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Sample records for bronchoepithelium egfp expression

  1. Successful expression of heterologous egfp gene in the mitochondria of a photosynthetic eukaryote Chlamydomonas reinhardtii.

    Science.gov (United States)

    Hu, Zhangli; Zhao, Zhonglin; Wu, Zhihua; Fan, Zhun; Chen, Jun; Wu, Jinxia; Li, Jiancheng

    2011-09-01

    The efficient expression of exogenous gene in mitochondria of photosynthetic organism has been an insurmountable problem. In this study, the pBsLPNCG was constructed by inserting the egfp gene into a site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA of Chlamydomonas reinhardtii CC-124 and introduced into the mitochondria of respiratory deficient dum-1 mutation of C. reinhardtii CC-2654. Sequencing and DNA Southern analyses revealed that egfp gene had been integrated into the mitochondrial genome of transgenic algae as expected and no other copy of egfp existed in their nucleic genome. Both the fluorescence detection and Western blot analysis confirmed the presence of eGFP protein in the transgenic algae; it indicated that the egfp gene was successfully expressed in the mitochondria of C. reinhardtii. PMID:21664493

  2. BAC-Dkk3-EGFP Transgenic Mouse: An In Vivo Analytical Tool for Dkk3 Expression

    Directory of Open Access Journals (Sweden)

    Yuki Muranishi

    2012-01-01

    Full Text Available Dickkopf (DKK family proteins are secreted modulators of the Wnt signaling pathway and are capable of regulating the development of many organs and tissues. We previously identified Dkk3 to be a molecule predominantly expressed in the mouse embryonic retina. However, which cell expresses Dkk3 in the developing and mature mouse retina remains to be elucidated. To examine the precise expression of the Dkk3 protein, we generated BAC-Dkk3-EGFP transgenic mice that express EGFP integrated into the Dkk3 gene in a BAC plasmid. Expression analysis using the BAC-Dkk3-EGFP transgenic mice revealed that Dkk3 is expressed in retinal progenitor cells (RPCs at embryonic stages and in Müller glial cells in the adult retina. Since Müller glial cells may play a potential role in retinal regeneration, BAC-Dkk3-EGFP mice could be useful for retinal regeneration studies.

  3. Using the Tg(nrd:egfp/albino zebrafish line to characterize in vivo expression of neurod.

    Directory of Open Access Journals (Sweden)

    Jennifer L Thomas

    Full Text Available In this study, we used a newly-created transgenic zebrafish, Tg(nrd:egfp/albino, to further characterize the expression of neurod in the developing and adult retina and to determine neurod expression during adult photoreceptor regeneration. We also provide observations regarding the expression of neurod in a variety of other tissues. In this line, EGFP is found in cells of the developing and adult retina, pineal gland, cerebellum, olfactory bulbs, midbrain, hindbrain, neural tube, lateral line, inner ear, pancreas, gut, and fin. Using immunohistochemistry and in situ hybridization, we compare the expression of the nrd:egfp transgene to that of endogenous neurod and to known retinal cell types. Consistent with previous data based on in situ hybridizations, we show that during retinal development, the nrd:egfp transgene is not expressed in proliferating retinal neuroepithelium, and is expressed in a subset of retinal neurons. In contrast to previous studies, nrd:egfp is gradually re-expressed in all rod photoreceptors. During photoreceptor regeneration in adult zebrafish, in situ hybridization reveals that neurod is not expressed in Müller glial-derived neuronal progenitors, but is expressed in photoreceptor progenitors as they migrate to the outer nuclear layer and differentiate into new rod photoreceptors. During photoreceptor regeneration, expression of the nrd:egfp matches that of neurod. We conclude that Tg(nrd:egfp/albino is a good representation of endogenous neurod expression, is a useful tool to visualize neurod expression in a variety of tissues and will aid investigating the fundamental processes that govern photoreceptor regeneration in adults.

  4. Gene gun bombardment-mediated expression and translocation of EGFP-tagged GLUT4 in skeletal muscle fibres in vivo

    DEFF Research Database (Denmark)

    Lauritzen, Hans P M M; Reynet, Christine; Schjerling, Peter;

    2002-01-01

    enhanced green fluorescent protein (EGFP) labelling technique with physical transfection methods in vivo: intramuscular plasmid injection or gene gun bombardment. During optimisation experiments with plasmid coding for the EGFP reporter alone EGFP-positive muscle fibres were counted after collagenase...... treatment of in vivo transfected flexor digitorum brevis (FDB) muscles. In contrast to gene gun bombardment, intramuscular injection produced EGFP expression in only a few fibres. Regardless of the transfection technique, EGFP expression was higher in muscles from 2-week-old rats than in those from 6-week......-old rats and peaked around 1 week after transfection. The gene gun was used subsequently with a plasmid coding for EGFP linked to the C-terminus of GLUT4 (GLUT4-EGFP). Rats were anaesthetised 5 days after transfection and insulin given i.v. with or without accompanying electrical hindleg muscle stimulation...

  5. Stable EGFP Gene Expression in C6 Glioma Cell Line after Transduction with HIV-1-based Lentiviral Vector

    Institute of Scientific and Technical Information of China (English)

    JIN Gui-shan; LIU Fu-sheng; CHAI Qi; WANG Jian-jao; LI Jun-hua

    2008-01-01

    Objective:To establish a stable C6/EGFP glioma cell line for studies on glioma. Methods:The C6 glioma cell line was transfected with the human immunodeficiency virus type Ⅰ(HIV-1)based lentivirus vector containing two enhancer-promoters CMV and EF1α.Enhanced green fluorescent protein(EGFP)-positive C6 cells were sorted out by fluorescence-activated cell sort.Expression of EGFP was observed by fluorescent microscopy.EGFP gene in C6 genome was assessed by Polymerase chain reaction(PCR)and DNA sequencing.Original and transfected cells were compared biologically and cytomorphologically. Results:Lentivirus vector transfection produced up to 40% EGFP-positive cells.After fluorescence-activated cell sort selection,a pure cell line C6/EGFP was established.PCR and DNA sequencing revealed integration of EGFP gene in C6 cell genome.Analysis of cell characteristics revealed no difference between transfected and original cells. Conclusion:A C6/EGFP cell line expressing EGFP as a marker is established,in which the EGFP gene is integrated into the genome.This cell line can be served as a promising tool for further basic research and gene therapy studies.

  6. Effects of clinorotation on COL1A1- EGFP gene expression

    Institute of Scientific and Technical Information of China (English)

    DAI; Zhongquan; LI; Yinghui; DING; Bai; ZHANG; Yuguo; LIU

    2004-01-01

    Bone-formation related gene plays a critical role in bone loss induced by space microgravity, however the exact mechanism is unclear. In this study, we aim to investigate the effect of microgravity on the activity of α 1(I) collagen (COL1A1) gene promoter and the expression of osteoblast-related genes. COL1A1 promoter was digested by restriction enzymes resulting in three DNA fragments. The fragments were ligated with the enhanced green fluorescent protein report gene, and subcloned into expression vectors. ROS17/2.8 cells transfected by these vectors were screened by G418, and enhanced green fluorescent protein (EGFP) positive colonies were isolated and cultured under clinostat condition. EGFP and Collagen type I expression level were detected by fluorescence intensity analysis and immunocytochemistry methods respectively. The results showed that the expression of EGFP and collagen type I was increased 24 h, 48 h after the cells were cultured under stimulated microgravity, illustrating that the activity of COL1A1 promoter might be increased. In conclusion, osteoblasts can compensatively increase the expression of type I collagen by enhancing the activity of COL1A1 promoter under short-term simulated microgravity conditions.

  7. The effect of housing temperature on the growth of CT26 tumor expressing fluorescent protein EGFP

    Science.gov (United States)

    Yuzhakova, Diana V.; Shirmanova, Marina V.; Lapkina, Irina V.; Serebrovskaya, Ekaterina O.; Lukyanov, Sergey A.; Zagaynova, Elena V.

    2016-04-01

    To date, the effect of housing temperature on tumor development in the immunocompetent mice has been studied on poorly immunogenic cancer models. Standard housing temperature 20-26°C was shown to cause chronic metabolic cold stress and promote tumor progression via suppression of the antitumor immune response, whereas a thermoneutral temperature 30-31°C was more preferable for normal metabolism of mice and inhibited tumor growth. Our work represents the first attempt to discover the potential effect of housing temperature on the development of highly immunogenic tumor. EGFP-expressing murine colon carcinoma CT26 generated in Balb/c mice was used as a tumor model. No statistically significant differences were shown in tumor incidences and growth rates at 20°C, 25°C and 30°C for non-modified CT26. Maintaining mice challenged with CT26-EGFP cells at 30°C led to complete inhibition of tumor development. In summary, we demonstrated that the housing temperature is important for the regulation of growth of highly immunogenic tumors in mice through antitumor immunity.

  8. [Construction of ADAMTS13-pEGFP-N1 vector and its expression in HeLa cells].

    Science.gov (United States)

    Ling, Jing; Ma, Zhen-Ni; Su, Jian; Ruan, Chang-Geng

    2013-02-01

    This study was aimed to construct a pEGFP-N1 vector of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motifs 13) so as to pave the way for further studying its synthesis and secretion. Human full-length cDNA sequence of ADAMTS13 was acquired by polymerase chain reaction (PCR) with Phusion(®) High-Fidelity (NEB), then the PCR product was double digested with EcoRI and XhoI. After digestion, the ADAMTS13 cDNA sequence was purified and recombined with the pEGFP-N1 vector. The DNA sequence analysis showed that ADAMTS13 was ligated to the pEGFP-N1 vector correctly. After transient expression in HeLa cells, the expression of EGFP could be detected by fluorescent microscopy, and the expression of ADAMTS13 protein could be detected by SDS-PAGE and Western blot. It is concluded that the ADAMTS13-pEGFP-N1 vector is successfully constructed, and it can be widely used in further research on the mechanism of the synthesis and secretion of ADAMTS13. PMID:23484705

  9. Construction of the mammalian expressing vector pEGFP-N1-P53 and its expression successful in chicken fibroblast cells and blastoderm.

    Science.gov (United States)

    Song, Z; Li, Z H; Lei, X Q; Xu, T S; Zhang, X H; Li, Y J; Zhang, G M; Xi, S M; Yang, Y B; Wei, Z G

    2015-01-01

    The enhanced green fluorescent protein (EGFP) pEGFP-N1-P53 eukaryotic expression vector, which contains the human tumor suppressor p53, was constructed and transfected into chicken fibroblast cells and stage-X blastoderm to analyze the transfection efficiency. The complementary DNA of the human p53 gene was cloned by reverse transcription-polymerase chain reaction from human peripheral blood and inserted into the pEGFP-N1 vector by HindIII and BamHI double digestion. The pEGFP-N1-P53 vector was transfected into chicken embryo fibroblasts by Lipofectamine 2000 liposomes, and the transfection efficiency was analyzed by fluorescence microscope after 36 h of transfection. The stage-X blastoderm was also transfected by blastoderm injection using Lipofectamine 2000 liposomes at room temperature after 12-24 h; then hatching occurred until seventh day, and the transfection efficiency was analyzed by fluorescence microscope in the dead embryo. A total of 90 hatching eggs were transfected by the pEGFP-N1-P53 vector, and 20 chicken embryos expressed the reporter gene, which indicated that recombinant pEGFP-N1-P53 could be transfected and expressed in stage-X blastoderm by liposomes. Chicken embryo fibroblasts were transfected and expressed the reporter gene. The pEGFP-N1-P53 vector was constructed successfully and could be transfected and expressed in chicken embryo fibroblasts and stage-X blastoderms efficiently. PMID:25730031

  10. Potential utility of eGFP-expressing NOG mice (NOG-EGFP as a high purity cancer sampling system

    Directory of Open Access Journals (Sweden)

    Shima Kentaro

    2012-06-01

    Full Text Available Abstract Purpose It is still technically difficult to collect high purity cancer cells from tumor tissues, which contain noncancerous cells. We hypothesized that xenograft models of NOG mice expressing enhanced green fluorescent protein (eGFP, referred to as NOG-EGFP mice, may be useful for obtaining such high purity cancer cells for detailed molecular and cellular analyses. Methods Pancreato-biliary cancer cell lines were implanted subcutaneously to compare the tumorigenicity between NOG-EGFP mice and nonobese diabetic/severe combined immunodeficiency (NOD/SCID mice. To obtain high purity cancer cells, the subcutaneous tumors were harvested from the mice and enzymatically dissociated into single-cell suspensions. Then, the cells were sorted by fluorescence-activated cell sorting (FACS for separation of the host cells and the cancer cells. Thereafter, the contamination rate of host cells in collected cancer cells was quantified by using FACS analysis. The viability of cancer cells after FACS sorting was evaluated by cell culture and subsequent subcutaneous reimplantation in NOG-EGFP mice. Results The tumorigenicity of NOG-EGFP mice was significantly better than that of NOD/SCID mice in all of the analyzed cell lines (p  Conclusions This method provides a novel cancer sampling system for molecular and cellular analysis with high accuracy and should contribute to the development of personalized medicine.

  11. Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

    Directory of Open Access Journals (Sweden)

    Veenhuis Marten

    2005-10-01

    Full Text Available Abstract Background Native as well as recombinant bacterial cell surface layer (S-layer protein of Geobacillus (G. stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099 of the S-layer protein and EGFP (enhanced green fluorescent protein, in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells. Results Upon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S. cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE promoter. Conclusion The mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of

  12. Direct evaluation of the effect of gene dosage on secretion of protein from yeast Pichia pastoris by expressing EGFP.

    Science.gov (United States)

    Liu, Hailong; Qin, Yufeng; Huang, Yuankai; Chen, Yaosheng; Cong, Peiqing; He, Zuyong

    2014-02-28

    Increasing the gene copy number has been commonly used to enhance the protein expression level in the yeast Pichia pastoris. However, this method has been shown to be effective up to a certain gene copy number, and a further increase of gene dosage can result in a decrease of expression level. Evidences indicate the gene dosage effect is product-dependent, which needs to be determined when expressing a new protein. Here, we describe a direct detection of the gene dosage effect on protein secretion through expressing the enhanced green fluorescent protein (EGFP) gene under the direction of the α-factor preprosequence in a panel of yeast clones carrying increasing copies of the EGFP gene (from one to six copies). Directly examined under fluorescence microscopy, we found relatively lower levels of EGFP were secreted into the culture medium at one copy and two copies, substantial improvement of secretion appeared at three copies, plateau happened at four and five copies, and an apparent decrease of secretion happened at six copies. The secretion of EGFP being limiting at four and five copies was due to abundant intracellular accumulation of proteins, observed from the fluorescence image of yeast and confirmed by western blotting, which significantly activated the unfolded protein response indicated by the up-regulation of the BiP (the KAR2 gene product) and the protein disulfide isomerase. This study implies that tagging a reporter like GFP to a specific protein would facilitate a direct and rapid determination of the optimal gene copy number for high-yield expression. PMID:24225373

  13. Construction of the Eukaryotic Expression Vector with EGFP and hVE GF121 Gene and its Expression in Rat Mesenchymal Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Su Li; Chen Yunzhen; Zhang Xiaogang; She Qiang

    2005-01-01

    Objectives To construct a recombinant plasmid carrying enhanced green fluorescent protein (EGFP) and human vascular endothelial growth factor (VEGF) 121 gene and detect its expression in rat mesenchymal stem cells (MSCs). Methods Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFPC1. After being identified with PCR, double enzyme digestion and DNA sequencing. The recombinant plasmid pEGFP/hVEGF121 was transferred into rat MSCs with lipofectamine. The expression of EGFP/VEGF121 fusion protein were detected with fluorescence microscope and immunocytochemical staining respectively. Results The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluorescence microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48 h after transfection.Conclusions The recombinant plasmid carrying EGFP and human VEGF was successfully constructed and expressed positively in rat MSCs. It offers a promise tool for further research on differentiation of MSCs and VEGF gene therapy for ischemial cardiovascular disease.

  14. Construction of a fusion protein expression vector MK-EGFP and its subcellular localization in different carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Li-Cheng Dai; Di-Yong Xu; Xing Yao; Li-Shan Min; Ning Zhao; Bo-Ying Xu; Zheng-Ping Xu; Yong-Liang Lu

    2006-01-01

    AIM: To construct an expression plasmid encoding human wild-type midkine (MK) and enhanced green fluorescence protein (EGFP) fusion protein (MK-EGFP), and to analyze the subcellular localization of MK in different carcinoma cell lines.METHODS: Two kinds of MK coding sequences with or without signal peptide were cloned into plasmid pEGFP-N2, and the recombinant plasmids constructed were introduced into HepG2, MCF7 and DU145 cells,respectively, by transfection. With the help of laser scanning confocal microscopy, the expression and subcellular localization of MK-GFP fusion protein could be detected.RESULTS: Compared with the GFP control, in which fluorescence was detected diffusely over the entire cell body except in the nucleolus, both kinds of fusion protein MK-GFP were localized exclusively to the nucleus and accumulated in the nucleolus in the three kinds of cancer cell lines.CONCLUSION: This study reveals the specific nucleolar translocation independent of signal peptide, which may be involved in the mechanism that MK works. It provides valuable evidence for further study on the functions of MK in nucleus and its possible mechanisms, in which ribosomal RNA transcription and ribosome assembly are involved.

  15. Analysis of Relevant Parameters for Autophagic Flux Using HeLa Cells Expressing EGFP-LC3.

    Science.gov (United States)

    Muñoz-Braceras, Sandra; Escalante, Ricardo

    2016-01-01

    Macroautophagy (called just autophagy hereafter) is an intracellular degradation machinery essential for cell survival under stress conditions and for the maintenance of cellular homeostasis. The hallmark of autophagy is the formation of double membrane vesicles that engulf cytoplasmic material. These vesicles, called autophagosomes, mature by fusion with endosomes and lysosomes that allows the degradation of the cargo. Autophagy is a dynamic process regulated at multiple steps. Assessment of autophagy is not trivial because the number autophagosomes might not necessarily reflect the real level of autophagic degradation, the so-called autophagic flux. Here, we describe an optimized protocol for the analysis of relevant parameters of autophagic flux using HeLa cells stably expressing EGFP-LC3. These cells are a convenient tool to determine the influence of the downregulation or overexpression of specific proteins in the autophagic flux as well as the analysis of autophagy-modulating compounds. Western blot analysis of relevant parameters, such as the levels of EGFP-LC3, free EGFP generated by autophagic degradation and endogenous LC3·I-II are analyzed in the presence and absence of the autophagic inhibitor chloroquine. PMID:27613046

  16. Expression of EGFP/SDCT1 fusion protein, subcellular localization signal analysis, tissue distribution and electrophysiological function study

    Institute of Scientific and Technical Information of China (English)

    BAI Xueyuan; CHEN Xiangmei; FEN Zhe; WU Di; HOU Kai; CHENG Genyang; PENG Lixia

    2004-01-01

    Full-length cDNA gene of sodium-dependent dicarboxylate co-transporter protein 1 (SDCT1) is cloned from normal human kidney tissue and inserted into EGFP (enhanced green fluorescent protein) expression vector along with N-terminal and C-terminal truncated SDCT1 genes, so to construct the eukaryotic expression vectors of EGFP/SDCT1 fusion proteins, which are transfected into human renal tubular epithelial cells (HKC). Subcellular localizations of these fusion proteins are observed by laser confocal microscope to determine the localization signal of the SDCT1 protein. Duplex PCR analysis validates that the fusion protein genes have been integrated into the genome of HKC. Western blot indicates that the fusion proteins have been expressed in HKC. Confocal microscopy analysis shows that human SDCT1 predominantly locates on the plasma membrane, which is consistent with the results predicted by bioinformatics approach; in HKC transfected with N-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed on the plasma membrane; in HKC transfected with C-terminal truncated SDCT1 gene, the green fluorescence is mainly distributed in the cytoplasm. EGFP/SDCT1 mRNAs obtained by in vitro transcription are microinjected into Xenopus laevis oocytes for expression and the trans-membrane currents are measured by using two-microelectrode voltage-clamp technique. Na+ inward currents are present on cellular membrane of the injected oocytes. Immunohistochemical staining shows that human SDCT1 proteins are expressed on lumen membrane of the renal proximal tubule, but are negative in distal tubule, collecting duct, renal interstitium and glomerulus. The above-mentioned studies suggest that human SDCT1 protein is located on the lumen membrane of the renal proximal tubule, the C-terminal sequence of the SDCT1 is required for delivery and targeting localization, and the plasma membrane localization signal of the SDCT1 protein maybe locate in the C-terminal sequence.

