• Title/Summary/Keyword: Gene Transfer

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Insect Cell Surface Expression of Hemagglutinin (HA) of Egyptian H5N1 Avian Influenza Virus Under Transcriptional Control of Whispovirus Immediate Early-1 Promoter

  • Gadalla, M.R.;El-Deeb, A.H.;Emara, M.M.;Hussein, H.A.
    • Journal of Microbiology and Biotechnology
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    • v.24 no.12
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    • pp.1719-1727
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    • 2014
  • In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

Induction of CEA-specific Cytotoxic T Lymphocytes by Murine Dendritic Cells Expressing CEA (CEA 발현 수지상 세포를 이용한 CEA 특이 살해 T 세포의 유도)

  • Won, Eun-Ha;Kim, Chang-Hyun;Park, Mi-Young;Cho, Hyun-Il;Oh, Seong-Taek;Hong, Yong-Kil;Kim, Tai-Gyu
    • IMMUNE NETWORK
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    • v.3 no.4
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    • pp.295-301
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    • 2003
  • Background: Carcinoembryonic antigen (CEA) is well-known soluble tumor marker frequently detectable in peripheral blood of carcinoma patients and considered as good target for antigen-specific immunotherapy. In this study, we used a replication-deficient adenovirus containing CEA to study CTL induction in vitro after adenovirus-mediated gene transfer into DC. Methods: DC were obtained from mouse bone marrow and cultured with IL-4 and GM-CSF. For measuring CTL activity, splenocytes were harvested from the mice, which were immunized with DC that had been infected AdV-CEA or pulsed with CEA peptide. Untreated DC was used as a control. Splenocytes were re-stimulated in vitro with DC pulsed with CEA peptide for 7 days and CTL activity with CEA peptide-pulsed EL-4 cells were assessed in a standard $^{51}Cr$-release assay. The frequencies of antigen-specific cytokine-secreting T cell were determined with $mIFN-{\gamma}$ELISPOT. Results: DC infected with recombinant adenovirus expressing CEA induced CEA-specific CTL responses in vivo. Splenocyte induced from mice immunized with AdV-CEA-infected DC increase in the number of $IFN-{\gamma}$ secreting T cells compared with those from mice immunized with CEA peptide-pulsed DC. Conclusion: These results suggested that DC infected with recombinant adenovirus has advantages over other forms of vaccination and could provide an alternative approach vaccination therapies.

Plant Regeneration and Transformation of Kentucky Bluegrass(Poa pratensis L.) via the Plant Tissue Culture (조직배양을 이용한 Kentucky bluegrass(Poa pratensis L.)의 식물체 재분화 및 형질전화 조건의 검토)

  • Miki Kusano;Koichi Tohyama;Bae, Chang-Hyu;Riu, Key-Zyung;Lee, Hyo-Yeon
    • Journal of Plant Biotechnology
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    • v.30 no.2
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    • pp.115-121
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    • 2003
  • In this study, plant regeneration and Agrobacterium tumefaciens-mediated transformation Kentucky bluegrass(Poa pratensis L.) were evaluated. Three different types of calli were produced depending on the combinations of growth regulators. They were non-friable brown or gray-colored callus (type I), compact, friable and yellow or white-colored callus (typeII), and soft, watery translucent callus with differentiated structure (typeIII). The highest regenerable organogenic callus (typeII) was obtained on the medium containing 1mg/L, 2,4-D and 0.1mg/L BA. Additionally, the production of typeII calli increased significantly when AgNO$_3$ was added to the callus induction and growth medium. The highest frequency of multiple shout formation from typeII callus was obtained on MS medium containing 1mg/L BA and 1mg/L Thidiazuron(TDZ). The organogenic calli(typeII) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pIG121Hm with $\beta$-glucuronidase gene, and various factors were found to influence the transfer-DNA delivery efficiency. The highest transient GUS activity was observed on typeIIcallus. In the present work, we reported the first transient GUS activity of Kentucky bluegrass mediated by Agrobacterium tumefaciens. Our system may contribute to genetic improvement for breed-recalcirtrant grass species, Kentucky bluegrass.

