations.

Transgene Expression of Biologically Active Human Follicle-Stimulating Hormone in Milk and Histological Analysis

  • Myoung-Ok Kim;Kil-Soo Kim;Eun-Ju Lee;Sung-Hyun Kim;Jun-Hong Park;Kyoungin-Cho;Boo-Kyung Jung;Hee-Chul Kim;Sol-ha Hwang
    • Proceedings of the KSAR Conference
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    • 2004.06a
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    • pp.214-214
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    • 2004
  • Follicle stimulating hormone (FSH) is a pituitary glycoprotein composed of two post translationally modified subunits, which must properly assemble to be biologically active. We developed a transgenic (TG) mouse model that overexpresses the human Follicle-Stimulating hormone (FSH) -subunit under the bovine -casein promoter, displaying in females an excessive levels express in mammary gland. (omitted)

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