  17. Establishment of a hepatocellular carcinoma cell line expressing dual reporter genes: sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP)

    International Nuclear Information System (INIS)

    Dual reporter gene imaging has several advantages for more sophisticated molecular imaging studies such as gene therapy monitoring. Herein, we have constructed hepatoma cell line expressing dual reporter genes of sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP), and the functionalities of the genes were evaluated in vivo by nuclear and optical imaging. A pRetro-PN vector was constructed after separating NIS gene from pcDNA-NIS. RSV-EGFP-WPRE fragment separated from pLNRGW was cloned into pRetro-PN vector. The final vector expressing dual reporter genes was named pRetro-PNRGW. A human hepatoma (HepG2) cells were transfected by the retrovirus containing NIS and EGFP gene (HepG2-NE). Expression of NIS gene was confirmed by RT-PCR, radioiodine uptake and efflux studies. Expression of EGFP was confirmed by RT-PCR and fluorescence microscope. The HepG2 and HepG2-NE cells were implanted in shoulder and hindlimb of nude mice, then fluorescence image, gamma camera image and I-124 microPET image were undertaken. The HepG2-NE cell was successfully constructed. RT-PCR showed NIS and EGFP mRNA expression. About 50% of cells showed fluorescence. The iodine uptake of NIS-expressed cells was about 9 times higher than control. In efflux study, T1/2 of HepG2-NE cells was 9 min. HepG2-NE xenograft showed high signal-to-background fluorescent spots and higher iodine-uptake compared to those of HepG2 xenograft. A hepatoma cell line expressing NIS and EGFP dual reporter genes was successfully constructed and could be used as a potential either by therapeutic gene or imaging reporter gene

  18. Establishment of a hepatocellular carcinoma cell line expressing dual reporter genes: sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP)

    Energy Technology Data Exchange (ETDEWEB)

    Kwak, Won Jung; Koo, Bon Chul; Kwon, Mo Sun [Kyungpook National University School of Medicine, Daegu (Korea, Republic of)] (and others)

    2007-06-15

    Dual reporter gene imaging has several advantages for more sophisticated molecular imaging studies such as gene therapy monitoring. Herein, we have constructed hepatoma cell line expressing dual reporter genes of sodium iodide symporter (NIS) and enhanced green fluorescence protein (EGFP), and the functionalities of the genes were evaluated in vivo by nuclear and optical imaging. A pRetro-PN vector was constructed after separating NIS gene from pcDNA-NIS. RSV-EGFP-WPRE fragment separated from pLNRGW was cloned into pRetro-PN vector. The final vector expressing dual reporter genes was named pRetro-PNRGW. A human hepatoma (HepG2) cells were transfected by the retrovirus containing NIS and EGFP gene (HepG2-NE). Expression of NIS gene was confirmed by RT-PCR, radioiodine uptake and efflux studies. Expression of EGFP was confirmed by RT-PCR and fluorescence microscope. The HepG2 and HepG2-NE cells were implanted in shoulder and hindlimb of nude mice, then fluorescence image, gamma camera image and I-124 microPET image were undertaken. The HepG2-NE cell was successfully constructed. RT-PCR showed NIS and EGFP mRNA expression. About 50% of cells showed fluorescence. The iodine uptake of NIS-expressed cells was about 9 times higher than control. In efflux study, T{sub 1/2} of HepG2-NE cells was 9 min. HepG2-NE xenograft showed high signal-to-background fluorescent spots and higher iodine-uptake compared to those of HepG2 xenograft. A hepatoma cell line expressing NIS and EGFP dual reporter genes was successfully constructed and could be used as a potential either by therapeutic gene or imaging reporter gene.

  19. Human parainfluenza virus type 3 (HPIV-3); Construction and rescue of an infectious, recombinant virus expressing the enhanced green fluorescent protein (EGFP).

    Science.gov (United States)

    The ability to rescue an infectious, recombinant, RNA virus from a cDNA clone, has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this protocol, the process of inserting enhanced green fluorescent protein (EGFP) gene into the human parainfluenza vi...

  20. A BAC transgenic Hes1-EGFP reporter reveals novel expression domains in mouse embryos

    DEFF Research Database (Denmark)

    Klinck, Rasmus; Füchtbauer, Ernst-Martin; Ahnfelt-Rønne, Jonas;

    2011-01-01

    Expression of the basic helix-loop-helix factor Hairy and Enhancer of Split-1 (Hes1) is required for normal development of a number of tissues during embryonic development. Depending on context, Hes1 may act as a Notch signalling effector which promotes the undifferentiated and proliferative stat...... the role of Hes1 in a number of different research areas including organ specification, development and regeneration....

  1. In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

    Science.gov (United States)

    Sadeghi, Somayeh; Seyed, Negar; Etemadzadeh, Mohammad-Hossein; Abediankenari, Saeid; Rafati, Sima; Taheri, Tahereh

    2015-01-01

    Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-γ/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency. PMID:26323836

  2. Construction of human eukaryotic expression vector pEGFP-C2-PRDX3 and its expression in human medulloblastoma DAOY cells%人pEGFP-C2-PRDX3真核表达载体构建与在人髓母细胞瘤DAOY细胞中表达

    Institute of Scientific and Technical Information of China (English)

    王晓玫; 刘汉勇; 金红涛; 成志强; 彭全洲; 胡锦涛; 高利昆; 许静; 张石芬; 曾照成

    2012-01-01

    目的 构建人pEGFP-C2-PRDX3真核表达载体,并检测其在人髓母细胞瘤DAOY细胞中的表达.方法 从人髓母细胞瘤DAOY细胞中提取总RNA,采用RT-PCR法对编码PRDX3的基因片段进行扩增,应用基因重组技术,将目的 片段亚克隆到pEGFP-C2真核表达载体,经PCR、酶切和测序鉴定后,用脂质体法将重组质粒转染入DAOY细胞,分别采用RT-PCR法、Western blot法检测PRDX3在DAOY细胞中的表达.结果 双酶切及测序结果验证PRDX3片段正确插入pEGFP-C2质粒,转染后的PRDX3基因mRNA及蛋白质水平均明显上调.结论 成功构建了具有报告基因-增强绿色荧光蛋白(EGFP)基因的真核表达载体pEGFP-C2-PRDX3,为进一步研究PRDX3基因在人髓母细胞瘤中的生物学功能奠定了基础.%Purpose To construct a human cukaryotie expression vector pEGFP-G2-PRDX3 and to investigate its expression in human modulloblastoma DAOY ceils. Methods Total RNA was extracted from human medullohlastoma DAOY cells and then the target se-qnenee of human PRDX3 gene was amplified by reverse transcription-polymerase chain reaction ( RT-PCR ). With the technique of gem re-arrangement, PRDX3 gene was subelonod into pEGFP-C2 plasmid. After identification with PCR, restriction enzyme digestion and sequence analysis, the reeomhinant plasmid was transfeetcd into DAOY ceils with the cationic loposome. RT-PCR, Western blotting were used to detect the PRDX3 mRNA and protein expression of DAOY cells. Results Correct construction of pEGFP-C2-PRDX3 was ieie:ntified by means the double digestion and sequeneing. The expression of mRNA and protoin of PRDX3 gene was significantly up-regulated after transfction. Conclusions The eukaryeitic expression vector pEGFP-C2-PRDX3 with a reporter gene-enhanee green flu oreseent protoin ( EGFP) gene is sueecssfully eonstrueted and would eontribute to the further studies on the role of PRDX3 in human medulloblastoma.

  3. SL7207/pEGFP-N1-AQP9及SL7207/pshRNA-AQP9重组沙门菌的构建及表达%Construction and expression of recombinant Salmonella SL7207/pEGFP-N1-AQP9 and SL7207/pshRNA-AQP9

    Institute of Scientific and Technical Information of China (English)

    王川; 亢渝俊; 姜政; 王丕龙

    2012-01-01

    Objective To transform human aquaglyceroporin 9(AQP9) recombinant eukaryotic expression plasmid pEGFP Nl AQP9 and interference plasmid pshRNA AQP9 into attenuated Salmonella strain SL7207 by electroporation and detect their ex pression,in order to lay the foundation for elucidating the human AQP9 in pathogenesis of nonalcoholic fatty liver disease (NAFLD). Methods AQP9 gene was obtained by RT PCR from human hepatic tissue and cloned into pEGFP Nl vector,recombi nant eukaryotic expression plasmid pEGFP Nl AQP9 was constructed,designed and synthesized human AQP9 SiRNA,annealed to double stranded ShRNA, inserted to pGenesil 1 plasmid, interference plasmid pshRNA AQP9 was constructed, and then trans formed into attenuated Salmonella strain SL7207 by electroporation,the expression were detected by PCR and Western Blot,and we detected their stability. Results Recombinant plasmid pEGFP Nl AQP9 and pshRNA AQP9 were constructed, transformed into attenuated Salmonella strain SL7207,after transformed into SL7207,could express in recombinant Salmonella. Conclusion Recom binant Salmonella SL7207/pEGFP Nl AQP9 and SL7207/pshRNA AQP9 are successfully constructed,lay the foundation for fur ther study on the gene therapy of NAFLD with AQP9.%目的 将人水甘油通道蛋白9(AQP9)真核表达质粒pEGFP-N1-AQP9及干扰质粒pshRNA-AQP9通过电穿孔转化减毒沙门菌株SL7207,并验证其表达情况,为阐明AQP9在非酒精性脂肪肝病(NAFLD)中的作用奠定基础.方法 从人肝脏组织中通过RT-PCR获得AQP9基因,克隆到载体pEGFP-N1上,构建真核表达质粒pEGFP-N1-AQP9,设计合成针对人AQP9的siRNA,退火形成双链shRNA,并插入pGenesil-1质粒中,构建干扰质粒pshRNA-AQP9,电穿孔转化减毒沙门菌株SL7207,通过PCR、Western blot验证其表达情况,并检测其稳定性.结果 成功构建pEGFP-N1-AQP9及pshRNA-AQP9重组质粒,将其转化SL7207后,能在重组沙门菌中表达.结论 成功构建SL7207/pEGFP-N1-AQP9及SL7207/psh

  4. Construction and Expression of Prokaryotic Expression Vector pET28a-EGFP%原核表达载体pET28a-EGFP的构建与表达

    Institute of Scientific and Technical Information of China (English)

    季爱加; 宁喜斌

    2011-01-01

    An enhanced green fluorescent protein ( ECFP) gene fragment in plasmid PEGFP-N3 as a template was used to amplify and obtained the EGFP gene fragment by PCR, and then designed a primer to introduce EcoR I and Hind Ⅲ Bites lo its both ends. After treated with double restriction enzyme, the introduced enzymatic site ECFP gene fragment, and pET28a plasmid, a recombinant expression plasmid pET28a-EGFP was obtained uung Tt ligase. The pET28a-EGFP was transformed into competence cells of E. Coli BL21 with heal shock method. The inducer of isopro-pyl β-D-l-thiogalactopyranoside (IPTG) was added to induce EGFP expression, as the optical density under 600 nm OD600 =0.4 of the B. Coli LB (Luria-Bertani) culture medium. The results indicated that the recombinant plasmtd enzymatic sites characterization and sequencing was correct. Under the natural light, the transformant colonies assumed green in LB solid medium (contains 1 mmol/L IPTG and SO μg/mL kanamycin ( Kan) ). When excited with blue-ray under fluorescence microscope, these recombinants emitting green fluorescence could clearly be observed. The successfully constructed prokaryotic expression vector pET28a-EGFP that expressed effectively in E. Coli BL21 will provide certain theoretical and technical supports for marking food borne pathogens as fluorescent markers in the future.%以质粒PEGFP-N3中增强型绿色荧光蛋白(Enhanced Green Fluorescent protein,EGFP)基因片段为模板,利用PCR技术扩增得到EGFP基因片段,并设计引物在其2端引入酶切位点EcoRⅠ和HindⅢ,对引入酶切位点的EGFP片段和pET28a质拉进行双酶切处理后,利用T4连接醇连接得到了重组质粒pET28a-EGFP.利用热击法把得到的重组质粒pET28a-EGFP特化至E.coli BL21( Escherichia coli BL21)感受态细胞中,当大肠埃希菌LB(Luria-Bertani)培养液在600 nm下的光密度值OD600 =0.4时,通过添加异丙基硫代β-D-半乳糖苷(IPTG)作为诱导剂诱导EGFP表达.结果表明:重组质

  5. Generation of stable L. major(+EGFP-LUC) and simultaneous comparison between EGFP and luciferase sensitivity.

    Science.gov (United States)

    Taheri, Tahereh; Saberi Nik, Hana; Seyed, Negar; Doustdari, Fatemeh; Etemadzadeh, Mohammad-Hossein; Torkashvand, Fatemeh; Rafati, Sima

    2015-03-01

    Because of the lack of an accurate and sensitive tool to evaluate the parasitemia level, treatment or prevention of leishmaniasis remains an important challenge worldwide. To monitor and track leishmanial infection by two parameters in real time, we generated stably transgenic Leishmania that express a bi-reporter protein as fused EGFP and firefly luciferase. Using two reporter genes (egfp-luc) simultaneously increases the experimental sensitivity for detection/diagnosis, and in vitro quantification of parasites as well as real-time infection in mice. Through different specific tools, EGFP and LUC signals from the parasite were detectable and measurable within a mammalian host and promastigotes. Here, the LUC protein provided a higher level of sensitivity than did EGFP, so that infection was detectable at an earlier stage of the disease in the footpad (injection site) and lymph nodes by bioluminescence. These results depicted that: (1) both quantitative reporter genes, EGFP and LUC, could be simultaneously used to detect parasitemia in vitro and in vivo and (2) sensitivity of firefly luciferase was 10-fold higher than that of EGFP in promastigotes. PMID:25637784

  6. 人血管性血友病因子裂解蛋白酶pEGFP-N1真核表达载体的构建及其在HeLa细胞中的表达%Construction of ADAMTS13-pEGFP-N1 Vector and Its Expression in HeLa Cells

    Institute of Scientific and Technical Information of China (English)

    凌婧; 马珍妮; 苏建; 阮长耿

    2013-01-01

    This study was aimed to construct a pEGFP-Nl vector of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motifs 13) so as to pave the way for further studing its synthesis and secretion. Human full-length cDNA sequence of ADAMTS13 was acquired by polymerase chain reaction (PCR) with Phusion? High-Fidelity(NEB) ,then the PCR product was double digested with EcoR I and Xho I . After digestion, the ADAMTS13 cDNA sequence was purified and recombined with the pEGFP-Nl vector. The DNA sequence analysis showed that ADAMTS13 was ligated to the pEGFP-Nl vector correctly. After transient expression in HeLa cells, the expression of EGFP could be detected by fluorescent microscopy, and the expression of ADAMTS13 protein could be detected by SDS-PAGE and Western blot. It is concluded that the ADAMTS13-pEGFP-N1 vector is successfully constructed, and it can be widely used in further research on the mechanism of the synthesis and secretion of ADAMTS13.%本研究旨在构建人血管性血友病因子裂解蛋白酶(ADAMTS13)的pEGFP-N1真核表达载体,为进一步研究ADAMTS13在细胞内合成及其分泌提供有力工具.通过PCR方法获取目的基因片段,并在目的基因两端加上限制性酶切位点.限制性内切酶酶切后,连接至pEGPF-N1真核表达载体.连接后获得质粒进行酶切鉴定及DNA测序验证,并转染HeLa细胞.通过荧光显微镜检测绿色荧光蛋白表达,Western blot方法鉴定所得蛋白.结果表明,酶切鉴定及DNA测序确认目的基因与载体正确连接,在荧光显微镜下观察到绿色荧光.Western blot显示,转染后HeLa细胞表达ADAMTS13蛋白.结论:成功构建了ADAMTS13-pEGFP-N1真核表达载体,为进一步研究ADAMTS13合成、分泌及其代谢的生理学机制提供了研究工具.

  7. Expression of pEGFP-bFGF by human epidermal cells after transfected with pEGFP-bFGF%碱性成纤维细胞生长因子基因转染人表皮细胞及其表达

    Institute of Scientific and Technical Information of China (English)

    伊海英; 孙晓艳; 付小兵; 任明姬; 张广田

    2009-01-01

    Objective To investigate whether human epidermal ceils could be transfected with the eukaryotic expressive vector of pEGFP-C3-bFGF and express the exogenous gene.Methods pEGFP-C3-bFGF was transferred into the human epidermal cells by using liposome-mediatedmethod.Subsequent-ly, the phenotypic changes of epidermal cells were detected by using immunohistochemical staining and im-munofluorescent analysis 36 h later.For controls, epidermal cells were cultured simultaneously in the ab-sence of exogenous gene transfection.Results Specific green fluorescence could be observed by the fluo-rescence microscopy in the gene positive groups.Immunohistochemical staining revealed that the expression levels of CK19 and CK14 were up-regulated, while those of CK10 significantly down-regulated.Meanwhile,immunofluorescent analysis indicated that β1 integrin was expressed on the membrane of positive cells in lower level,CK19 and CK14 were expressed on the membrane and in the cytoplasm of those cells, and CK10 was negative.Conclusion The pEGFP-C3-bFGF is successfully transferred into human epidermal ceils, which can induce epidermal cells to reverse their differentiated process and thus express some undif-ferentiated proteins of epidermal stem cells.%目的 观察碱性成纤维细胞生长因子基因真核表达载体(pEGFP-C3-bFGF)能否转染体外培养的人表皮细胞并表达.方法 采用脂质体(LipofectamineTM 2000)转染法,将pEGFP-C3-bFGF转染至人表皮细胞系(HEKa细胞)中,在荧光显微镜下观察其瞬时表达及细胞形态.以同期培养的表皮细胞作为阴性对照,免疫细胞化学染色和免疫荧光标记法检测实验组及对照组中β1整合素、CKl9、CK14及CK10在表皮细胞中表达的差异.结果 荧光显微镜下观察基因转染率为31.6%,转染的细胞体积小,呈圆形或圆梭形,核质比大.免疫细胞化学染色结果显示,实验组细胞中CK19、CK14表达增强,CK10表达减弱.免疫荧光染色结果

  8. Construction and identification of a green fluorescent protein expression vector carrying the HBx gene (pHBx-EGFP) and its expression in hepatocellular carcinoma cell line Bel 7402%pHBx-EGFP载体构建及其在肝癌Bel 7402细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    李伟; 朱明月; 鲁琰; 朱丽琴; 董栩; 陈栘; 李孟森

    2013-01-01

    AIM:To construct an eukaryotic expression vector carrying hepatitis B virus X (HBx) gene and enhanced green fluorescent protein gene (pHBx-EGFP),and to express it transiently in hepatocellular carcinoma (HCC) Bel 7402 cells for observing the expression and cellular localization of HBx-EGFP fusion protein and providing an experimental tool for investigating the function of HBx gene.METHODS:pcDNA3.1-HBx was used to amplify the HBx gene fragment by polymerase chain reaction (PCR).Recombinant DNA technology was used to insert the HBx gene into the eukaryotic expression vector pEGFP to obtain a recombinant vector pHBx-EGFP.After the recombinant vector or pEGFP was transfected into Bel 7402 cells for 24 h,the expression and subcellular location of HBx-EGFP was detected under an inverted fluorescence microscope,and the expression of HBx protein in total cellular proteins was detected by Western blot.RESULTS:Restriction digestion and DNA sequence analyses verified that the recombinant plasmid was constructed successfully.After the HBx-EGFP recombinant plasmid was transfected into Bel 7402 ceils,it was found that HBx-EGFP was present in the perinuclear region,while EGFP was distributed throughout the cells.Western blot analysis demonstrated that EGFP and HBx were expressed efficiently.CONCLUSION:A recombinant eukaryotic fluorescent expression vector carrying the HBx gene (pHBx-EGFP) has been constructed successfully,which could express EGFP and HBx in Bel 7402 cells.%目的:构建乙型肝炎病毒(hepatitis B virus,HBV)-X蛋白(hepatitis B virus x protein,HBx)和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)真核融合蛋白表达载体(pHBx-EGFP),转染肝癌Bel 7402细胞,观察其表达和定位,为进一步研究HBx功能提供工具.方法:以pcDNA3.1-HBx为模板,应用PCR和DNA重组技术构建增强型绿色荧光蛋白HBx-EGFP融合蛋白表达载体pHBx-EGFP,经脂质体转染Bel 7402细胞,应用倒置荧光显微镜观察融合蛋白表

  9. Construction and identification of eukaryotic expression vector of AMH%真核表达载体pIRES2-EGFP-AMH的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    严丽丽; 胡蓉; 李娟; 王飞苗

    2014-01-01

    目的 构建人AMH基因的真核表达载体pIRES2-EGFP-AMH.方法 从COV434细胞中获取AMH基因片段,经EcoRI、BglH双酶切定向克隆至载体pIRES2-EGFP,构建pIRES2-EGFP-AMH.经双酶切和测序鉴定后转染COV434细胞,通过荧光定量PCR和细胞免疫荧光技术检测AMH基因的表达变化.结果 重组人AMH真核表达质粒经酶切和测序鉴定正确,转染后能显著提高AMH mRNA和蛋白的表达.结论 人AMH真核表达载体构建成功,为进一步研究人AMH基因的功能提供初步的实验基础.