The importation of genetically modified crops and its environmental impacts in Korea

  • Han, Sung Min;Kim, Young Tae;Won, Ok Jae;Choi, Kyung Hwa;Rho, Young Hee;Park, Kee Woong
    • Korean Journal of Agricultural Science
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    • v.43 no.2
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    • pp.215-220
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    • 2016
  • The global cultivation area of genetically modified crops (GM crops) has been increasing every year. Cultivation of GM crops is not only beneficial to the economy but also has positive effects on the environment in decreasing the use of agrochemicals, chemical fertilizers, and agricultural machinery. However, there have been controversies about the admixture of GM crops and non-GM crops and the unintentional release of GM crops to the environment. Especially in Korea, where consumption of agricultural products is import-dependent, the economic importance of GM crops has been a significant issue. The Act on import and distribution of GM crops was established in 2001 to start the management of GM crops in Korea. Recently, the imported amount of GM crops to Korea has reached over 10 million tons and is increasing very rapidly; consequently, the potential environmental impact of GM crops is becoming a big issue in Korea. In Japan, the discovery of imported GM canola plants around ports in 2005 raised awareness of the unintentional release of GM crops. In Korea, GM maize plants were also found in port and feed factory surroundings from 2005 to 2007. It is now necessary to monitor imported GM crops by tracing distribution, transport process for practical environmental risk assessment. Possible gene transfer from GM crops to non-GM crops should also be investigated in the cultivation area and the surroundings as well.

Plant Regeneration from Cryopreserved Embryogenic Cell Suspension Cultures of Cucumber (초저온 보존된 오이 배발생세포 현탁배양으로부터 식물체 재분화)

  • Kim, Seok-Won;In, Dong-Soo;Jung, Won-Joong;Woo, Je-Wook;Jung, Min;Yoo, Jang-Ryul
    • Korean Journal of Plant Tissue Culture
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    • v.25 no.6
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    • pp.501-505
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    • 1998
  • Conditions for high frequency plant regeneration from cryopreserved embryogenic cell suspension cultures derived from hypocotyl explants of cucumber (Cucumis sativus L.) are described. Cells cryoprotected with a mixture of 2 M DMSO and 0.4 M sucrose exhibited a regeneration frequency of 85%. However, cells cryoprotected with different concentrations of glycerol showed no regeneration after cryopreservation. Pretreatment of cells in a high osmotic medium was not necessary to the process. Upon transfer to MS medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid, regenerated calli gave rise to numerous somatic embryos, then underwent development into plantlets.

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Beam Control Method of Multiple Array Antenna Using The Modified Genetic Algorithm (변형된 유전자 알고리즘을 이용한 Multiple Array 안테나의 빔 제어방식)

  • Hyun, Kyo-Hwan;Jung, Kyung-Kwon;Eom, Ki-Hwan
    • Journal of the Institute of Electronics Engineers of Korea SC
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    • v.44 no.2 s.314
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    • pp.39-45
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    • 2007
  • This paper presents a novel scheme that quickly searches for the sweet spot of multiple array antennas, and locks on to it for high-speed millimeter wavelength transmissions, when communications to another antenna array are disconnected. The proposed method utilizes a modified genetic algorithm, which selects a superior initial group through preprocessing in order to solve the local solution in agenetic algorithm. TDD (Time Division Duplex) is utilized as the transfer method and data controller for the antenna. Once the initial communication is completed for the specific number of individuals, no longer antenna's data will be transmitted until each station processes GA in order to produce the next generation. After reproduction, individuals of the next generation become the data, and communication between each station is made again. Simulation results of 1:1, 1:2, 1:5 array antennas confirmed the efficiency of the proposed method. The 16bit split is 8bit, but it has similar performance as 16bit gene.

Anti-metastasis Activity of Black Rice Anthocyanins Against Breast Cancer: Analyses Using an ErbB2 Positive Breast Cancer Cell Line and Tumoral Xenograft Model

  • Luo, Li-Ping;Han, Bin;Yu, Xiao-Ping;Chen, Xiang-Yan;Zhou, Jie;Chen, Wei;Zhu, Yan-Feng;Peng, Xiao-Li;Zou, Qiang;Li, Sui-Yan
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.15
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    • pp.6219-6225
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    • 2014
  • Background: Increasing evidence from animal, epidemiological and clinical investigations suggest that dietary anthocyanins have potential to prevent chronic diseases, including cancers. It is also noteworthy that human epidermal growth factor receptor 2 (ErbB2) protein overexpression or ErbB2 gene amplification has been included as an indicator for metastasis and higher risk of recurrence for breast cancer. Materials and Methods: The present experiments investigated the anti-metastasis effects of black rice anthocyanins (BRACs) on ErbB2 positive breast cancer cells in vivo and in vitro. Results: Oral administration of BRACs (150 mg/kg/day) reduced transplanted tumor growth, inhibited pulmonary metastasis, and decreased lung tumor nodules in BALB/c nude mice bearing ErbB2 positive breast cancer cell MDA-MB-453 xenografts. The capacity for migration, adhesion, motility and invasion was also inhibited by BRACs in MDA-MB-453 cells in a concentration dependent manner, accompanied by decreased activity of a transfer promoting factor, urokinase-type plasminogen activator (u-PA). Conclusions: Together, our results indicated that BRACs possess anti-metastasis potential against ErbB2 positive human breast cancer cells in vivo and in vitro through inhibition of metastasis promoting molecules.