  10. Distinct expression/function of potassium and chloride channels contributes to the diverse volume regulation in cortical astrocytes of GFAP/EGFP mice

    Czech Academy of Sciences Publication Activity Database

    Benešová, Jana; Rusňáková, Vendula; Honsa, Pavel; Pivoňková, Helena; Džamba, Dávid; Kubista, Mikael; Anděrová, Miroslava

    2012-01-01

    Roč. 7, č. 1 (2012), e29725. E-ISSN 1932-6203 R&D Projects: GA ČR GAP303/10/1338 Grant ostatní: GA ČR(CZ) GD309/08/H079 Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50520701 Institutional support: RVO:86652036 ; RVO:68378041 Keywords : cell volume regulation * GFAP/EGFP * ischemia Subject RIV: FH - Neurology; FH - Neurology (BTO-N) Impact factor: 3.730, year: 2012

  11. 人源载体介导的minidystrophin-EGFP融合基因在Cos-7细胞中的表达%A minidystrophin-EGFP fusion gene expressed in Cos-7 cells mediated by human source vector

    Institute of Scientific and Technical Information of China (English)

    梁羽; 梁德生; 薛志刚; 龙志高; 邬玲仟; 潘乾; 胡艺俏; 戴和平; 夏昆; 夏家辉

    2005-01-01

    目的构建微小肌营养不良蛋白(minidystrophin)和增强绿色荧光蛋白(enhanced green fluoresce protein, EGFP)融合基因的人源载体,观察该载体在Cos-7细胞中的表达.方法以正常人肌营养不良基因cDNA(GenBank NM004006)为模板,通过PCR克隆的方法构建minidystrophin基因,融合EGFP基因后连接到人源载体pHrneo,大量提取重组质粒,转染Cos-7细胞,通过逆转录聚合酶链反应、荧光显微镜观察等方法检测该载体在细胞内的表达.结果成功构建pHrnDysG载体,转染Cos-7后,逆转录聚合酶链反应可扩增出735 bp特异条带,荧光显微镜观察可见表达蛋白分布于细胞膜上.结论 pHrn载体介导的minidystrophin基因可以在真核细胞表达,并被有效地转运至细胞膜,可望用于杜氏肌营养不良基因治疗的研究.%Objective To construct a human source vector containing minidystrophin-EGFP fusion gene and investigate its expression in Cos-7 cells. Methods The recombinant human source vector named pHrnDysG was constructed with PCR-clone methods. Three fragments of dystrophin gene were PCR amplified from normal human dystrophin gene cDNA (GenBank NM04006). These three fragments were ligated to generate a minidystrophin gene. The enhanced green fluorescent protein (EGFP) gene was fused to the C terminal of the minidystrophin gene, and then the pHrnDysG was finally obtained by cloning the fusion gene to pHrneo. Fluorescence microscope and RT-PCR were used to detect the expression of minidystrophin-EGFP fusion gene after the recombinant construct was transfected into Cos-7 cells by lipofectamine. Results Restrictive enzyme digestion analysis and sequencing confirmed that pHrnDysG vector was constructed successfully. After the recombinant pHrnDysG was transfected to Cos-7 cells, RT-PCR demonstrated that the fusion gene was successfully transcribed, and the green fluorescence was observed at the cell membrane. Conclusion The minidystrophin-EGFP fusion gene

  12. 重组质粒pEGFP-N1-Twist的构建、表达及其对SKOV3细胞克隆形成能力的影响%Construction of recombinant pEGFP-N1-Twist plasmid and its stable expression in SKOV3 cells

    Institute of Scientific and Technical Information of China (English)

    尉春艳; 张熙; 孙学军; 彭慧霞; 史玉霞; 王桂贤

    2014-01-01

    目的 构建含有Twist基因的重组质粒pEGFP-N1-Twist,为进一步研究上皮性卵巢癌的发生及转移机制奠定基础.方法 生物合成Twist cDNA基因序列,定向克隆至pEGFP-N1表达载体内,将pEGFP-N1-Twist转染入SKOV3细胞系,用Real-time PCR和Western blot检测Twist基因的表达情况;通过克隆形成实验检测pEGFP-N1-Twist对SKOV3细胞的作用.结果 Twist基因的cDNA已克隆到真核细胞表达载体pEGFP-N1中,将pEGFP-N1-Twist转染入SKOV3细胞后,Twist基因/蛋白的表达明显上调,转染组较对照组克隆形成明显增加(P<0.05).结论 成功构建了Twist基因的真核表达载体并能在细胞内稳定表达,Twist基因能增强SKOV3细胞的克隆形成能力.

  13. 真核表达载体pEGFP-C2-miR-126-sponge的构建及其表达活性研究%Construction of pEGFP-C2-miR-126-sponge eukaryotic expression vector and its expression activity

    Institute of Scientific and Technical Information of China (English)

    胡燕; 廖珍媛; 李永菊; 陈超; 朱顺飞; 熊思东; 徐林

    2014-01-01

    目的 构建靶向miR-126的真核表达载体pEGFP-C2-miR-126-海绵体(sponge),并探讨其下调miR-126的表达对肝癌HepG2细胞体外生长的影响,为后续深入研究miR-126在肝癌发生中的作用提供前期实验基础.方法 合成miR-126-sponge,构建pEGFP-C2-miR-126-sponge真核表达载体,体外瞬时转染肝癌细胞HepG2,荧光显微镜下观察EGFP的表达情况;Real-time PCR检测细胞中miR-126的表达水平;MTT法和克隆形成实验检测细胞的增殖变化;划痕实验观察细胞的体外迁移能力改变.结果 成功构建pEGFP-C2-miR-126-sponge真核表达载体(命名为p-miR-126-sp);Real-time PCR结果显示,与对照组(p-Cont)相比,p-miR-126-sp组中miR-126表达水平显著降低(P<0.05);MTT检测发现,p-miR-126-sp组中细胞增殖数目明显减少(P<0.05);此外,p-miR-126-sp组的克隆形成数和细胞迁移数均明显减少(P<0.05).结论 miR-126-sponge可显著下调miR-126的表达水平进而抑制肝癌细胞HepG2的体外增殖及迁移能力.

  14. EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice.

    Science.gov (United States)

    Seif, Samira; Kazemi, Fereshteh; Gholami, Elham; Seyed, Negar; Taslimi, Yasaman; Habibzadeh, Sima; Azarian, Bahareh; Jamshidi, Shahram; Hashemi, Mehrdad; Rafati, Sima; Taheri, Tahereh

    2016-05-01

    Optical reporter genes such as green fluorescent protein (GFP) and luciferase are efficiently and widely used in monitoring and studying the protective/therapeutic potential of candidate agents in leishmaniasis. But several observations and controversial reports have generated a main concern, whether enhanced GFP (EGFP) affects immune response. To address this issue, we studied the immunogenicity of EGFP in vivo by two lines of stably transfected parasites (Leishmania major (EGFP) or L. major (EGFP-LUC)) in BALB/c model and/or as a recombinant protein (rEGFP) produced in vitro by bacteria in parallel. Disease progression was followed by footpad swelling measurements and parasite burden in draining lymph nodes using microtitration assay and real-time PCR, and immune responses were also evaluated in spleen. EGFP-expressing parasites generated larger swellings in comparison with wild-type (L. major) while mice immunized with rEGFP and challenged with wild-type parasite were quite comparable in footpad swelling with control group without significant difference. However, both conventional and molecular approaches revealed no significant difference in parasite load between different groups. More importantly, no significant inflammatory responses were detected in groups with higher swelling size measured by interferon-γ (IFN-γ), interleukin (IL)-10, IL-5, and nitric oxide against frozen and thawed lysate of parasite as stimulator. Altogether, these results clearly revealed that EGFP protein expressed in prokaryotic and eukaryotic hosts is not an immunological reactive molecule and acts as a neutral protein without any side effects in mice. So, EGFP expressing Leishmania could be a safe and reliable substitution for wild-types that simplifies in situ follow-up and eliminates the animal scarification wherever needed during the study. PMID:26685673

  15. The viral RNA-based transfection of enhanced green fluorescent protein (EGFP) in the parasitic protozoan Trichomonas vaginalis.

    Science.gov (United States)

    Li, Wei; Ding, He; Zhang, Xinxin; Cao, Lili; Li, Jianhua; Gong, Pengtao; Li, He; Zhang, Guocai; Li, Shuhong; Zhang, Xichen

    2012-03-01

    Here we have developed methods to transiently and stably transfect the human pathogenic protist Trichomonas vaginalis. The viral RNA-based transfection vector pTVV-EGFP/NEO was constructed by using enhanced green fluorescent protein gene (EGFP) and neomycin resistance gene (NEO) in tandem to replace the whole gene encoding region of T. vaginalis virus (TVV). The in vitro transcripts of linearized pTVV-EGFP/NEO were electroporated into trophozoites and the transfectants transiently expressed EGFP after 16 h postincubation. Stable expression of EGFP was persistently detected by fluorescence microscopy and by RT-PCR in transfected trophozoites under G418 selection. Our study provides a novel and valuable approach for genetic study of T. vaginalis. PMID:21861063

  16. Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Xiaoxia [Key Laboratory of Aquatic Biodiversity and Conservation of the Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); University of Chinese Academy of Sciences, Beijing (China); Chen, Xiaowen; Jin, Xia; He, Jiangyan [Key Laboratory of Aquatic Biodiversity and Conservation of the Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Yin, Zhan, E-mail: zyin@ihb.ac.cn [Key Laboratory of Aquatic Biodiversity and Conservation of the Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Ningbo Laboratory, State Key Laboratory of Freshwater Ecology and Biotechnology (China)

    2014-07-01

    The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were all identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO{sub 4}, dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. - Highlights: • Identification of the promoter of zebrafish glycoprotein subunit α (gsuα) gene • Generation of stable transmission gsuα:EGFP transgenic zebrafish reporter • Demonstration of the recapitulation of the gsuα:EGFP and endogenous gsuα expression • Suggestion of the gsuα:EGFP transgenic zebrafish as a novel platform for EDC study.

  17. Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

    International Nuclear Information System (INIS)

    The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were all identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO4, dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. - Highlights: • Identification of the promoter of zebrafish glycoprotein subunit α (gsuα) gene • Generation of stable transmission gsuα:EGFP transgenic zebrafish reporter • Demonstration of the recapitulation of the gsuα:EGFP and endogenous gsuα expression • Suggestion of the gsuα:EGFP transgenic zebrafish as a novel platform for EDC study

  18. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Masahito [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Umeyama, Kazuhiro [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); International Cluster for Bio-Resource Research, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Matsunari, Hitomi [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Takayanagi, Shuko [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Haruyama, Erika; Nakano, Kazuaki; Fujiwara, Tsukasa; Ikezawa, Yuka [Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa 214-8571 (Japan); Nakauchi, Hiromitsu [Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, Tokyo University, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); and others

    2010-11-05

    Research highlights: {yields} EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. {yields} ZFNs induced targeted mutations in porcine primary cultured cells. {yields} Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  19. Knockout of exogenous EGFP gene in porcine somatic cells using zinc-finger nucleases

    International Nuclear Information System (INIS)

    Research highlights: → EGFP gene integrated in porcine somatic cells could be knocked out using the ZFN-KO system. → ZFNs induced targeted mutations in porcine primary cultured cells. → Complete absence of EGFP fluorescence was confirmed in ZFN-treated cells. -- Abstract: Zinc-finger nucleases (ZFNs) are expected as a powerful tool for generating gene knockouts in laboratory and domestic animals. Currently, it is unclear whether this technology can be utilized for knocking-out genes in pigs. Here, we investigated whether knockout (KO) events in which ZFNs recognize and cleave a target sequence occur in porcine primary cultured somatic cells that harbor the exogenous enhanced green fluorescent protein (EGFP) gene. ZFN-encoding mRNA designed to target the EGFP gene was introduced by electroporation into the cell. Using the Surveyor nuclease assay and flow cytometric analysis, we confirmed ZFN-induced cleavage of the target sequence and the disappearance of EGFP fluorescence expression in ZFN-treated cells. In addition, sequence analysis revealed that ZFN-induced mutations such as base substitution, deletion, or insertion were generated in the ZFN cleavage site of EGFP-expression negative cells that were cloned from ZFN-treated cells, thereby showing it was possible to disrupt (i.e., knock out) the function of the EGFP gene in porcine somatic cells. To our knowledge, this study provides the first evidence that the ZFN-KO system can be applied to pigs. These findings may open a new avenue to the creation of gene KO pigs using ZFN-treated cells and somatic cell nuclear transfer.

  20. Generation of mt:egfp transgenic zebrafish biosensor for the detection of aquatic zinc and cadmium.

    Science.gov (United States)

    Liu, Lili; Yan, Yanchun; Wang, Jian; Wu, Wei; Xu, Lei

    2016-08-01

    Zebrafish embryo toxicity testing has become a popular method for detecting environmental pollutions. However, the present research showed that zebrafish embryos exhibited no visible paramorphia, malformation, or mortality when exposed to heavy metals in a range above environmental standard limits, indicating that zebrafish embryos are an imprecise model for monitoring environmental heavy metals concentrations above regulatory limits. Aiming to obtain a biosensor for aquatic heavy metals, a metal-sensitive vector including zebrafish metallothionein (MT) promoter and enhanced green fluorescent protein (EGFP) was reconstructed and microinjected into 1-cell stage zebrafish embryos. The authors obtained an mt:egfp transgenic zebrafish line sensitive to aquatic zinc and cadmium. A quantitative experiment showed that zinc and cadmium treatment significantly induced the expression of EGFP in a dose- and time-dependent manner. In particular, EGFP messenger RNA levels increased remarkably when exposed to heavy metals above the standard limits. The results suggest that the transgenic zebrafish is a highly sensitive biosensor for detecting environmental levels of zinc and cadmium. Environ Toxicol Chem 2016;35:2066-2073. © 2016 SETAC. PMID:26752424

  1. Zebrafish sp7:EGFP: a transgenic for studying otic vesicle formation, skeletogenesis, and bone regeneration

    Science.gov (United States)

    DeLaurier, April; Eames, B. Frank; Blanco-Sánchez, Bernardo; Peng, Gang; He, Xinjun; Swartz, Mary E.; Ullmann, Bonnie; Westerfield, Monte; Kimmel, Charles B.

    2010-01-01

    Summary We report the expression pattern and construction of a transgenic zebrafish line for a transcription factor involved in otic vesicle formation and skeletogenesis. The zinc finger transcription factor sp7 (formerly called osterix) is reported as a marker of osteoblasts. Using bacterial artificial chromosome (BAC)-mediated transgenesis, we generated a zebrafish transgenic line for studying skeletal development, Tg(sp7:EGFP)b1212. Using a zebrafish BAC, EGFP was introduced downstream of the regulatory regions of sp7 and injected into 1 cell-stage embryos. In this transgenic line, GFP expression reproduces endogenous sp7 gene expression in the otic placode and vesicle, and in forming skeletal structures. GFP-positive cells were also detected in adult fish, and were found associated with regenerating fin rays post-amputation. This line provides an essential tool for the further study of zebrafish otic vesicle formation and the development and regeneration of the skeleton. PMID:20506187

  2. Fluorescence lifetime dynamics of eGFP in protein aggregates with expanded polyQ

    Science.gov (United States)

    Ghukasyan, Vladimir; Hsu, Chih-Chun; Liu, Chia-Rung; Kao, Fu-Jen; Cheng, Tzu-Hao

    2009-02-01

    Expanding a polyglutamine (polyQ) stretch at the N-terminus of huntingtin protein is the main cause of the neurodegenerative disorder Huntington's disease (HD). Expansion of polyQ above 39 residues has an inherent propensity to form amyloid-like fibrils and aggregation of the mutant protein is found to be a critical component for abnormal pathology of HD. Using yeast Saccharomyces cerevisiae as a model system, we have observed a decrease in fluorescence lifetime of the enhanced green fluorescence protein (eGFP) fused to 97 successive glutamine residues (97Q). Compared to the sample expressing evenly distributed eGFP, the 97Q-eGFP fusion proteins show the formation of grain-like particles and the reduction of eGFP lifetime by ~250 ps as measured by time-correlated single-photon counting technique (TCSPC). More importantly, this phenomenon does not appear in Hsp104-deficient cells. The gene product of HSP104 is required for the formation of polyQ aggregates in yeast cells; therefore, the cellular 97Q-eGFP become soluble and evenly distributive in the absence of Hsp104. Under this condition, the lifetime value of 97Q-eGFP is close to the one exhibited by eGFP alone. The independence of the effect of the environmental parameters, such as pH and refraction index is demonstrated. These data indicate that the fluorescence lifetime dynamics of eGFP is linked to the process of polyQ protein aggregation per se.

  3. Use of TSHβ:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Cheng [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Graduate University of Chinese Academy of Sciences, Beijing (China); Jin, Xia; He, Jiangyan [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Yin, Zhan, E-mail: zyin@ihb.ac.cn [Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China)

    2012-07-15

    Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic–pituitary–thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit β (TSHβ) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSHβ in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSHβ promoter, the similar responsive patterns between the expression levels of TSHβ:EGFP and endogenous TSHβ mRNA in the pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSHβ:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system. Highlights: ► The promoter of zebrafish TSHβ gene has been identified. ► The stable TSHβ:EGFP transgenic zebrafish reporter germline has been generated. ► The EGFP in the transgenic fish recapitulated the pattern of pituitary TSHβ mRNA. ► The transgenic zebrafish may be an in vivo model for EDC assessment.

  4. Use of TSHβ:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals

    International Nuclear Information System (INIS)

    Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic–pituitary–thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit β (TSHβ) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSHβ in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSHβ promoter, the similar responsive patterns between the expression levels of TSHβ:EGFP and endogenous TSHβ mRNA in the pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSHβ:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system. Highlights: ► The promoter of zebrafish TSHβ gene has been identified. ► The stable TSHβ:EGFP transgenic zebrafish reporter germline has been generated. ► The EGFP in the transgenic fish recapitulated the pattern of pituitary TSHβ mRNA. ► The transgenic zebrafish may be an in vivo model for EDC assessment.

  5. An E.coil SOS-EGFP biosensor for fast and sensitive detection of DNA damaging agents

    Institute of Scientific and Technical Information of China (English)

    Zhilan Chen; Meiling Lu; Dandan Zou; Hailin Wang

    2012-01-01

    An E.coli SOS-EGFP biosensor which expresses enhanced green fluorescent protein as a reporter protein under the control of recA gene promoter in SOS response was constructed for detection of DNA damage and evaluation of DNA damaging chemicals.The chemicals that may cause substantial DNA damage will trigger SOS response in the constructed bacterial biosensor,and then the reporter egfp gene under the control of recA promoter is stimulated to express as a fluorescent protein,allowing fast and sensitive fluorescence detection.Interestingly,this biosensor can be simultaneously applied for evaluation of genotoxicity and cytotoxicity.The SOS-EGFP bacterial biosensor provides a sensitive,specific and simple method for detecting known and potential DNA damaging chemicals.

  6. Construction of EGFP-tagged rBCG of E.tenella and distribution in chickens

    Institute of Scientific and Technical Information of China (English)

    WANG QiuYue; LI JianHua; ZHANG XiChen; LIU ChengWu; CAO LiLi; REN KeYan; GONG PengTao; CAI YaNan

    2009-01-01

    Chicken coccidiosis is a major parasitic disease with substantial economic burden to the poultry in-dustry. Enhanced green fluorescent protein (EGFP) tagged recombinant Bacille Calmette-Guerin (rBCG), as a fusion protein with coccidian rhomboid antigen was constructed to track rBCG in vivo in chickens in this study. Immunization of chickens with one dose of rBCG pMV361-Rho/EGFP induced humoral immune response. The colonization of rBCG in liver, spleen, lung, kidney and caecum was observed by laser confocal microscopy. Real-time quantitative RT-PCR showed s rise expression level of rhomboid protein on the 7th day and a peak on the 14th day and disappearance on the 28th day after immunization. These results have significant implications for the development of rBCG vaccines against avian coccidiosis.

  7. Construction of EGFP-tagged rBCG of E.tenella and distribution in chickens

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Chicken coccidiosis is a major parasitic disease with substantial economic burden to the poultry industry.Enhanced green fluorescent protein(EGFP) tagged recombinant Bacille Calmette-Guerin(rBCG),as a fusion protein with coccidian rhomboid antigen was constructed to track rBCG in vivo in chickens in this study.Immunization of chickens with one dose of rBCG pMV361-Rho/EGFP induced humoral immune response.The colonization of rBCG in liver,spleen,lung,kidney and caecum was observed by laser confocal microscopy.Real-time quantitative RT-PCR showed a rise expression level of rhomboid protein on the 7th day and a peak on the 14th day and disappearance on the 28th day after immunization.These results have significant implications for the development of rBCG vaccines against avian coccidiosis.

  8. Development of Neutralization Assay Using an eGFP Chikungunya Virus

    Directory of Open Access Journals (Sweden)

    Cheng-Lin Deng

    2016-06-01

    Full Text Available Chikungunya virus (CHIKV, a member of the Alphavirus genus, is an important human emerging/re-emerging pathogen. Currently, there are no effective antiviral drugs or vaccines against CHIKV infection. Herein, we construct an infectious clone of CHIKV and an eGFP reporter CHIKV (eGFP-CHIKV with an isolated strain (assigned to Asian lineage from CHIKV-infected patients. The eGFP-CHIKV reporter virus allows for direct visualization of viral replication through the levels of eGFP expression. Using a known CHIKV inhibitor, ribavirin, we confirmed that the eGFP-CHIKV reporter virus could be used to identify inhibitors against CHIKV. Importantly, we developed a novel and reliable eGFP-CHIKV reporter virus-based neutralization assay that could be used for rapid screening neutralizing antibodies against CHIKV.

  9. The flavonoid luteolin worsens chemical-induced colitis in NF-kappaB(EGFP transgenic mice through blockade of NF-kappaB-dependent protective molecules.

    Directory of Open Access Journals (Sweden)

    Thomas Karrasch

    Full Text Available BACKGROUND: The flavonoid luteolin has anti-inflammatory properties both in vivo and in vitro. However, the impact of luteolin on experimental models of colitis is unknown. METHODOLOGY/PRINCIPAL FINDINGS: To address the therapeutic impact of luteolin, NF-kappaB(EGFP transgenic mice were fed a chow diet containing 2% luteolin- or isoflavone-free control chow (AIN-76, and acute colitis was induced using 3% dextran sodium sulfate (DSS. Additionally, development of spontaneous colitis was evaluated in IL-10(-/-;NF-kappaB(EGFP transgenic mice fed 2% luteolin chow diet or control chow diet. Interestingly, NF-kappaB(EGFP transgenic mice exposed to luteolin showed worse DSS-induced colitis (weight loss, histological scores compared to control-fed mice, whereas spontaneous colitis in IL-10(-/-;NF-kappaB(EGFP mice was significantly attenuated. Macroscopic imaging of live resected colon showed enhanced EGFP expression (NF-kappaB activity in luteolin-fed mice as compared to control-fed animals after DSS exposure, while cecal EGFP expression was attenuated in luteolin-fed IL-10(-/- mice. Interestingly, confocal microscopy showed that EGFP positive cells were mostly located in the lamina propria and not in the epithelium. Caspase 3 activation was significantly enhanced whereas COX-2 gene expression was reduced in luteolin-fed, DSS-exposed NF-kappaB(EGFP transgenic mice as assessed by Western blot and immunohistochemical analysis. In vitro, luteolin sensitized colonic epithelial HT29 cells to TNFalpha-induced apoptosis, caspase 3 activation, DNA fragmentation and reduced TNFalpha-induced C-IAP1, C-IAP2 and COX-2 gene expression. CONCLUSIONS/SIGNIFICANCE: We conclude that while luteolin shows beneficial effects on spontaneous colitis, it aggravates DSS-induced experimental colitis by blocking NF-kappaB-dependent protective molecules in enterocytes.

  10. Effect of the HBV Capsid Assembly Inhibitor Bayer 41-4109 on the Intracellular Localization of EGFP-Core Fusion Proteins

    Directory of Open Access Journals (Sweden)

    Aris Haryanto

    2015-11-01

    Full Text Available Bayer 41-4109 is heteroarylpyrimidine (HAP which has been identified as potent of HBV capsid assemblyinhibitor. The present study was to study effect of Bayer 41-4109 treatment on the intracellular localization ofEGFP-Core fusion proteins into HepG2 cells. Three recombinant plasmids of pEGFP-Core with single, double andtriple NLS of HBV core (EGFP-Core 1C, 2C and 3C and two recombinant plasmids with single and triple NLS ofSV-40 (EGFP-Core 1 and 3 SV-40 were used in this work. After transient transfected into HepG2 cells and treatedwith Bayer 41-4109, the intracellular localization of expressed fusion proteins from all plasmid constructions weredetermined and quantified under confocal laser microscope. Results shown that Bayer 41-4109 treatment in HepG2cells inhibited the nuclear localization of EGFP-Core with single of triple HBV core NLS. As well as the constructionsof expressed fusion protein with single and triple SV-40 NLS (EGFP-Core 1 and 3 SV-40 NLS showeddecreasing the nuclear localization after treated with Bayer 41-4109, even not as strong as EGFP-Core 1C and 3CNLS. Bayer 41-4109 has been identified as a potent inhibitors of HBV replication which has multiple effects on HBVcapsid assembly. It may inhibit virus replication by inducing assembly inappropriately and by misdirectingassembly decreasing the stability of normal capsids.Keywords: HBV capsid, Bayer 41-4109, EGFP-Core fusion protein, HepG2 cell

  11. Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Zhang Yingang; Guo Xiong; Liu Zheng; Wang Shijie

    2007-01-01

    Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2 in mesenchymal stem cells. Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP2 vector by the optimized retroviral transduction protocol. Fluorescent microscopy's examination was to evaluate the results of the transduction, flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells. Bioactivity test from C2C12K4 cells was to show the expression and bio-activity of the fusion gene. Results Fluorescent microscopy showed the success of the transduction. By flow cytometer's analysis, the mean efficiency of the transduction with EGFP was (42.8±6.1)% SD. Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting, almost of those showed the expression of BMP2. Fluorescently and strongly bioactivity test for C2C12K4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control. Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting. Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP2, the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique, the expressed chimeric protein embraced the double bioactivity of EGFP and BMP2, and moreover, the expression had not attenuated over time.

  12. mglur6b:EGFP Transgenic zebrafish suggest novel functions of metabotropic glutamate signaling in retina and other brain regions.

    Science.gov (United States)

    Glasauer, Stella M K; Wäger, Robert; Gesemann, Matthias; Neuhauss, Stephan C F

    2016-08-15

    Metabotropic glutamate receptors (mGluRs) are mainly known for regulating excitability of neurons. However, mGluR6 at the photoreceptor-ON bipolar cell synapse mediates sign inversion through glutamatergic inhibition. Although this is currently the only confirmed function of mGluR6, other functions have been suggested. Here we present Tg(mglur6b:EGFP)zh1, a new transgenic zebrafish line recapitulating endogenous expression of one of the two mglur6 paralogs in zebrafish. Investigating transgene as well as endogenous mglur6b expression within the zebrafish retina indicates that EGFP and mglur6b mRNA are not only expressed in bipolar cells, but also in a subset of ganglion and amacrine cells. The amacrine cells labeled in Tg(mglur6b:EGFP)zh1 constitute a novel cholinergic, non-GABAergic, non-starburst amacrine cell type described for the first time in teleost fishes. Apart from the retina, we found transgene expression in subsets of periventricular neurons of the hypothalamus, Purkinje cells of the cerebellum, various cell types of the optic tectum, and mitral/ruffed cells of the olfactory bulb. These findings suggest novel functions of mGluR6 besides sign inversion at ON bipolar cell dendrites, opening up the possibility that inhibitory glutamatergic signaling may be more prevalent than currently thought. J. Comp. Neurol. 524:2363-2378, 2016. © 2016 Wiley Periodicals, Inc. PMID:27121676

  13. Surface display of monkey metallothionein {alpha} tandem repeats and EGFP fusion protein on Pseudomonas putida X4 for biosorption and detection of cadmium

    Energy Technology Data Exchange (ETDEWEB)

    He, Xiaochuan; Chen, Wenli; Huang, Qiaoyun [Huazhong Agricultural Univ., Wuhan (China). State Key Lab. of Agricultural Microbiology

    2012-09-15

    Monkey metallothionein {alpha} domain tandem repeats (4mMT{alpha}), which exhibit high cadmium affinity, have been displayed for the first time on the surface of a bacterium using ice nucleation protein N-domain (inaXN) protein from the Xanthomonas campestris pv (ACCC - 10049) as an anchoring motif. The shuttle vector pIME, which codes for INAXN-4mMT{alpha}-EGFP fusion, was constructed and used to target 4mMT{alpha} and EGFP on the surface of Pseudomonas putida X4 (CCTCC - 209319). The surface location of the INAXN-4mMT{alpha}-EGFP fusion was further verified by western blot analysis and immunofluorescence microscopy. The growth of X4 showed resistance to cadmium presence. The presence of surface-exposed 4mMT{alpha} on the engineered strains was four times higher than that of the wild-type X4. The Cd{sup 2+} accumulation by X4/pIME was not only four times greater than that of the original host bacterial cells but was also remarkably unaffected by the presence of Cu{sup 2+} and Zn{sup 2+}. Moreover, the surface-engineered strains could effectively bind Cd{sup 2+} under a wide range of pH levels, from 4 to 7. P. putida X4/pIME with surface-expressed 4mMT{alpha}-EGFP had twice the cadmium binding capacity as well as 1.4 times the fluorescence as the cytoplasmic 4mMTa-EGFP. These results suggest that P. putida X4 expressing 4mMT{alpha}-EGFP with the INAXN anchor motif on the surface would be a useful tool for the remediation and biodetection of environmental cadmium contaminants. (orig.)

  14. Feasibility of dual reporter gene in rat myoblast cell line using human sodium iodide symporter (hNIS) and enhanced green fluorescent protein (EGFP) gene

    International Nuclear Information System (INIS)

    To develop a non-invasive combined imaging method of gamma camera and optical imaging to assess rat myoblast cell line, H9c2, we constructed retrovirus containing hNIS and EGFP gene, and transfected to rat myoblast cell and monitored hNIS and EGFP expression. Rat myoblast cell line, H9C2, was transfected with hNIS and EGFP gene using retrovirus (H9C2-NG). The expression of hNIS and EGFP gene was determined by RT-PCR and fluorescence microscopy, respectively. The uptake and efflux of I-125 were measured in the transfected and wild type cell lines. Each cell line was injected to 4 flank sites (H9c2: 1X107 or 2X107, H9C2-NG: 1X107 or 2X107) in nude mouse. Scintigraphic image was performed at 3h, 1 day after H9C2 and H9C2-NG cell inoculation. We performed gamma camera and animal PET imaging to evaluate NIS expression. Also, GFP image obtained using optical imaging system. The expression of hNIS and EGFP gene was confirmed by RT-PCR. In iodide uptake, H9C2-NG cells accumulated 274.52.2 pmol/ mg protein at 30 min. But wild type cell line did not uptake iodide. In fluorescent microscopy, H9C2-NG cells were highly fluorescent than that of H9C2 cells. In iodide efflux study, 50% of radioactivity flowed out during the first 10min. Scintigraphy showed increased uptake of Tc-99m in H9c2-NG than in H9C2 for 1 day. Also, H9C2-NG cells showed high signal-to-background fluorescent spots in animal body. In this study, NIS and EGFP reporter gene were successfully transfected by a retrovirus in myoblast cell line, and the transfected cell can be easily visualized in vivo. These results suggest that NIS and EGFP gene has an excellent feasibility as a reporter gene, and it can be used to monitor cell trafficking for monitoring

  15. Feasibility of dual reporter gene in rat myoblast cell line using human sodium iodide symporter (hNIS) and enhanced green fluorescent protein (EGFP) gene

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Yong Jin; Lee, You La; Ahn, Sohn Joo; Choi, Chang Ik; Lee, Sang Woo; Ahn, Byeong Cheol; Lee, Jae Tae [School of Medicine, Kyungpook National University, Daegu (Korea, Republic of)

    2007-07-01

    To develop a non-invasive combined imaging method of gamma camera and optical imaging to assess rat myoblast cell line, H9c2, we constructed retrovirus containing hNIS and EGFP gene, and transfected to rat myoblast cell and monitored hNIS and EGFP expression. Rat myoblast cell line, H9C2, was transfected with hNIS and EGFP gene using retrovirus (H9C2-NG). The expression of hNIS and EGFP gene was determined by RT-PCR and fluorescence microscopy, respectively. The uptake and efflux of I-125 were measured in the transfected and wild type cell lines. Each cell line was injected to 4 flank sites (H9c2: 1X107 or 2X107, H9C2-NG: 1X107 or 2X107) in nude mouse. Scintigraphic image was performed at 3h, 1 day after H9C2 and H9C2-NG cell inoculation. We performed gamma camera and animal PET imaging to evaluate NIS expression. Also, GFP image obtained using optical imaging system. The expression of hNIS and EGFP gene was confirmed by RT-PCR. In iodide uptake, H9C2-NG cells accumulated 274.52.2 pmol/ mg protein at 30 min. But wild type cell line did not uptake iodide. In fluorescent microscopy, H9C2-NG cells were highly fluorescent than that of H9C2 cells. In iodide efflux study, 50% of radioactivity flowed out during the first 10min. Scintigraphy showed increased uptake of Tc-99m in H9c2-NG than in H9C2 for 1 day. Also, H9C2-NG cells showed high signal-to-background fluorescent spots in animal body. In this study, NIS and EGFP reporter gene were successfully transfected by a retrovirus in myoblast cell line, and the transfected cell can be easily visualized in vivo. These results suggest that NIS and EGFP gene has an excellent feasibility as a reporter gene, and it can be used to monitor cell trafficking for monitoring.

  16. Thalidomide Effects in Patients with Hereditary Hemorrhagic Telangiectasia During Therapeutic Treatment and in Fli-EGFP Transgenic Zebrafish Model

    Institute of Scientific and Technical Information of China (English)

    Hong-Ling Peng; Yi-Fang Yi; Shun-Ke Zhou; Si-Si Xie; Guang-Sen Zhang

    2015-01-01

    Background: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease characterized by recurrent epistaxis,mucocutaneous telangiectasia, and arteriovenous malformations.The efficacy of traditional treatments for HHT is very limited.The aim of this study was to investigate the therapeutic role of thalidomide in HHT patients and the effect in FLI-EGFP transgenic zebrafish model.Methods: HHT was diagnosed according to Shovlin criteria.Five HHT patients were treated with thalidomide (100 mg/d).The Epistaxis Severity Score (ESS), telangiectasia spots, and hepatic computed tomography angiography (CTA) were used to assess the clinical efficacy of thalidomide.The Fli-EGFP zebrafish model was investigated for the effect of thalidomide on angiogenesis.Dynamic real-time polymerase chain reaction assay, ELISA and Western blotting from patient's peripheral blood mononuclear cells and plasma were used to detect the expression of transforming growth factor beta 3 (TGF-β3) messenger RNA (mRNA) and vascular endothelial growth factor (VEGF) protein before and after 6 months of thalidomide treatment.Results: The average ESS before and after thalidomide were 6.966 ± 3.093 and 1.799 ± 0.627, respectively (P =0.009).The "telangiectatic spot" on the tongue almost vanished;CTA examination of case 2 indicated a smaller proximal hepatic artery and decreased or ceased hepatic artery collateral circulation.The Fli-EGFP zebrafish model manifested discontinuous vessel development and vascular occlusion (7 of 10 fishes), and the TGF-β3 mRNA expression of five patients was lower after thalidomide therapy.The plasma VEGF protein expression was down-regulated in HHT patients.Conclusions: Thalidomide reverses telangiectasia and controls nosebleeds by down-regulating the expression of TGF-β3 and VEGF in HHT patients.It also leads to vascular remodeling in the zebrafish model.

  17. Fluorescence-Activated Cell Sorting of EGFP-Labeled Neural Crest Cells From Murine Embryonic Craniofacial Tissue

    Directory of Open Access Journals (Sweden)

    Saurabh Singh

    2005-01-01

    Full Text Available During the early stages of embryogenesis, pluripotent neural crest cells (NCC are known to migrate from the neural folds to populate multiple target sites in the embryo where they differentiate into various derivatives, including cartilage, bone, connective tissue, melanocytes, glia, and neurons of the peripheral nervous system. The ability to obtain pure NCC populations is essential to enable molecular analyses of neural crest induction, migration, and/or differentiation. Crossing Wnt1-Cre and Z/EG transgenic mouse lines resulted in offspring in which the Wnt1-Cre transgene activated permanent EGFP expression only in NCC. The present report demonstrates a flow cytometric method to sort and isolate populations of EGFP-labeled NCC. The identity of the sorted neural crest cells was confirmed by assaying expression of known marker genes by TaqMan Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR. The molecular strategy described in this report provides a means to extract intact RNA from a pure population of NCC thus enabling analysis of gene expression in a defined population of embryonic precursor cells critical to development.

  18. In vitro cultivation of rat bone marrow mesenchymal stem cells and establishment of pEGFP/Ang-1 transfection method

    Institute of Scientific and Technical Information of China (English)

    Xiu-Qun; Zhang; Long; Wang; Shu-Li; Zhao; Wei; Xu

    2014-01-01

    Objective:To obtain the bone marrow mesenchymal stem cells(BMSCs).complete phenotypic identification and successfully transfecl rat BMSCs by recombinant plasmid pF.GFP/Ang-1.Methods:BMSCs were isolated from bone marrow using density gradient centrifugation method and adherence screening method,and purified.Then the recombinant plasmid pEGFP/Ang-1was used to transfect BMSCs and the positive clones were obtained by the screen of C418 and observed under light microscopy inversely.Green fluorescent exhibited by protein was enhanced to measure the change time of the expression amount of Ang-1.Results:BMSCs cell lines were obtained successfully by adherence screening method and density gradient ccntrifugation.Ang-1 recombinant plasmid was transfected smoothly into rat BMSCs,which can express Ang-1 for 3 d and decreased after 7 d.Conclusions:Adherence screening method und density gradient ceiilrifugation can be effective methods lo obtain BMSCs with high purity and rapid proliferation.Besides,the expression of transfected recombinant plasmid pEGFP/Ang-1 in rat BMSCs is satisfactory.

  19. Wavelength dependent specific plasmon resonance coupling of single silver nanoparticles with EGFP

    Science.gov (United States)

    Lee, Kerry J.; Huang, Tao; Nallathamby, Prakash D.; Xu, Xiao-Hong Nancy

    2015-10-01

    Noble metal nanoparticles (NPs) possess unique plasmonic properties, enabling them to serve as sub-diffraction light sources and nano- antennae for a wide range of applications. Here we report the specific interaction of single Ag NPs with single EGFP molecules and a high dependence of their interaction upon localized-surface-plasmon-resonance (LSPR) spectra of single Ag NPs and EGFP. The LSPR spectra of single red Ag NPs show a stunning 60 nm blue-shift during their incubation with EGFP, whereas they remain unchanged during their incubation with bovine serum albumin (BSA). Interestingly, the peak wavelengths of the LSPR spectra of green and blue Ag NPs remain essentially unchanged during their incubation with either EGFP or BSA. These interesting findings suggest that plasmon-resonance-energy-transfer (PRET) from single Ag NPs to EGFP might follow a two-photon excitation mechanism to excite EGFP and the fluorescence of the excited EGFP might couple with the plasmon of single NPs leading to a blue-shift of the red NPs. These distinctive phenomena are only observed by real-time single NP spectroscopic measurements. This study offers exciting new opportunities to design new sensing and imaging tools with high specificity and sensitivity to study long-range molecular interactions and dynamic events in single live cells, and to probe the underlying molecular mechanisms of PRET.