Adenovirus vector-mediated FAM176A overexpression induces cell death in human H1299 non-small cell lung cancer cells

  • Xie, Hong;Hu, Jia;Pan, Huan;Lou, Yaxin;Lv, Ping;Chen, Yingyu
    • BMB Reports
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    • v.47 no.2
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    • pp.104-109
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    • 2014
  • FAM176A (family with sequence similarity 176 member A) is a novel molecule related to programmed cell death. A decreased expression of FAM176A has been found in several types of human tumors in including lung cancers. In the present study, we investigated the biological activities of FAM176A on the human non-small cell lung cancer cell line H1299 cells. We constructed a recombinant adenovirus 5-FAM176A vector (Ad5-FAM176A) and evaluated the expression and anti-tumor activities in vitro. Cell viability analysis revealed that the adenovirus-mediated increase of FAM176A inhibited the growth of the tumor cells in a dose- and time-dependent manner. This inhibitory effect was mediated by both autophagy and apoptosis that involved caspase activation. In addition, cell cycle analysis suggested that Ad5-FAM176A could induce cell cycle arrest at the G2/M phase, all of which suggested that adenovirus-mediated FAM176A gene transfer might present a new therapeutic approach for lung cancer treatment.

Seasonal and Spatial Diversity of Picocyanobacteria Community in the Great Mazurian Lakes Derived from DGGE Analyses of 16S rDNA and cpcBA-IGS Markers

  • Jasser, Iwona;Krolicka, Adriana;Jakubiec, Katarzyna;Chrost, Ryszard J.
    • Journal of Microbiology and Biotechnology
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    • v.23 no.6
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    • pp.739-749
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    • 2013
  • The seasonal and spatial diversity of picocyanobacteria (Pcy) in lakes of the Great Mazurian Lakes (GLM) system was examined by DGGE analysis of molecular markers derived from the 16S-23S internal transcribed spacer (ITS) of the ribosomal operon and the phycocyanin operon (cpcBA-IGS). The study of nine lakes, ranging from mesotrophy to hypereutrophy, demonstrated seasonal variance of Pcy. The richness and Shannon diversity index calculated on the basis of both markers were higher in spring and lower in early and late summer. No statistically significant relationships were found between the markers and trophic status of the studied lakes or Pcy abundance. There were, however, statistically significant relationships between the diversity indices and sampling time. The analysis pointed to a different distribution of the two markers. The ITS marker exhibited more unique sequences in time and space, whereas a greater role for common and ubiquitous sequences was indicated by the cpcBA-IGS data. Examination of the Pcy community structure demonstrated that communities were grouped in highly similar clusters according to sampling season/time rather than to the trophic status of the lake. Our results suggest that time is more important than trophic status in shaping the diversity and structure of Pcy communities. The seasonal changes in picocyanobacteria and differences in diversity and community structures are discussed in the context of well-established ecological hypotheses: the PEG model, intermediate disturbance hypothesis (IDH), and horizontal gene transfer (HGT).

Expression of Green Fluorescent Protein in Both Spodoptera frugiperda Cells and Bombyx mori Larvae by Ac-Bm Hybrid Virus

  • Jin, Byung-Rae;Yoon, Hyung-Joo;Yun, Eun-Young;Kang, Seok-Woo;Cho, Eun-Sook;Kang, Seok-Kwon
    • The Journal of Korean Society of Virology
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    • v.28 no.3
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    • pp.225-232
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    • 1998
  • We have expressed GFP in Sf9 and Bm5 cells or Bombyx mori larvae by using Ac-Bm hybrid virus capable of replicating in both Bm5 and Sf9 cells. Genomic DNA of Ac-Bm hybrid virus expressing ${\beta}$-galactosidase was cotransfected with baculovirus transfer vector containing GFP gene, pBacPAK-GFP in Sf9 cells. The Ac-Bm hybrid virus harboring GFP was named as Ac-Bm hybrid virus-GFP. The Ac-Bm hybrid virus-GFP-infected insect cells were easily selected by detecting the emission of GFP from each well of cell culture dish on the UV illuminator. GFP produced by Ac-Bm hybrid virus-GFP in Sf9 and Bm5 cells or B. mori larvae was confirmed by SDS-PAGE and Western blot analysis using GFP antibody. In addition, B. mori larvae infected with Ac-Bm hybrid virus-GFP was apparently appeared fluorescence from the whole body at S days postinoculation. The fluorescence of GFP from the hemolymph and fat body of B. mori larvae infected with Ac-Bm hybrid virus-GFP was also observed by fluorescence microscope. In conclusion, our results demonstrated that in baculovirus expression vector system, use of Ac-Bm hybrid virus have an additional advantage of expanded host range for producing recombinant proteins.

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