  20. A cationic cholesterol based nanocarrier for the delivery of p53-EGFP-C3 plasmid to cancer cells.

    Science.gov (United States)

    Misra, Santosh K; Naz, Sarwat; Kondaiah, Paturu; Bhattacharya, Santanu

    2014-01-01

    The p53 protein mediated anti-tumor strategy is limited due to the lack of suitable delivery agent with insignificant immunogenic response, serum compatibility, and early and easy detection of the transfected cell population. To overcome these problems, we generated a p53-EGFP-C3 fusion construct which expressed easily detectable green fluorescence protein (GFP) and allowed an estimation of p53 mediated anti-tumor activity. A mixture of cationic cholesterol gemini (Chol-5L) with natural lipid, DOPE (molar ratio 1:4), acronymed as Chol-5LD, formed a nano-liposome as characterized by various physical methods. The prepared clone was evaluated for the expression of GFP and functional p53 in HeLa and two additional cell lines with varied p53 status namely, H1299 (p53(-/-)) and HEK293T (p53(+/+)). Transfected cells were screened using RT-PCR, Western blotting, FACS analysis, MTT, Trypan blue assay and visualized under a fluorescence microscope. The p53-EGFP-C3 fusion protein induced apoptosis in cancer cells as evident from DNA fragmentation, cell cycle analysis, Annexin-V staining and PARP cleavage assays. The transfection and apoptosis induction efficiency of Chol-5LD was significantly higher than commercial reagents Lipofectamine2000 and Effectene irrespective of the cell lines examined. Further it significantly decreases the xenograft tumor volume in nude mice tumors via apoptosis as observed in H&E staining. PMID:24211075

  1. 视锥视杆细胞同源盒基因的慢病毒载体的构建%The construction of the recombinant CRX gene-containing lentiviral vectors LV-Rho-EGFP/CRX

    Institute of Scientific and Technical Information of China (English)

    李伟; 高玲; 卢光琇; 刘刚; 王建; 周民稳

    2012-01-01

    Objective To investigate the transfection efficiency of the recombinant cone-rod hemeobox ( CRX ) gene-containing lentiviral vector ( LV-Rho-EGFP/CRX ) drived by the Rhodopsin promoter in vitro. Methods The lentiviral vector( LV-Rho-EGFP/CRX )was reconstructed by replace of the EGFP gene fragment in the lentiviral vectors LV-Rho-EGFP with the EGFP/CRX gene fragment, which was amplified by PCR from pEGFP-C3/CRX vector. It was analyzed by restriction digestion and bidirectional sequencing. The recombination lentiviral particles were produced by the packaging 293T cell by using the Ca3( P04 )2 method, the culture supernatant was harvested and the titration of them were calculated by the limiting dilution method. The photoreceptor-deriving human retinoblastoma cell line HXO-RB44 cells were transduced by the recombinant lentiviral particles, after 48 h the GFP expression was observed under the fluorescence microscope. Results The recombinant lentiviral vector LV-Rho-EGFP/CRX had been successfully constructed, the titration of lentivirus particles was 2 × 104 IU/ml. The HXO-RB44 cells could be transduced by the recombinant lentiviral particles( 2 × 104 IU/ml ). After 48 h coculture, some HXO-RB44 cells could express GFP. Conclusions The recombination lentiviral vector LV-Rho-EGFP/CRX was successfully constructed. The photoreceptor-deriving HXO-RB44 cells could be transduced by lentiviral particles with the titration 2 × 10 IU/ml in a certain degree.%目的 构建视紫红质启动子(rhodopsin,Rho)驱动的、大鼠视锥视杆细胞同源盒基因(cone-rod homeobox gene,CRX)为目的基因的重组慢病毒载体LV-Rho-EGFP/CRX,研究其在体外的转染效率.方法 用PCR法扩增质粒pEGFP-C3/CRX中的EGFP/CRX基因片段,取代慢病毒载体LV-Rho-EGFP中的EGFP片段,构建表达载体LV-Rho-EGFP/CRX,经酶切和双向测序验证.用磷酸钙沉淀法四质粒共转染293T细胞,包装、收集病毒,利用有限稀释法测定病毒滴度.用慢病毒原

  2. Systematic colocalization errors between acridine orange and EGFP in astrocyte vesicular organelles

    OpenAIRE

    Nadrigny, F.; Li, D.; Kemnitz, K; Ropert, N.; Koulakoff, A; Rudolph, S.; Vitali, M.; Giaume, C.; Kirchhoff, F.; Oheim, M

    2007-01-01

    Dual-color imaging of acridine orange (AO) and EGFP fused to a vesicular glutamate transporter or the vesicle-associated membrane proteins 2 or 3 has been used to visualize a supposedly well-defined subpopulation of glutamatergic astrocytic secretory vesicles undergoing regulated exocytosis. However, AO metachromasy results in the concomitant emission of green and red fluorescence from AO-stained tissue. Therefore, the question arises whether AO and EGFP fluorescence can be distinguished reli...

  3. A Sensitive and Rapid Assay for Investigating Vertical Transmission of Hepatitis B Virus via Male Germ Line Using EGFP Vector as Reporter

    Science.gov (United States)

    Ahmed, Mohamed Morsi M.; Huang, Tian-Hua; Xie, Qing-Dong

    2008-01-01

    Hepatitis B virus (HBV) constitutes a serious menace to man. DNA recombination and sequencing, interspecific in vitro fertilization, single-embryo PCR and RT-PCR were employed to establish a sensitive and rapid assay for exploring the vertical transmission of viruses via male germ line. Plasmid pIRES2-EGFP-HBs which expressed enhanced green fluorescent protein as reporter for the expression of hepatitis B virus S gene was successfully constructed and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize with zona-free hamster ova. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBs DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive control (PCR) indicating expression of pIRES2-EGFP-HBs, and not observed in the embryos without green fluorescence and negative controls (PCR and RT-PCR) indicating no pIRES2-EGFP-HBs in the cells. The advantages and application foreground of this assay for study on vertical transmission of viruses such as HCV, HIV, HPV, and SARS via germ line were discussed. PMID:18670607

  4. Gene transfer and expression of enhanced green fluorescent protein in variant HT-29c cells

    Institute of Scientific and Technical Information of China (English)

    Min Wang; Lars Boenicke; Bradley D. Howard; Ilka Vogel; Hoiger Kalthoff

    2003-01-01

    AIM: To study the expression of enhanced green fluorescent protein (EGFP) gene in retrovirally transduced variant HT29 cells.METHODS: The retroviral vector prkat EGFP/neo was constructed and transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (selected from HT-29 cells) were transduced by a retroviral vector encoding the GEFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transduced with the EGFP bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter (FACS) analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransduced and transduced cells in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo.RESULTS: After transduced, HT-29c cells displayed a stable and long-term EGFP expression under the nonselective conditionsin vitro. After cells were successively cultured to passage 50 in vitro, EGFP expression was still at a high level. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransduced parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed.CONCLUSION: An EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo.These cells with EGFP are a valuable tool forin vivo research of tumor metastatic spread.

  5. [Stable expression of recombinant human podoplanin in Chinese hamster ovary (CHO) cells].

    Science.gov (United States)

    Qu, Le; Zhao, Xingpeng; Fu, Jianxin; Xia, Lijun; Dai, Lan; Ruan, Changgeng; Zhao, Yiming

    2016-01-01

    Objective To construct podoplanin (PDPN) eukaryotic expression plasmid PDPN-pEGFP-N1, establish Chinese hamster ovary (CHO) cell line stably expressing recombinant human PDPN and investigate its biological activity. Methods PDPN cDNA was cloned from HEK293 cells by reverse transcription PCR and recombinant DNA technology and inserted into plasmid pEGFP-N1 labeled by enhanced green fluorescent protein (EGFP). The recombinant vector was identified by PCR, restriction enzyme digestion and DNA sequencing, and then transfected into CHO cells. Recombinant PDPN-EGFP was observed by fluorescent microscopy and CHO cell line with the high expression of PDPN-EGFP was selected by flow cytometry. Recombinant PDPN was detected by Western blotting and the biological activity of the cell line was determined by platelet aggregation assay. Results DNA sequencing and restriction enzyme digestion proved that the gene of PDPN was inserted successfully into pEGFP-N1 plasmid. After stable transfection of the recombinant plasmid into CHO cells, CHO with EGFP could be seen under a fluorescent microscope. The CHO cell line with the high expression of recombinant PDPN-EGFP was obtained after sorting by flow cytometry. Western blotting showed that the recombinant PDPN was expressed on the cell surface. The over-expressing PDPN-EGFP CHO cells were able to induce human platelet aggregation. Conclusion The CHO cell line with the stable and high expression of recombinant PDPN-EGFP has been constructed successfully, and it could induce platelet aggregation. PMID:26728373

  6. Noninvasive optical diagnostics of enhanced green fluorescent protein expression in skeletal muscle for comparison of electroporation and sonoporation efficiencies

    Science.gov (United States)

    Tamošiūnas, Mindaugas; Kadikis, Roberts; Saknīte, Inga; Baltušnikas, Juozas; Kilikevičius, Audrius; Lihachev, Alexey; Petrovska, Ramona; Jakovels, Dainis; Šatkauskas, Saulius

    2016-04-01

    We highlight the options available for noninvasive optical diagnostics of reporter gene expression in mouse tibialis cranialis muscle. An in vivo multispectral imaging technique combined with fluorescence spectroscopy point measurements has been used for the transcutaneous detection of enhanced green fluorescent protein (EGFP) expression, providing information on location and duration of EGFP expression and allowing quantification of EGFP expression levels. For EGFP coding plasmid (pEGFP-Nuc Vector, 10 μg/50 ml) transfection, we used electroporation or ultrasound enhanced microbubble cavitation [sonoporation (SP)]. The transcutaneous EGFP fluorescence in live mice was monitored over a period of one year using the described parameters: area of EGFP positive fibers, integral intensity, and mean intensity of EGFP fluorescence. The most efficient transfection of EGFP coding plasmid was achieved, when one high voltage and four low voltage electric pulses were applied. This protocol resulted in the highest short-term and long-term EGFP expression. Other electric pulse protocols as well as SP resulted in lower fluorescence intensities of EGFP in the transfected area. We conclude that noninvasive multispectral imaging technique combined with fluorescence spectroscopy point measurements is a suitable method to estimate the dynamics and efficiency of reporter gene transfection in vivo.

  7. Embryo development, fetal growth and postnatal phenotype of eGFP lambs generated by lentiviral transgenesis.

    Science.gov (United States)

    Crispo, M; Vilariño, M; dos Santos-Neto, P C; Núñez-Olivera, R; Cuadro, F; Barrera, N; Mulet, A P; Nguyen, T H; Anegón, I; Menchaca, A

    2015-02-01

    Lentiviral technology has been recently proposed to generate transgenic farm animals more efficiently and easier than traditional techniques. The objective was to evaluate several parameters of lambs obtained by lentiviral transgenesis in comparison with non-transgenic counterparts. In vitro produced embryos were microinjected (TG group) at two-cell stage with a lentiviral construct containing enhanced green fluorescent protein (eGFP) gene, while embryos produced by in vitro fertilization (IVF group) or intrauterine insemination (IUI group) were not microinjected. Microinjection technique efficiently generated eight-cell transgenic embryos (97.4%; 114/117). Development rate on day 5 after fertilization was similar for TG (39.3%, 46/117) and IVF embryos (39.6%, 44/111). Pregnancy rate was detected in 50.0% (6/12) of recipient ewes with TG embryos, in 46.7% (7/15) with IVF embryos, and in 65.0% (13/20) of IUI ewes (P = NS). Nine lambs were born in TG group, six lambs in IVF group, and 16 lambs in IUI group. All TG lambs (9/9) were GFP positive to real-time PCR and eight (88.9%) showed a strong and evident GFP expression in mucosae, eyes and keratin tissues. Fetal growth monitored every 15 day by ultrasonography did not show significant differences. Transgenic lambs neither differ in morphometric variables in comparison with non transgenic IVF lambs within 3 months after birth. Transmission of the transgene to the progeny was observed in green fluorescent embryos produced by IVF using semen from the TG founder lambs. In conclusion, this study demonstrates the high efficiency of lentiviral technology to produce transgenic sheep, with no clinic differences in comparison with non transgenic lambs. PMID:25048992

  8. Genetic Transformation of the Codling Moth, Cydia pomonella L., with piggyBac EGFP

    Science.gov (United States)

    Genetic transformation of the codling moth, Cydia pomonella, was accomplished through embryo microinjection with a plasmid-based piggyBac vector containing the enhanced green fluorescent protein (EGFP) gene. Sequencing of the flanking regions around the inserted construct results in identification o...

  9. Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

    Directory of Open Access Journals (Sweden)

    Kawakami Minoru

    2011-11-01

    Full Text Available Abstract Background Neural crest cells (NCCs are embryonic, multipotent stem cells. Their long-range and precision-guided migration is one of their most striking characteristics. We previously reported that P0-Cre/CAG-CAT-lacZ double-transgenic mice showed significant lacZ expression in tissues derived from NCCs. Results In this study, by embedding a P0-Cre/CAG-CAT-EGFP embryo at E9.5 in collagen gel inside a culture glass slide, we were able to keep the embryo developing ex vivo for more than 24 hours; this development was with enough NCC fluorescent signal intensity to enable single-cell resolution analysis, with the accompanying NCC migration potential intact and with the appropriate NCC response to the extracellular signal maintained. By implantation of beads with absorbed platelet-derived growth factor-AA (PDGF-AA, we demonstrated that PDGF-AA acts as an NCC-attractant in embryos. We also performed assays with NCCs isolated from P0-Cre/CAG-CAT-EGFP embryos on culture plates. The neuromediator 5-hydroxytryptamine (5-HT has been known to regulate NCC migration. We newly demonstrated that dopamine, in addition to 5-HT, stimulated NCC migration in vitro. Two NCC populations, with different axial levels of origins, showed unique distribution patterns regarding migration velocity and different dose-response patterns to both 5-HT and dopamine. Conclusions Although avian species predominated over the other species in the NCC study, our novel system should enable us to use mice to assay many different aspects of NCCs in embryos or on culture plates, such as migration, division, differentiation, and apoptosis.

  10. Intranuclear Localization of EGFP-mouse PPARγ1 in Bovine Fibroblast Cells

    Directory of Open Access Journals (Sweden)

    Sorayya Ghasemi

    2010-01-01

    Full Text Available Objective: The aim of this study was to clone PPARγ1 cDNA in an appropriate mammalianexpression vector, with a chimeric cDNA form, encompassing PPARγ with enhanced greenfluorescent protein (EGFP cDNA. This recombinant plasmid will be used for further analysesto investigate the molecular mechanism of PPARγ1 for neural differentiation process.Moreover, the nuclear localization of the PPARγ1 protein linked to EGFP marker was chasedby using transient transfection of a constructed plasmid into bovine fibroblast cells.Materials and Methods: Total RNA was extracted from the fatty tissue of an adult mouse.Using specific pair primers, PPARγ1 cDNA was synthesized and amplified to producethe entire length of ORF. RT-PCR products containing PPARγ1 cDNA were treated byenzymatic digestion and inserted into the pEGFP-C1 downstream from EGFP cDNA. Theconstructed vector was used for transformation into bacterial competent cells. Positivecolonies which showed inserted PPARγ1 cDNA were selected for plasmid preparationsand additional analysis was performed to ensure that PPARγ1 cDNA was inserted properly.Finally, to confirm the intracellular localization of EGFP-PPARγ1, bovine fibroblastcells were transfected with the recombinant plasmid.Results: Our results from enzymatic digestion and sequencing confirmed, as expected, thatPPARγ1 cDNA was amplified and cloned correctly. This cDNA gene encompassed 1428 bp.The related product was entered into the nucleus of bovine fibroblasts after transfection ofits cDNA.

  11. Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang

    Science.gov (United States)

    Junghans, Cornelia; Schmitt, Franz-Josef; Vukojević, Vladana; Friedrich, Thomas

    2016-02-01

    Measurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular crowding, making it difficult to follow the track of individual molecules carrying a fluorescence label. Novel switchable fluorescence proteins such as Dreiklang [1], are, in principle, promising tools to study the diffusion behavior of individual molecules in situations of molecular crowding due to excellent spectral control of the ON- and OFF-switching process. In this work, we expressed an integral membrane transport protein, the Na,K-ATPase comprising the human α2-subunit carrying an N-terminal eGFP or Dreiklang tag and human β1-subunit, in HEK293T cells and measured autocorrelation curves by fluorescence correlation spectroscopy (FCS). Furthermore,we measured diffusion times and diffusion constants of eGFP and Dreiklang by FCS, first, in aqueous solution after purification of the proteins upon expression in E. coli, and, second, upon expression as soluble proteins in the cytoplasm of HEK293T cells. Our data show that the diffusion behavior of the purified eGFP and Dreiklang in solution as well as the properties of the proteins expressed in the cytoplasm are very similar. However, the autocorrelation curves of eGFP- and Dreiklanglabeled Na,K-ATPase measured in the plasma membrane exhibit marked differences, with the Dreiklang-labeled construct showing shorter diffusion times. This may be related to an additional, as yet unrecognized quenching process that occurs on the same time

  12. Generation of Col2a1-EGFP iPS Cells for Monitoring Chondrogenic Differentiation

    OpenAIRE

    Saito, Taku; Yano, Fumiko; Mori, Daisuke; Ohba, Shinsuke; Hojo, Hironori; Otsu, Makoto; Eto, Koji; Nakauchi, Hiromitsu; Tanaka, Sakae; Chung, Ung-il; Kawaguchi, Hiroshi

    2013-01-01

    Induced pluripotent stem cells (iPSC) are a promising cell source for cartilage regenerative medicine; however, the methods for chondrocyte induction from iPSC are currently developing and not yet sufficient for clinical application. Here, we report the establishment of a fluorescent indicator system for monitoring chondrogenic differentiation from iPSC to simplify screening for effective factors that induce chondrocytes from iPSC. We generated iPSC from embryonic fibroblasts of Col2a1-EGFP t...

  13. Quantification of Astrocyte Volume Changes during Ischemia in the Cortox of EGFP/GFAP Mice.

    Czech Academy of Sciences Publication Activity Database

    Benešová, Jana; Anděrová, Miroslava; Hock, Miroslav; Neprašová, Helena; Prajerová, Iva; Chvátal, Alexandr

    Magdeburg : Institute for Neurobiochemistry, 2006. [International Symposium on Neuroprotection and Neurorepair /4./. 03.05.2006-06.05.2006, Magdeburg] R&D Projects: GA ČR GA305/03/1172; GA ČR GA305/06/1316; GA ČR GA305/06/1464; GA MŠk LC554 Institutional research plan: CEZ:AV0Z50390512 Keywords : EGFP/GFAP Subject RIV: FH - Neurology

  14. Characterization of a Dmd EGFP reporter mouse as a tool to investigate dystrophin expression

    OpenAIRE

    Petkova, Mina V.; Morales-Gonzales, Susanne; Relizani, Karima; Gill, Esther; Seifert, Franziska; Radke, Josefine; Stenzel, Werner; Garcia, Luis; Amthor, Helge; Schuelke, Markus

    2016-01-01

    Background Dystrophin is a rod-shaped cytoplasmic protein that provides sarcolemmal stability as a structural link between the cytoskeleton and the extracellular matrix via the dystrophin-associated protein complex (DAPC). Mutations in the dystrophin-encoding DMD gene cause X-linked dystrophinopathies with variable phenotypes, the most severe being Duchenne muscular dystrophy (DMD) characterized by progressive muscle wasting and fibrosis. However, dystrophin deficiency does not only impair th...

  15. Plasmid-based genetic modification of human bone marrow-derived stromal cells: analysis of cell survival and transgene expression after transplantation in rat spinal cord

    Directory of Open Access Journals (Sweden)

    Van Tendeloo Viggo FI

    2007-12-01

    Full Text Available Abstract Background Bone marrow-derived stromal cells (MSC are attractive targets for ex vivo cell and gene therapy. In this context, we investigated the feasibility of a plasmid-based strategy for genetic modification of human (hMSC with enhanced green fluorescent protein (EGFP and neurotrophin (NT3. Three genetically modified hMSC lines (EGFP, NT3, NT3-EGFP were established and used to study cell survival and transgene expression following transplantation in rat spinal cord. Results First, we demonstrate long-term survival of transplanted hMSC-EGFP cells in rat spinal cord under, but not without, appropriate immune suppression. Next, we examined the stability of EGFP or NT3 transgene expression following transplantation of hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP in rat spinal cord. While in vivo EGFP mRNA and protein expression by transplanted hMSC-EGFP cells was readily detectable at different time points post-transplantation, in vivo NT3 mRNA expression by hMSC-NT3 cells and in vivo EGFP protein expression by hMSC-NT3-EGFP cells was, respectively, undetectable or declined rapidly between day 1 and 7 post-transplantation. Further investigation revealed that the observed in vivo decline of EGFP protein expression by hMSC-NT3-EGFP cells: (i was associated with a decrease in transgenic NT3-EGFP mRNA expression as suggested following laser capture micro-dissection analysis of hMSC-NT3-EGFP cell transplants at day 1 and day 7 post-transplantation, (ii did not occur when hMSC-NT3-EGFP cells were transplanted subcutaneously, and (iii was reversed upon re-establishment of hMSC-NT3-EGFP cell cultures at 2 weeks post-transplantation. Finally, because we observed a slowly progressing tumour growth following transplantation of all our hMSC cell transplants, we here demonstrate that omitting immune suppressive therapy is sufficient to prevent further tumour growth and to eradicate malignant xenogeneic cell transplants. Conclusion In this study, we

  16. Quantification of Astrocyte Volume Changes during Ischemia in the Cortox of EGFP/GFAP Mice

    Czech Academy of Sciences Publication Activity Database

    Benešová, Jana; Anděrová, Miroslava; Hock, Miroslav; Neprašová, Helena; Prajerová, Iva; Chvátal, Alexandr

    Magdeburg : Organizátor symposia, 2006. s. 14-14. [International symposium on Neuroprotection and Neurorepair: Cerebral Ischemia and Stroke /4./. 03.05.2006-06.05.2006, Magdeburg] R&D Projects: GA ČR GA305/03/1172; GA ČR GA305/06/1316; GA ČR GA305/06/1464; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50390512 Keywords : EGFP/GFAP * Ischemia Subject RIV: FH - Neurology http://www.neurorepair-2006.de/nr/files/Program.pdf

  17. Quantification for total demethylation potential of environmental samples utilizing the EGFP reporter gene.

    Science.gov (United States)

    Qian, Yan; Wang, Xiao-li; Lv, Zhan-lu; Tysklind, Mats; Guo, Chen; Liang, Bao; Wu, Jia-bing; Yang, Yong-jian; Yang, Yi-shu; Wang, Fei-fei; Duan, Xiao-li; Ma, Jin; Wei, Yong-jie; Wang, Chun-hui; Yang, Li-xin; Zhang, Jin-liang; Shi, Xiao-ming; Wang, Xian-liang

    2016-04-01

    The demethylation potential of pollutants is arguably an innate component of their toxicity in environmental samples. A method was developed for determining the total demethylation potential of food samples (TDQ). The demethylation epigenetic toxicity was determined using the Hep G2 cell line transfected with pEGFP-C3 plasmids containing a methylated promoter of the EGFP reporter gene. The total demethylation potential of the sample extracts (the 5-AZA-CdR demethylation toxic equivalency) can be quantified within one week by using a standard curve of the 5-AZA-CdR demethylation agent. To explore the applicability of TDQ for environmental samples, 17 groundwater samples were collected from heavy polluted Kuihe river and the total demethylation potentials of the sample extracts were measured successfully. Meaningful demethylation toxic equivalencies ranging from 0.00050 to 0.01747μM were found in all groundwater sample extracts. Among 19 kinds of inorganic substance, As and Cd played important roles for individual contribution to the total demethylation epigenetic toxicity. The TDQ assay is reliable and fast for quantifying the DNA demethylation potential of environmental sample extracts, which may improve epigenetic toxicity evaluations for human risk assessment, and the consistent consuming of groundwater alongside the Kuihe river pose unexpected epigenetic health risk to the local residents. PMID:26774982

  18. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    Science.gov (United States)

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  19. Truncated forms of viral VP2 proteins fused to EGFP assemble into fluorescent parvovirus-like particles

    Directory of Open Access Journals (Sweden)

    Vuento Matti

    2006-12-01

    Full Text Available Abstract Fluorescence correlation spectroscopy (FCS monitors random movements of fluorescent molecules in solution, giving information about the number and the size of for example nano-particles. The canine parvovirus VP2 structural protein as well as N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids were fused to the C-terminus of the enhanced green fluorescent protein (EGFP. The proteins were produced in insect cells, purified, and analyzed by western blotting, confocal and electron microscopy as well as FCS. The non-truncated form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic radii of 7, 20 and 14 nm, respectively. These results show that the non-truncated EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as much as 40 amino acids were able to form virus-like particles (VLPs. The fluorescent VLP, harbouring VP2 truncated by 23 amino acids, showed a somewhat larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast, the construct containing EGFP-VP2 truncated by 14 amino acids was not able to assemble into VLP-resembling structures. Formation of capsid structures was confirmed by confocal and electron microscopy. The number of fluorescent fusion protein molecules present within the different VLPs was determined by FCS. In conclusion, FCS provides a novel strategy to analyze virus assembly and gives valuable structural information for strategic development of parvovirus-like particles.

  20. Construction of plasmid vector pAFP-HSVtk-IRES2-EGFP and its effect on the cytotoxicity of ganciclovir to hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Lai Zhiyong; Qin Qin; Yu Baofeng; Xie Jun; Gao Ranpeng; Zhang Tiantian; Li Chunfeng

    2014-01-01

    Background Herpes simplex virus thymidine kinase phosphorylates ganciclovir to ganciclovir monophosphate,which is then converted to ganciclovir triphosphate by endogenous cellular nucleoside kinases.The ganciclovir triphosphate acts as a DNA chain terminator due to the lack of a functional 3'-OH group and terminates the process of DNA replication,hence leading to cell apoptosis.At present,HSVtk gene usually acts as suicide gene to kill tumor cells.The aim of this study was to investigate the selective cytotoxicity of the herpes simplex virus thymidine kinase/ganciclovir (HSVtK/GCV) suicide gene system controlled by the α-fetoprotein (AFP) promoter on hepatocellular carcinoma (HCC) cells in vitro.Methods pAFP-HSVtk-IRES2-EGFP recombinant plasmid vectors driven by the AFP promoter were constructed.HL-7702 liver cells,HUH-7 HCC,and HepG2 HCC were transfected with the recombinant plasmids.HSVtK gene expression was detected using Western blotting analysis.HepG2 cells line stably expressing HSVtk gene was selected by G418 reagent.The cytotoxicity of HSVtK/GCV suicide gene system on hepatoma cells was measured by CCK-8 reagents when different doses of ganciclovir were added.Results Plasmid pAFP-TK-IRES2-EGFP-expressed HSVtk gene was constructed successfully.HSVtk gene expression level was significantly higher in AFP-positive hepatoma cells than in AFP-negative liver cells.After G418 selection,a HepG2 cells line stably expressing HSVtk gene was acquired.With the increase of the dose of ganciclovir the optical density at 450 nm of HepG2 cells stably expressing HSVtk gene gradually decreased (P <0.05).Conclusion The HSVtK gene-specific expression in hepatoma cells as well as the cytotoxicity of the suicide gene system in HepG2 cells provided the basis for the targeted gene therapy of HCC.

  1. Rat bone marrow mesenchymal stem cells in vitro cultivation and establishing pEGFP/Ang-1 transfection method%大鼠骨髓间充质干细胞体外培养与pEGFP/Ang-1转染方法的建立

    Institute of Scientific and Technical Information of China (English)

    李福智; 侯阳; 李晓明; 房艳; 臧东钰

    2014-01-01

    Objective To train the successful bone marrow mesenchymal stem cells ( BMSCs ) and phenotypic characterization , and investigate the beneficial effects of recombinant plasmid ( pEGFP/Ang-1) transfected rats BMSCs .Methods Using adherence screening and density gradient centrifugation methods , purified rats BMSCs were obtained .After transfection with pEGFP/Ang-1, the BMSCs were screened with G418, and the positive clones were sequentially confirmed by expression of enhanced green fluorescent protein (GFP) with fluorescence microscope, and expression of angiogenin-1(Ang-1) was tested by Western blot.Results Using adherence screening and density gradient centrifugation methods , purified rats BMSCs were successfully obtained .The successfully stable transfection of pEGFP-N1-Ang-1 in BMSCs had be stably expressed Ang-1 3 days,later started to diminish.Conclusions The successful use of adherent culture method and density gra-dient centrifugation of rat BMSCs and the recombinant plasmid pEGFP /Ang-1 being transfected into the rat BMSCs could effectively express .%目的:探讨培养大鼠骨髓间充质干细胞(BMSCs)细胞株、表型鉴定及利用重组质粒(pEGFP/Ang-1)转染大鼠 BMSCs细胞株的实验方法。方法采用贴壁筛选法和密度梯度离心法获取BMSCs,纯化大鼠BMSCs后,以重组质粒pEGFP/Ang-1转染,G 418筛挑阳性克隆,倒置荧光显微镜下观察增强型绿色荧光蛋白的表达,检测Ang-1表达量的时间变化。结果采用贴壁筛选法和密度梯度离心法成功获取BMSCs细胞株,成功将血管生成素-1重组质粒(pEGFP/Ang-1)转染大鼠BMSCs,3 d可稳定表达Ang-1,7 d以后开始减弱。结论成功运用贴壁筛选法和密度梯度离心法分离培养的大鼠BMSCs,并重组质粒pEGFP/Ang-1转染大鼠BMSCs可有效表达。

  2. Transfection of HeLa-cells with pEGFP plasmid by impedance power-assisted electroporation

    DEFF Research Database (Denmark)

    Glahder, Jacob; Norrild, Bodil; Persson, Mikael B;

    2005-01-01

    Bioimpedance spectrometry was applied to study cell viability and pEGFP plasmid-transfection efficiency in electroporation (EP) of 20,000 HeLa cells with 0.3 microg DNA in 90 microl low conductivity 0.32 M sucrose medium of pH 7.5. Monopolar rectangular pulses, of field strength 75 V/mm, and pulse...

  3. BKV agnoprotein interacts with α-soluble N-ethylmaleimide-sensitive fusion attachment protein, and negatively influences transport of VSVG-EGFP.

    Directory of Open Access Journals (Sweden)

    Mona Johannessen

    Full Text Available BACKGROUND: The human polyomavirus BK (BKV infects humans worldwide and establishes a persistent infection in the kidney. The BK virus genome encodes three regulatory proteins, large and small tumor-antigen and the agnoprotein, as well as the capsid proteins VP1 to VP3. Agnoprotein is conserved among BKV, JC virus (JCV and SV40, and agnoprotein-deficient mutants reveal reduced viral propagation. Studies with JCV and SV40 indicate that their agnoproteins may be involved in transcription, replication and/or nuclear and cellular release of the virus. However, the exact function(s of agnoprotein of BK virus remains elusive. PRINCIPAL FINDINGS: As a strategy of exploring the functions of BKV agnoprotein, we decided to look for cellular interaction partners for the viral protein. Several partners were identified by yeast two-hybrid assay, among them α-SNAP which is involved in disassembly of vesicles during secretion. BKV agnoprotein and α-SNAP were found to partially co-localize in cells, and a complex consisting of agnoprotein and α-SNAP could be co-immunoprecipitated from cells ectopically expressing the proteins as well as from BKV-transfected cells. The N-terminal part of the agnoprotein was sufficient for the interaction with α-SNAP. Finally, we could show that BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter suggesting that agnoprotein may modulate exocytosis. CONCLUSIONS: We have identified the first cellular interaction partner for BKV agnoprotein. The most N-terminal part of BKV agnoprotein is involved in the interaction with α-SNAP. Presence of BKV agnoprotein negatively interferes with secretion of VSVG-EGFP reporter.

  4. TALEN/CRISPR-mediated eGFP knock-in add-on at the OCT4 locus does not impact differentiation of human embryonic stem cells towards endoderm.

    Directory of Open Access Journals (Sweden)

    Nicole A J Krentz

    Full Text Available Human embryonic stem cells (hESCs have great promise as a source of unlimited transplantable cells for regenerative medicine. However, current progress on producing the desired cell type for disease treatment has been limited due to an insufficient understanding of the developmental processes that govern their differentiation, as well as a paucity of tools to systematically study differentiation in the lab. In order to overcome these limitations, cell-type reporter hESC lines will be required. Here we outline two strategies using Transcription Activator Like Effector Nucleases (TALENs and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-CRISPR-Associated protein (Cas to create OCT4-eGFP knock-in add-on hESC lines. Thirty-one and forty-seven percent of clones were correctly modified using the TALEN and CRISPR-Cas9 systems, respectively. Further analysis of three correctly targeted clones demonstrated that the insertion of eGFP in-frame with OCT4 neither significantly impacted expression from the wild type allele nor did the fusion protein have a dramatically different biological stability. Importantly, the OCT4-eGFP fusion was easily detected using microscopy, flow cytometry and western blotting. The OCT4 reporter lines remained equally competent at producing CXCR4+ definitive endoderm that expressed a panel of endodermal genes. Moreover, the genomic modification did not impact the formation of NKX6.1+/SOX9+ pancreatic progenitor cells following directed differentiation. In conclusion, these findings demonstrate for the first time that CRISPR-Cas9 can be used to modify OCT4 and highlight the feasibility of creating cell-type specific reporter hESC lines utilizing genome-editing tools that facilitate homologous recombination.

  5. Solvent dependence of two-photon absorption spectra of the enhanced green fluorescent protein (eGFP) chromophore

    Science.gov (United States)

    Hosoi, Haruko; Tayama, Ryo; Takeuchi, Satoshi; Tahara, Tahei

    2015-06-01

    Two-photon absorption spectra of 4‧-hydroxybenzylidene-2,3-dimethylimidazolinone, a model chromophore of enhanced green fluorescent protein (eGFP), were measured in various solvents. The two-photon absorption band of its anionic form is markedly blue-shifted from the corresponding one-photon absorption band in all solvents. Moreover, the magnitude of the blue shift varies largely depending on the solvent, which does not accord with the assignment of the two-photon absorption band to the transitions to the vibrationally excited S1 state. Our finding is readily rationalized by considering overlapping contributions of the S1 ← S0 and S2 ← S0 transitions, suggesting the involvement of the S2 state also in two-photon fluorescence of eGFP.

  6. Effect of recombinant plasmid pEGFP-N1-NPRL2 on the biological characteristics of human gastric cancer ceils%重组质粒pEGFP-N1-NPRL2对人胃癌细胞生物学特性的影响

    Institute of Scientific and Technical Information of China (English)

    胡在敏; 王川; 季文; 刘波; 蔡璨; 姜政

    2012-01-01

    目的:探讨pEGFP-N1-NPRL2真核表达载体对SGC-7901胃癌细胞体外增殖、细胞周期、凋亡、侵袭及迁移的影响.方法:构建pEGFP-N1-NPRL2真核表达载体,并进行酶切及测序鉴定.脂质体介导转染入SGC-7901胃癌细胞,应用荧光显微镜、RT-PCR、Western blot法检测NPRL2的基因及蛋白水平情况,CCK8法检测细胞增殖的变化,流式细胞仪分析细胞周期和凋亡率的变化,Transwell实验检测NPRL2对细胞迁移及侵袭能力的影响.结果:成功构建了pEGFP-N1-NPRL2真核表达载体.重组质粒转染SGC-7901细胞后,通过荧光显微镜观察到绿色荧光的表达,RT-PCR和Westernblot法检测到NPRL2 mRNA及蛋白的表达,CCK8法检测发现NPRL2能够明显抑制肿瘤细胞增殖(P<0.05),细胞周期分析显示NPRL2使细胞停在G0 ~ G1期(P<0.05),细胞凋亡结果显示NPRL2能抑制细胞凋亡(P<0.05),TransweU侵袭和迁移实验显示NPRL2使细胞侵袭迁移能力均降低(P<0.05).结论:NPRL2可抑制肿瘤细胞的增殖、周期、侵袭及迁移,并促进凋亡.%AIM: To investigate the effect of eukaryotic expression vector pEGFP-Nl-NPRL2 on the proliferation, cell cycle, apoptosis, invasion and migration of human gastric carcinoma SGC-7901 cells in vitro. METHODS; The eukaryotic expression vector pEGFP-Nl-NPRL2 was constructed and confirmed by enzyme digestion and sequencing analysis. Then, it was transfected into SGC-7901 cells via the liposome. The expression of NPRL2 mRNA and protein was detected by RT-PCR, fluorescent microscopy and Western blotting, respectively. The proliferation of SGC-7901 was tested by CCK8, the cell cycle and apoptosis rate by flow cytometry and the effects of NPRL2 on the cell invasion and migration by Transwell (Boyden Chamber) assay. RESULTS; The eukaryotic expression vector pEGFP-Nl-NPRL2 was constructed successfully and transfected into SGC-7901 cells. The expression of green fluorescent protein was observed using a fluorescence

  7. A single amino acid change resulting in loss of fluorescence of eGFP in a viral fusion protein confers fitness and growth advantage to the recombinant vesicular stomatitis virus

    Energy Technology Data Exchange (ETDEWEB)

    Dinh, Phat X.; Panda, Debasis; Das, Phani B.; Das, Subash C.; Das, Anshuman [School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0900 (United States); The Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0900 (United States); Pattnaik, Asit K., E-mail: apattnaik2@unl.edu [School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0900 (United States); The Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0900 (United States)

    2012-10-25

    Using a recombinant vesicular stomatitis virus encoding eGFP fused in-frame with an essential viral replication protein, the phosphoprotein P, we show that during passage in culture, the virus mutates the nucleotide C289 within eGFP of the fusion protein PeGFP to A or T, resulting in R97S/C amino acid substitution and loss of fluorescence. The resultant non-fluorescent virus exhibits increased fitness and growth advantage over its fluorescent counterpart. The growth advantage of the non-fluorescent virus appears to be due to increased transcription and replication activities of the PeGFP protein carrying the R97S/C substitution. Further, our results show that the R97S/C mutation occurs prior to accumulation of mutations that can result in loss of expression of the gene inserted at the G-L gene junction. These results suggest that fitness gain is more important for the recombinant virus than elimination of expression of the heterologous gene.

  8. Dynamic Expression of Lgr6 in the Developing and Mature Mouse Cochlea

    Directory of Open Access Journals (Sweden)

    Yanping eZhang

    2015-05-01

    Full Text Available The Wnt/β-catenin signaling pathway plays important roles in mammalian inner ear development. Lgr5, one of the downstream target genes of the Wnt/β-catenin signaling pathway, has been reported to be a marker for inner ear hair cell progenitors. Lgr6 shares approximately 50% sequence homology with Lgr5 and has been identified as a stem cell marker in several organs. However, the detailed expression profiles of Lgr6 have not yet been investigated in the mouse inner ear. Here, we first used Lgr6-EGFP-Ires-CreERT2 mice to examine the spatiotemporal expression of Lgr6 protein in the cochlear duct during embryonic and postnatal development. Lgr6-EGFP was first observed in one row of prosensory cells in the middle and basal turn at embryonic day 15.5 (E15.5. From E18.5 to postnatal day 3 (P3, the expression of Lgr6-EGFP was restricted to the inner pillar cells (IPCs. From P7 to P15, the Lgr6-EGFP expression level gradually decreased in the IPCs and gradually increased in the inner border cells (IBCs. At P20, Lgr6-EGFP was only expressed in the IBCs, and by P30 Lgr6-EGFP expression had completely disappeared. Next, we demonstrated that Wnt/β-catenin signaling is required to maintain the Lgr6-EGFP expression in vitro. Finally, we demonstrated that the Lgr6-EGFP-positive cells isolated by flow cytometry could differentiate into myosin 7a-positive hair cells after 10 days in-culture, and this suggests that the Lgr6-positive cells might serve as the hair cell progenitor cells in the cochlea.

  9. Expression of heterologous genes from an IRES translational cassette in replication competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Duch, Mogens R.; Carrasco, M L;

    1999-01-01

    of neo or the enhanced green fluorescence protein gene (EGFP). Akv-MLV's with IRES-neo and IRES-EGFP cassettes replicated with titers of about 10(6) infectious units/ml while SL3-3-MLV with IRES-neo gave about 10(3)-fold lower titers. Interestingly, RNA analysis showed a drastic reduction in the amount...... of spliced env mRNA for the SL3-3 derived vector relative to the Akv derived vectors, seemingly contributing to its low replication capacity. The EGFP expressing Akv-MLV was genetically stable for multiple rounds of infection; marker-cassette deletion revertants appeared after several replication rounds...

  10. Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line

    Directory of Open Access Journals (Sweden)

    Behra Martine

    2012-01-01

    Full Text Available Abstract Background Because of the structural and molecular similarities between the two systems, the lateral line, a fish and amphibian specific sensory organ, has been widely used in zebrafish as a model to study the development/biology of neuroepithelia of the inner ear. Both organs have hair cells, which are the mechanoreceptor cells, and supporting cells providing other functions to the epithelium. In most vertebrates (excluding mammals, supporting cells comprise a pool of progenitors that replace damaged or dead hair cells. However, the lack of regenerative capacity in mammals is the single leading cause for acquired hearing disorders in humans. Results In an effort to understand the regenerative process of hair cells in fish, we characterized and cloned an egfp transgenic stable fish line that trapped tnks1bp1, a highly conserved gene that has been implicated in the maintenance of telomeres' length. We then used this Tg(tnks1bp1:EGFP line in a FACsorting strategy combined with microarrays to identify new molecular markers for supporting cells. Conclusions We present a Tg(tnks1bp1:EGFP stable transgenic line, which we used to establish a transcriptional profile of supporting cells in the zebrafish lateral line. Therefore we are providing a new set of markers specific for supporting cells as well as candidates for functional analysis of this important cell type. This will prove to be a valuable tool for the study of regeneration in the lateral line of zebrafish in particular and for regeneration of neuroepithelia in general.

  11. [Eukaryonization of T7 RNA polymerase prokaryotic expression system and development of its couple expression system].

    Science.gov (United States)

    Zheng, Hai-Xue; Jin, Ye; Yin, Shuang-Hui; Guo, Hui-Chen; Shang, You-Jun; Bai, Xing-Wen; Liu, Xiang-Tao; Xie, Qing-Ge

    2007-09-01

    To make transcription of the target gene be driven by T7 RNA polymerase (T7 RNAP) in the eukaryotic cells, and the transcripts be CAP-independent translated. Firstly, the T7 RNAP was introduced into eukaryotic cells by two methods: (1) the BHK-21 cells were contransfected by the plasmid expressing T7 RNAP and pIERS-EGFP-ET vector; (2) by transfection of the cell line stably expressing T7 RNAP. The internal ribosome entry site (IRES) element from FMDV was cloned into the downstream of the T7 promoter sequence of the prokaryotic expressing vector pET-40a-c (+), resulted in the plasmid would express the transcripts carrying the IERS element at its 5' end. The enhanced green fluorescent protein (EGFP) gene was cloned into the downstream of the IERS element, resulted in plasmid pIERS-EGFP-ET. Then, the two kinds of cells expressing T7 RANP were transfected by pIERS-EGFP-ET. The green fluorescence in the transfected cells was observed under a fluorescence microscope equipped with a video documentation system. And the expressional efficiency was analyzed with flow cytometry (FCM). The results show that the IRES element from FMDV has the role of initiating CAP-independent translation, and lay foundation for researching function of the element and interrelated proteins. It would be potential for expressing target gene by the T7 RNAP couple expression system. PMID:18051880

  12. In vitro construction of a recombinant human embryonic brain-derived neurotrophin-4 gene and pEGFP-N1 vector

    Institute of Scientific and Technical Information of China (English)

    Jintao Li; Qi Yan; Xingbao Zhu; Dan Xu; Tinghua Wang; Huatang Zhang; Jia Liu

    2009-01-01

    BACKGROUND: Neurotrophin-4 (NT-4) can promote neuronal growth, development, differentiation, maturation, and survival. NT-4 can also improve recovery and regeneration of injured neurons, but cannot pass through the blood-brain barrier, which limits its activity in the central nervous system. Delivering NT-4 into the central nervous system via cells or vectors may have therapeutic benefit.OBJECTIVE: To construct a recombinant vector with a human embryonic brain-derived NT-4 gene and pEGFP-N1.DESIGN, TIME AND SETTING: Neural genetic engineering experiment. The study was performed at the Neuroscience Institute of Kunming Medical College between October 2007 and March 2008.MATERIALS: The pEGFP-N1 plasmid vector was provided by Kunming Institute of Zoology, Chinese Academy of Sciences; embryonic brain tissues were provided by the First Affiliated Hospital of Kunming Medical College. TRIzol RNA extraction Kit was purchased from Sigma (USA), One Step RNA PCR Kit (AMV) etc. were from Takara (Dalian, China).METHODS: Total RNA was extracted from human embryonic brain tissues using Trizol. The agarose gel electrophoresis showed two bands: 18 S and 28 S, which were essential subunits of total RNA. The human NT-4 DNA was obtained via RT-PCR and inserted into the pEGFP-N1 vector using ligation and transformation reaction.MAIN OUTCOME MEASURES: The sequencing results of the DNA in the recombinant of NT-4-pEGFP-N1.RESULTS: The NT-4-pEGFP-N1 vector was sequence-verified and showed the expected molecular weight.CONCLUSION: The recombinant of NT-4-pEGFP-N1 was constructed successfully in vitro.

  13. Easy measurement of diffusion coefficients of EGFP-tagged plasma membrane proteins using k-space Image Correlation Spectroscopy

    DEFF Research Database (Denmark)

    Christensen, Eva Arnspang; Koffman, Jennifer Skaarup; Marlar, Saw; Weissman, Paul; Nejsum, Lene Niemann

    2014-01-01

    available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the...... correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS. First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope Then...... slopes" plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical...

  14. Synthesis, characterization, and cytotoxicity of the plasmid EGFP-p53 loaded on pullulan-spermine magnetic nanoparticles

    Science.gov (United States)

    Eslaminejad, Touba; Nematollahi-Mahani, Seyed Noureddin; Ansari, Mehdi

    2016-03-01

    Magnetic nanoparticles have been used as effective vehicles for the targeted delivery of therapeutic agents that can be controlled in their concentration and distribution to a desired part of the body by using externally driven magnets. This study focuses on the synthesis, characterization, and functionalization of pullulan-spermine (PS) magnetic nanoparticles for medical applications. Magnetite nanopowder was produced by thermal decomposition of goethite (FeOOH) in oleic acid and 1-octadecene; pullulan-spermine was deposited on the magnetite nanoparticles in the form of pullulan-spermine clusters. EGFP-p53 plasmid was loaded on functionalized iron oleate to transfer into cells. Synthesized nanoparticles were characterized by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), vibrating sample magnetometry (VSM), and transmission electron microscopy (TEM). The encapsulation efficiency and drug loading efficiency of the nanocomplexes were tested. FTIR studies showed the presence of oleic acid and 1-octadecene in the iron oleate nanopowder and verified the interaction between spermine and pullulan. The characteristic bands of PS in the spectrum of the pullulan-spermine-coated iron oleate (PSCFO) confirmed that PS covered the surface of the iron oleate particles. TEM studies showed the average size of the iron oleate nanopowder, the PSCFO, and the plasmid-carrying PSCFO (PSCFO/pEGFP-p53) to be 34±12 nm, 100±50 nm and 172±3 nm, respectively. Magnetic measurements revealed that magnetic saturation of the PSCFO was lower in comparison with the iron oleate nanopowder due to the presence of organic compounds in the former. In cytotoxicity tests performed using U87 cells as glioblastoma cells, a 92% survival rate was observed at 50 μg/μl of the plasmid-carrying PSCFO, with an IC50 value of 189 μg/μl.

  15. Controlled expression of enhanced green fluorescent protein and hepatitis B virus precore protein in mammalian cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    A novel tetracycline regulation expression system was used to regulate the expression of enhanced green fluorescent protein (EGFP) and hepatitis B virus precore protein in the mammalian cell lines with lipofectAMINE. Flow cytometry assays showed that application of the system resulted in about 18-fold induction of EGFP expression in CHO cell lines and 5-fold induction in SSMC-7721 cells and about 2-fold in the HEK293 cells. Furthermore, the effective use of this system for the controlled expression of HBV precore protein gene in hepatocellular carcinoma cells was tested.

  16. Production of Transgenic Korean Native Cattle Expressing Enhanced Green Fluorescent Protein Using a FIV-Based Lentiviral Vector Injected into MII Oocytes

    Institute of Scientific and Technical Information of China (English)

    Yong-Nan Xu; Joon-Ho Yoon; Dae-Hwan Ko; Teoan Kim; Nam-Hyung Kim; Sang-Jun Uhm; Bon-Chul Koo; Mo-Sun Kwon; Ji-Yeol Roh; Jung-Seok Yang; Hyun-Yong Choi; Young-Tae Heo; Xiang-Shun Cui

    2013-01-01

    The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins.Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer,but these approaches have been largely ineffective; however,a third approach,lentivirus-mediated transgenesis,has successfully produced transgenic livestock.In this study,we generated transgenic (TG) Korean native cattle using perivitelline space injection of viral vectors,which expressed enhanced green fluorescent protein (EGFP) systemically.Two different types of lentiviral vectors derived from feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV)carrying EGFP were injected into the perivitelline space of MII oocytes.EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group (47.5% ± 2.2% v.s.22.9% ± 2.9%).Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers.Ten heifers were successfully impregnated and delivered 10 healthy calves.All of these calves expressed EGFP as detected by in vivo imaging,PCR and Southern blotting.In addition,we established an EGFP-expressing cell line from TG calves,which was followed by nuclear transfer (NT).Recloned 8-cell embryos also expressed EGFP,and there were no differences in the rates of fusion,cleavage and development between cells derived from TG and non-TG calves,which were subsequently used for NT.These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle.Moreover,our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells.

  17. Identification of neurons that express ghrelin receptors in autonomic pathways originating from the spinal cord.

    Science.gov (United States)

    Furness, John B; Cho, Hyun-Jung; Hunne, Billie; Hirayama, Haruko; Callaghan, Brid P; Lomax, Alan E; Brock, James A

    2012-06-01

    Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with

  18. Construction and Characterization of Insect Cell-Derived Influenza VLP: Cell Binding, Fusion, and EGFP Incorporation

    Directory of Open Access Journals (Sweden)

    Yi-Shin Pan

    2010-01-01

    Full Text Available We have constructed virus-like particles (VLPs harboring hemagglutinin (HA, neuraminidase (NA, matrix protein 1 (M1 ,and proton channel protein (M2 using baculovirus as a vector in the SF9 insect cell. The size of the expressed VLP was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy. Recognition of HA on the VLP surface by the HA2-specific monoclonal antibody IIF4 at acidic pH, as probed by surface plasmon resonance, indicated the pH-induced structural rearrangement of HA. Uptake of the particle by A549 mediated by HA-sialylose receptor interaction was visualized by the fluorescent-labeled VLP. The HA-promoted cell-virus fusion activity was illustrated by fluorescence imaging on the Jurkat cells incubated with rhodamine-loaded VLP performed at fusogenic pH. Furthermore, the green fluorescence protein (GFP was fused to NA to produce VLP with a pH-sensitive probe, expanding the use of VLP as an antigen carrier and a tool for viral tracking.

  19. CNPase Expression in Olfactory Ensheathing Cells

    Directory of Open Access Journals (Sweden)

    Christine Radtke

    2011-01-01

    Full Text Available A large body of work supports the proposal that transplantation of olfactory ensheathing cells (OECs into nerve or spinal cord injuries can promote axonal regeneration and remyelination. Yet, some investigators have questioned whether the transplanted OECs associate with axons and form peripheral myelin, or if they recruit endogenous Schwann cells that form myelin. Olfactory bulbs from transgenic mice expressing the enhanced green fluorescent protein (eGFP under the control of the 2-3-cyclic nucleotide 3-phosphodiesterase (CNPase promoter were studied. CNPase is expressed in myelin-forming cells throughout their lineage. We examined CNPase expression in both in situ in the olfactory bulb and in vitro to determine if OECs express CNPase commensurate with their myelination potential. eGFP was observed in the outer nerve layer of the olfactory bulb. Dissociated OECs maintained in culture had both intense eGFP expression and CNPase immunostaining. Transplantation of OECs into transected peripheral nerve longitudinally associated with the regenerated axons. These data indicate that OECs in the outer nerve layer of the olfactory bulb of CNPase transgenic mice express CNPase. Thus, while OECs do not normally form myelin on olfactory nerve axons, their expression of CNPase is commensurate with their potential to form myelin when transplanted into injured peripheral nerve.

  20. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    Directory of Open Access Journals (Sweden)

    Weir Jerry P

    2007-05-01

    Full Text Available Abstract Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2 BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors.

  1. Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

    Directory of Open Access Journals (Sweden)

    Tripti Tamhane

    2015-12-01

    Full Text Available The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015 [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

  2. CONSTRUCTION AND IDENTIFICATION OF EUKARYOTIC VECTOR EXPRESSING SIRNA SPECIFIC FOR BACE

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Objective To generate eukaryotic expression vector of siRNA specific for β-site APP cleaving enzyme(BACE),and detect the interfering effect to the expression of BACE. Methods To clone the BACE targeting siRNA gene by PCR, the PCR products was inserted into the pUC19/EGFP-U6 plasmid. Then it was sub-cloned into the vector named pLXSN. The resultant plasmid was named pLXSN/EGFP-U6-siBACE, it was packaged into AmphoPack-293 cells by calcium phosphate transfection and collected the virus supernatant. The neuroblastoma cells SK-N-SH was infected with the pLXSN/EGFP-U6-siBACE retroviral vector, immunohistochemistry method was used to detect whether the pLXSN/EGFP-U6-siBACE infection can inhibit the expression of BACE of the neuroblastoma cells. Results The pLXSN/EGFP-U6-siBACE retroviral vector was constructed successfully and the siBACE can inhibit the BACE of the neuroblastma effectively. Conclusion The siRNA can inhibit the expression of the BACE gene, the endogenous production of BACE protein was decreased. It will lay the important foundation for using RNA technology to prevent the Alzheimer's disease.

  3. Characterization of Aromatase Expression in the Adult Male and Female Mouse Brain. I. Coexistence with Oestrogen Receptors α and β, and Androgen Receptors

    OpenAIRE

    Davor Stanić; Sydney Dubois; Hui Kheng Chua; Bruce Tonge; Nicole Rinehart; Malcolm K Horne; Wah Chin Boon

    2014-01-01

    Aromatase catalyses the last step of oestrogen synthesis. There is growing evidence that local oestrogens influence many brain regions to modulate brain development and behaviour. We examined, by immunohistochemistry, the expression of aromatase in the adult male and female mouse brain, using mice in which enhanced green fluorescent protein (EGFP) is transcribed following the physiological activation of the Cyp19A1 gene. EGFP-immunoreactive processes were distributed in many brain regions, in...

  4. 重组质粒pEGFP-N2/XPD和Pifithrin-α对肝癌细胞增殖及凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    丁浩; 马果果; 张吉翔

    2012-01-01

    目的 探讨人着色性干皮病基因D(xeroderma pigmentosum D,XPD)和P53对肝癌细胞HepG2.2.15增殖凋亡及P21等基因表达的影响.方法 用脂质体转染法将空载质粒pEGFP-N2和重组质粒pEGFP-N2/XPD转染HepG2.2.15,然后给予20 μmol/L的Pifithrin-α(P53抑制剂)孵育24 h.实验分为5组:空白对照组、pEGFP-N2组、pEGFP-N2/XPD组、pEGFP-N2/XPD+Pifithrin-α组和Pifithrin-α组.用荧光显微镜观察绿色荧光蛋白报告基因表达情况;用RT-PCR和Western blot检测XPD、P53、磷酸化P53(phosphorylated P53,p-P53)、P21和乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)的表达;用MTT法检测细胞活力;用流式细胞仪检测凋亡率.结果 在荧光显微镜下,可在转染了空载质粒pEGFP-N2和重组质粒pEGFP-N2/XPD的细胞中观察到绿色荧光,即转染成功;RT-PCR和Western blot检测发现,重组质粒pEGFP-N2/XPD的转染使XPD表达增高(P<0.001);XPD表达升高使P53、p-P53(ser-15)、P21表达增高,同时使得HBx表达降低,而Pifithrin-α能抑制XPD这一作用(P<0.001).MTT结果显示,XPD表达升高抑制了细胞活力,而Pifithrin-α能抑制XPD减弱细胞活力的作用(P<0.001).流式细胞仪结果显示,XPD表达增高引起了细胞凋亡率增加,而Pifithrin-α能抑制XPD诱导细胞凋亡的作用(P<0.001).结论 XPD能通过P53通路抑制肝癌细胞增殖并促进其凋亡,上调P21的表达以及下调HBx的表达.

  5. Analysis of the effects of blue light on morphofunctional status of in vitro cultured blastocysts from mice carrying gene of enhanced green fluorescent protein (EGFP).

    Science.gov (United States)

    Sakharova, N Yu; Mezhevikina, L M; Smirnov, A A; Vikhlyantseva, E F

    2014-05-01

    We studied the effect of blue light (440-490 nm) on the development of late blastocysts of mice carrying the gene of enhanced green fluorescent protein (EGFP). Exposure to blue light for 20 min reduced adhesive properties of blastocysts and their capacity to form primary colonies consisting of the cells of inner cell mass, trophoblast, and extraembryonic endoderm. The negative effects of blue light manifested in morphological changes in the primary colonies and impairment of differentiation and migration of cells of the trophoblast and extraembryonic endoderm. The problems of cell-cell interaction and inductive influences of the inner cell mass on other cell subpopulations are discussed. EGFP blastocysts were proposed as the model for evaluation of the mechanisms underlying the effects of blue light as the major negative factor of visible light used in in vitro experiments on mammalian embryos. PMID:24913583

  6. Three-Dimensional Imaging of Prox1-EGFP Transgenic Mouse Gonads Reveals Divergent Modes of Lymphangiogenesis in the Testis and Ovary

    OpenAIRE

    Svingen, Terje; François, Mathias; Wilhelm, Dagmar; Koopman, Peter

    2012-01-01

    The lymphatic vasculature forms a specialized part of the circulatory system, being essential for maintaining tissue fluid homeostasis and for transport of hormones, macromolecules, and immune cells. Although lymphatic vessels are assumed to play an important role in most tissues, their morphogenesis and function in the gonads remains poorly understood. Here we have exploited a lymphatic-specific Prox1-EGFP reporter mouse model and optical projection tomography technology to characterize both...

  7. Development of transgenic chickens expressing enhanced green fluorescent protein

    International Nuclear Information System (INIS)

    In this work we demonstrated the successful production of transgenic chickens expressing the enhanced green fluorescence protein (EGFP) gene. Replication-defective recombinant retroviruses produced from vesicular stomatitis virus G glycoprotein pseudotyped retrovirus vector system were injected beneath the blastoderm of non-incubated chicken embryos (stage X). From 129 injected eggs, 13 chicks hatched after 21 days of incubation. All hatched chicks were found to express vector-encoded EGFP gene, which was under the control of the Rous sarcoma virus promoter and boosted post-transcriptionally by woodchuck hepatitis virus post-transcriptional regulatory element sequence. Green fluorescent signals, indicative of the EGFP gene expression, were detected in various body parts, including head, limb, eye, toe, and several internal organs. Genomic incorporation of the transgene was also proven by Southern blot assay. Our results show the exceptional versatile effectiveness of the EGFP gene as a marker in the gene expression-related studies which therefore would be very helpful in establishing a useful transgenic chicken model system for studies on embryo development and for efficient production of transgenic chickens as bioreactors

  8. Construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3

    Institute of Scientific and Technical Information of China (English)

    Chunxia Peng; Xiaobei Yin; Mengda Li; Ting He; Genlin Li

    2013-01-01

    Neurotrophin-3 (NT-3) can promote the repair of central nervous system and retinal damage. In previous reports, NT-3 has been expressed by viral vectors. However, plasmid vectors have a safer profile compared with viral vectors in clinical studies. This study recombined amplified human retinal NT-3 with a eukaryotic expression plasmid containing green fluorescent protein (GFP) to construct an NT-3 expression plasmid, pEGFP-N1-NT-3. The transfection efficiency 48 hours after pEGFP-N1-NT-3 transfection to 293T cells was 50.06 ± 2.78%. Abundant NT-3-GFP was expressed in 293T cells as observed by fluorescence microscopy, suggesting the construct pEGFP-N1-NT-3 effectively expressed and secreted NT-3-GFP. Secretory vesicles containing NT-3-GFP were observed in a constant location in cells by laser scan confocal microscopy, indicating the expression and secretion processes of NT-3 in eukaryotic cells were in accordance with the physical synthesis processes of secreted proteins. Western blot assay showed that pro-NT-3-GFP had a molecular weight of 56 kDa, further confirming NT-3-GFP expression. At 48 hours after transfection, the concentration of NT-3 in culture medium was 22.3 ng/mL, suggesting NT-3 produced by pEGFP-N1-NT-3 was efficiently secreted. This study constructed a human retinal-derived NT-3 eukaryotic expression plasmid that efficiently expressed and secreted NT-3.

  9. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  10. [Expression analysis of green fluorescent protein in tissues and organs in α-1,3 galactosyltransferase knockout pigs].

    Science.gov (United States)

    Zhifang, Li; Chong, Feng; Huili, Ji; Ningning, Shi; Xiaofeng, Song; Qinli, Zhao; Chuan, Long; Dengke, Pan; Xiaogan, Yang

    2015-12-01

    The pig is an ideal source to provide organs because its organ size and physiology are similar to humans. However, an acute rejection will ensue after pig-to-human xenotransplantation. The α-1,3 galactosyltransferase gene knockout (GTKO) pigs were generated in recent years, and could solve the problem of hyperacute rejection. But due to lack of reporting genes, the rejection status of cells and organs post pig-to-human xenotransplantation cannot be visualized. In this study, we introduced the enhanced green fluorescent protein (EGFP) gene driven by the CAG promoter into GTKO porcine ear fibroblasts. Then we produced transgenic pigs expressing the EGFP gene by nuclear transfer technology. Expression levels of EGFP in different tissues and organs of the cloned pig were investigated by Nightsea DFP-1 Fluorescent Protein Flashlight, fluorescence microscope and quantitative PCR assays. The results showed that the protein and transcript of EGFP were expressed in all tissues and organs of the GTKO pig, but the expression was weak in the liver and central nervous system. In conclusion, we have successfully produced the transgenic GTKO pigs expressing EGFP in all tested tissues and organs, which builds up a good basis to track transplanted cells or tissues. PMID:26704946

  11. Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Jensen, Tanja B; Mathiesen, Christian K;

    2011-01-01

    To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A...... deletions in EGFP, while 2a(J6)¿40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5A¿40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those...... of these reporter viruses for high-throughput fluorescence- and luminescence-based studies of HCV-receptor interactions and serum-neutralizing antibodies was demonstrated. Finally, using RLuc viruses, we showed that the genotype-specific core-NS2 sequence did not influence the response to alfa-2b interferon (IFN...

  12. Transgenic approach to express the channelrhodopsin 2 gene in arginine vasopressin neurons of rats.

    Science.gov (United States)

    Ishii, Masahiro; Hashimoto, Hirofumi; Ohkubo, Jun-Ichi; Ohbuchi, Toyoaki; Saito, Takeshi; Maruyama, Takashi; Yoshimura, Mitsuhiro; Yamamoto, Yukiyo; Kusuhara, Koichi; Ueta, Yoichi

    2016-09-01

    Optogenetics provides a powerful tool to regulate neuronal activity by light-sensitive ion channels such as channelrhodopsin 2 (ChR2). Arginine vasopressin (AVP; also known as the anti-diuretic hormone) is a multifunctional hormone which is synthesized in the magnocellular neurosecretory cells (MNCs) of the hypothalamus. Here, we have generated a transgenic rat that expresses an AVP-ChR2-enhanced green fluorescent protein (eGFP) fusion gene in the MNCs of the hypothalamus. The eGFP fluorescence that indicates the expression of ChR2-eGFP was observed in the supraoptic nucleus (SON) and in the magnocellular division of the paraventricular nucleus (PVN) that is known to contain AVP-secreting neurons. The eGFP fluorescence intensities in those nuclei and posterior pituitary were markedly increased after chronic salt loading (2% NaCl in drinking water for 5days). ChR2-eGFP was localized mainly in the membrane of AVP-positive MNCs. Whole-cell patch-clamp recordings were performed from single MNCs isolated from the SON of the transgenic rats, and blue light evoked repetitive action potentials. Our work provides for the first time an optogenetic approach to selectively activate AVP neurons in the rat. PMID:27493075

  13. The Construction of the EGFP Enterovirus 71%肠道病毒71型(EV71)荧光病毒的构建

    Institute of Scientific and Technical Information of China (English)

    朱守海; 王璐璐

    2014-01-01

    人肠道病毒71型(Enterovirus,EV71)是引起手足口病的主要病原体。首先利用反向遗传学的方法,构建了EV71全长cDNA感染性克隆,经体外转录、转染RD细胞后成功获得了拯救病毒。随后,将增强型绿色荧光蛋白基因(Enhanced green fluorescent protein,EGFP)插入到EV71基因组中构建荧光病毒。结果表明,此荧光病毒不仅可以侵染、复制,在传代的过程中EGFP基因也保持了较好的稳定性,表明其可以作为一种报告病毒应用于高通量的EV71抗病毒药物筛选中。%Human Enterovirus 71(EV71)is one of the major agents causing hand, foot and mouth disease(HFMD). An infectious full-length cDNA clone of EV71 as well as the recuse virus were obtained using the reverse genetic techniques. Then, the EGFP gene was inserted into the EV71 genome to construct the reporter virus. The results show that the EGFP-EV71 virus not only are replication-competent and infectious, but the EGFP gene are stable during passaging, implicating the recombinant virus could be used as a reporter virus for high throughput EV71 antiviral drugs screening.

  14. EXPRESS

    International Nuclear Information System (INIS)

    This paper presents EXPRESS, an expert system developed for the automation of reliability studies. The first part consists in the description of the method for static thermohydraulic systems. In this step, the authors define the knowledge representation based on the two inference engines - ALOUETTE and LCR developed by EDF. They explain all the process to construct a fault tree from a topological and functional description of the system. Numerous examples are exhibited in illustration of the method. This is followed by the lessons derived from the studies performed on some safety systems of the PALUEL nuclear plant. The development of the same approach for electric power systems is described, insisting on the difference resulting from the sequential nature of these systems. Finally, they show the main advantages identified during the studies

  15. Infection of the upper respiratory tract of hamsters by the bovine parainfluenza virus type 3 BN-1 strain expressing enhanced green fluorescent protein.

    Science.gov (United States)

    Ohkura, Takashi; Minakuchi, Moeko; Sagai, Mami; Kokuho, Takehiro; Konishi, Misako; Kameyama, Ken-Ichiro; Takeuchi, Kaoru

    2015-02-01

    Bovine parainfluenza virus type 3 (BPIV3) is an important pathogen associated with bovine respiratory disease complex (BRDC). We have generated a recombinant BPIV3 expressing enhanced green fluorescent protein (rBPIV3-EGFP) based on the BN-1 strain isolated in Japan. After intranasal infection of hamsters with rBPIV3-EGFP, EGFP fluorescence was detected in the upper respiratory tract including the nasal turbinates, pharynx, larynx, and trachea. In the nasal turbinates, rBPIV3-EGFP attained high titers (>10(6) TCID50/g of tissue) 2-4 days after infection. Ciliated epithelial cells in the nasal turbinates and trachea were infected with rBPIV3-EGFP. Histopathological analysis indicated that mucosal epithelial cells in bronchi were shed by 6 days after infection, leaving non-ciliated cells, which may have increased susceptibility to bacterial infection leading to the development of BRDC. These data indicate that rBPIV3-EGFP infection of hamsters is a useful small animal model for studying the development of BPIV3-associated BRDC. PMID:25543964

  16. Modulating the expression level of secreted Wnt3 influences cerebellum development in zebrafish transgenics.

    Science.gov (United States)

    Teh, Cathleen; Sun, Guangyu; Shen, Hongyuan; Korzh, Vladimir; Wohland, Thorsten

    2015-11-01

    The boundaries of brain regions are associated with the tissue-specific secretion of ligands from different signaling pathways. The dynamics of these ligands in vivo and the impact of its disruption remain largely unknown. Using light and fluorescence microscopy for the overall imaging of the specimen and fluorescence correlation spectroscopy (FCS) to determine Wnt3 dynamics, we demonstrated that Wnt3 regulates cerebellum development during embryogenesis using zebrafish wnt3 transgenics with either tissue-specific expression of an EGFP reporter or a functionally active fusion protein, Wnt3EGFP. The results suggest a state of dynamic equilibrium of Wnt3EGFP mobility in polarized neuroepithelial-like progenitors in the dorsal midline and cerebellar progenitors on the lateral side. Wnt3EGFP is secreted from the cerebellum as shown by measurements of its mobility in the ventricular cavity. The importance of Wnt secretion in brain patterning was validated with the Porcn inhibitor Wnt-C59 (C59), which, when applied early, reduced membrane-bound and secreted fractions of Wnt3EGFP and led to a malformed brain characterized by the absence of epithalamus, optic tectum and cerebellum. Likewise, interference with Wnt secretion later on during cerebellar development negatively impacted cerebellar growth and patterning. Our work, supported by quantitative analysis of protein dynamics in vivo, highlights the importance of membrane-localized and secreted Wnt3 during cerebellum development. PMID:26395493

  17. Construction of recombinant ZNF230/GFP fused plasmids and their expression and cellular localization

    DEFF Research Database (Denmark)

    Xu, Wen-Ming; Zhang, Si-Zhong; Qiu, Wei-Min;

    2004-01-01

    cutting, the mutated human and mouse ZNF230(znf230) were inserted into mammalian expression plasmid pEGFP-N1. Thus we constructed the plasmid with fusion gene of ZNF230 and green fluorescent protein(GFP). Then the Cos cell was transfected with the fused gene by liposome. Fluorescence microscopy showed...

  18. Immune Responses in Pigs Induced by Recombinant DNA Vaccine Co-Expressing Swine IL-18 and Membrane Protein of Porcine Reproductive and Respiratory Syndrome Virus

    Directory of Open Access Journals (Sweden)

    Zhuang Ding

    2012-05-01

    Full Text Available In this study, two DNA vaccines, which express the membrane (M protein of porcine respiratory and reproductive syndrome virus (PRRSV (pEGFP-M and co-express both M and swine IL-18 (pEGFP-IL18-M, were constructed and their abilities to induce humoral and cellular responses in piglets were comparatively evaluated. Experimental results showed that both recombinant DNA vaccines could not elicit neutralizing antibodies in the immunized piglets. However, both DNA vaccines elicited Th1-biased cellular immune responses. Notably, pigs immunized with the plasmid pEGFP-IL18-M developed significantly higher levels of IFN-γ and IL-2 production response and stronger specific T-lymphocyte proliferation response than the pigs inoculated with the plasmids pEGFP-M and pEGFP-IL18 (P < 0.05. These results illustrated that co-expression of M and IL-18 proteins could significantly improve the potency of DNA vaccination on the activation of vaccine-induced virus-specific cell-mediated immune responses in pigs, which may be used as a strategy to develop a new generation of vaccines against highly pathogenic PRRSV.

  19. Transdifferention of some supporting cells in the cochlea induced by Ad5 atoh1/EGFP in the young adult guinea pigs%Ad5-atoh1/EGFP在体诱导近成年豚鼠耳蜗产生毛细胞样细胞

    Institute of Scientific and Technical Information of China (English)

    韩朝; 丛宁; 杨娟梅; 黄一波; 靳楷; 李雯

    2012-01-01

    Objective; To explore whether the Ad5-atohl/EGFP could transdifferent the supporting ceils into the new hair cells in young adult guinea pigs cochlea in vivo. Method: Twelve healthy pigmented guinea pigs weighted 200-250 g were included in this experiment. 5 ul of Ad5-El/E3 defected-atohl/EGFP were infused into the scala media through a hole made on the lateral wall of the cochlea. Six of the 12 animal were killed 2 weeks after the infusion operation. The others were killed 4 weeks after the operation. The whole mount of the basal membranes were directly observed under the fluorescence microscope for the expression of the EGFP (enhance green fluorescent protein) or for the expression of the hair cell specific marker and nuclear after staining with myosinVIIa rabbit polyclonal antibody and Dapi dye. Result:New cells with big nuclear, ellipse body and expressed with EGFP were found in the region near to the outmost row of the outer hair cells in 2 animal 2 weeks after the infusion. Moreover there were 3 animals with specific morphologic new cells in the location where ever been located by the outer hair cells and the region as 2 weeks animals 4 weeks after the infusion. Those cells were stained by myosinVIIa antibody. Conclusion: Atohl gene could transdifferent some supporting cells in the basal membrane into hair cell like cells in young adult guinea pigs in vivo. These supporting cells locate in the region of outer hair cells and the basal membrane which do not belong to the region of outer hair cells.%目的:在近成年豚鼠耳蜗内携带atoh1/EGFP基因的腺病毒是否能够将基膜上残留的支持细胞转分化为新的毛细胞.方法:选用体重在200~250g的健康花豚鼠12只,将构建同时表达atoh1和EGFP基因的E1/E3区缺失的人类免疫5型腺病毒5μl,通过耳蜗侧壁打孔灌注导入中阶内淋巴系统.其中6只于灌注2周后处死,6只灌注4周后处死,基膜铺片观察EGFP、毛细胞标记物myosinⅦa和Dapi

  20. Molecular and functional characterization of GAD67-expressing, newborn granule cells in mouse dentate gyrus

    Directory of Open Access Journals (Sweden)

    Carolina eCabezas

    2013-04-01

    Full Text Available Dentate gyrus granule cells (GCs have been suggested to synthesize both GABA and glutamate immediately after birth and under pathological conditions in the adult. Expression of the GABA synthesizing enzyme GAD67 by GCs during the first few weeks of postnatal development may then allow for transient GABA synthesis and synaptic release from these cells. Here, using the GAD67-EGFP transgenic strain G42, we explored the phenotype of GAD67-expressing GCs in the mouse dentate gyrus. We report a transient, GAD67-driven EGFP expression in differentiating GCs throughout ontogenesis. EGFP expression correlates with the expression of GAD and molecular markers of GABA release and uptake in 2-4 weeks postmitotic GCs. These rather immature cells are able to fire action potentials and are synaptically integrated in the hippocampal network. Yet they show physiological properties that differentiate them from mature GCs. Finally, GAD67-expressing GCs express a specific complement of GABAA receptor subunits as well as distinctive features of synaptic and tonic GABA signaling. Our results reveal that GAD67 expression in dentate gyrus granule cells is a transient marker of late differentiation that persists throughout life and the G42 strain may be used to visualize newborn GCs at a specific, well-defined differentiation stage.

  1. Construction and identification of recombinant vectors carrying herpes simplex virus thymidine kinase and cytokine genes expressed in gastric carcinoma cell line SGC7901

    Institute of Scientific and Technical Information of China (English)

    Jian-Hua Zhang; Ming-Xi Wan; Jia-Ying Yuan; Bo-Rong Pan

    2004-01-01

    AIM: To construct and identify the recombinant vectors carrying herpes simplex virus thymidine kinase (HSVoTK) and tumor necrosis factor alpha (TNF-α) or interleukin-2 (IL-2)genes expressed in gastric carcinoma cell line SGC7901.METHODS: The fragments of HSV-TK, internal ribosome entry sites (IRES) and TNF-α or TL-2 genes were inserted in a TK-IRES-TNF-α or TK-IRES-IL-2 order into pEGFP-N3 and pLXSN to generate the therapeutic vectors pEGFP-TT,pEGFP-TI, pL(TT)SN and pL(TI)SN respectively, which were structurally confirmed by the digestion analysis of restriction endonuclease. The former two plasmids were used for the transient expression of recombinant proteins in the target cells while pL(TT)SN and pL(TI)SN were transfected into SGC7901 cells by lipofectamine for the stable expression of objective genes through G418 selection. The protein products expressed transiently and stably in SGC7901 cells by the constructed vectors were confirmed by fluorescent microscopy and Western blot respectively.RESULTS: The inserted fragments in all constructed plasmids were structurally confirmed to be consistent with that of the published data. In the transient expression, both pEGFP-TT and pEGFP-TI were shown expressed in nearly 50% of the transfected SGC7901 cells. Similarly, the G418 selected vectors PL(-TT)SN and PL(TI)SN were confirmed to be successful in the stable expression of the objective proteins in the target cells.CONCLUSION: The constructed recombinant vectors in the present study that can express the suicide gene TK in combination with cytokines genes may serve as the potential tools to perform more effective investigations in future for the gene therapy of gastric carcinoma.

  2. Combination of lithium chloride and pEGFP-N1-BmK CT effectively decreases proliferation and migration of C6 glioma cells.

    Science.gov (United States)

    Fu, Yuejun; Jiao, Yanmei; Zheng, Shuhua; Liang, Aihua; Hu, Fengyun

    2016-03-01

    Deleterious invasiveness of glioma cells into the normal brain tissue is endorsed by its inherent ability to regulate the receptor-mediated adhesive properties, extracellular matrix degradation and remodeling and elevated secretory ability of metalloproteinase (MMPs) such as MMP-2. By doing so, it will create an intercellular space for the invasion of glioma cells. Here, we reported that combination of gene therapy Buthus martensii Karsch (BmK) CT, a type of scorpion toxin peptide, with lithium chloride (LiCl), clinically used as mood stabilizer, could inhibit the migration and invasion of C6 glioma cells. The results showed that concomitant administration of LiCl and pEGFP-N1-BmK CT on glioma cells would hamper pro-MMP2 secretion and in the meantime, inhibited its proliferation in a synergistic manner. These results try to extrapolate the potential interplay between the combined treatment of LiCl and BmK CT with signaling pathways β-catenin, MMP, GSK-3 in C6 glioma cells. This strategy can stand for a novel approach designated for the development of a new method for glioma therapy. PMID:25286828

  3. Rapid yeast estrogen bioassays stably expressing human estrogen receptors alpha and beta, and green fluorescent protein: a comparison of different compounds on both receptor types

    NARCIS (Netherlands)

    Bovee, T.F.H.; Helsdingen, J.R.; Rietjens, I.M.C.M.; Keijer, J.; Hoogenboom, L.A.P.

    2004-01-01

    Previously, we described the construction of a rapid yeast bioassay stably expressing human estrogen receptor (hER) and yeast enhanced green fluorescent protein (yEGFP) in response to estrogens. In the present study, the properties of this assay were further studied by testing a series of estrogenic

  4. Tightly regulated and homogeneous transgene expression in human adipose-derived mesenchymal stem cells by lentivirus with tet-off system.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Moriyama

    Full Text Available Genetic modification of human adipose tissue-derived multilineage progenitor cells (hADMPCs is highly valuable for their exploitation in therapeutic applications. Here, we have developed a novel single tet-off lentiviral vector platform. This vector combines (1 a modified tetracycline (tet-response element composite promoter, (2 a multi-cistronic strategy to express an improved version of the tet-controlled transactivator and the blasticidin resistance gene under the control of a ubiquitous promoter, and (3 acceptor sites for easy recombination cloning of the gene of interest. In the present study, we used the cytomegalovirus (CMV or the elongation factor 1 α (EF-1α promoter as the ubiquitous promoter, and EGFP was introduced as the gene of interest. hADMPCs transduced with a lentiviral vector carrying either the CMV promoter or the EF-1α promoter were effectively selected by blasticidin without affecting their stem cell properties, and EGFP expression was strictly regulated by doxycycline (Dox treatment in these cells. However, the single tet-off lentiviral vector carrying the EF-1α promoter provided more homogenous expression of EGFP in hADMPCs. Intriguingly, differentiated cells from these Dox-responsive cell lines constitutively expressed EGFP only in the absence of Dox. This single tet-off lentiviral vector thus provides an important tool for applied research on hADMPCs.

  5. Enhanced protein secretion from insect cells by co-expression of the chaperone calreticulin and translation initiation factor eIF4E

    NARCIS (Netherlands)

    Teng, C.Y.; Chang, S.L.; Oers, van M.M.; Wu, T.Y.

    2013-01-01

    Host protein synthesis is shut down in the lytic baculovirus expression vector system (BEVS). This also affects host proteins involved in routing secretory proteins through the endoplasmic reticulum (ER)-Golgi system. It has been demonstrated that a secretory alkaline phosphatase–EGFP fusion protein

  6. Expression of thymidine kinase mediated by a novel non-viral delivery system under the control of vascular endothelial growth factor receptor 2 promoter selectively kills human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Ying Wang; Hui-Xiong Xu; Ming-De Lu; Qing Tang

    2008-01-01

    AIM: To investigate the killing efficiency of a recombinant plasmid containing a thymidine kinase (TK) domain insert driven by the vascular endothelial growth factor receptor 2 (VEGFR2) promoter (KDR) on vascular endothelial cells.METHODS: The KDR-TK fragment was extracted from pBluescript 11 KDR-TK plasmid by enzymatic digestion with XhoI and Sa/I. The enhanced green fluorescence protein (EGFP) carrier was extracted from pEGFP by the same procedure. The KDR-TK was inserted into the pEGFP carrier to construct pEGFP-KDR-TK. Using ultrasound irradiation and microbubble,pEGFP-KDR-TK was transferred into human umbilical vein endothelial cells (HUVECs). The transient infection rate was estimated by green fluorescent protein (GFP)expression. Transfected HUVECs, non-transfected HUVECs, and HepG2 cells were cultured in the presence of different concentrations of ganciclovir (GCV), and the killing efficacy of HSV-TK/GCV was analyzed by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTr) assay.RESULTS: The recombinant pEGFP-KDR-TK was successfully constructed by inserting the KDR-TK fragment into the pEGFP carrier. Transfected HUVECs showed cytoplasmic green fluorescence, and the transient transfection rate was about 20.3%. Pools of G418-resistant cells exhibited a higher sensitivity to the prodrug/GCV compared to non-transfected HUVECs or non-transfected HepG2 cells, respectively.CONCLUSION: KDR promoter and the suicide gene/prodrug system mediated by diagnostic ultrasound combined with microbubble can significantly kill HUVECs.Such therapy may present a novel and attractive approach to target gene therapy on tumor vessels.

  7. 人类前列腺凋亡反应因子4基因真核表达载体的构建及其在K562细胞中的表达%Construction of human Par-4 eukaryotic expression vector and expression in K562 cells

    Institute of Scientific and Technical Information of China (English)

    秦洁; 王宏伟; 杨涛; 朱镭; 张丽

    2009-01-01

    目的 构建带有绿色荧光蛋白的人类前列腺凋亡反应因子4(Par-4)基因真核表达载体pIRES2-EGFP/Par-4,并转染K562细胞.方法 以pDNR/Par-4质粒为模板,PCR扩增Par-4基因,T-A克隆,亚克隆至pIRES2-EGFP上,并对重组表达载体进行酶切、测序鉴定.采用Superfect转染试剂转染K562细胞.提取细胞总蛋白,Western blotting柃测Par-4表达.结果 经限制性酶切鉴定及测序分析证实pIRES2-EGFP/Par-4载体序列止确;荧光显微镜下可见转染的K562细胞有绿色荧光蛋白的表达,Western blotting 证实转染细胞中存在Par-4蛋白的表达.结论 pIRES2-EGFP/Par-4表达载体构建成功,并在K562细胞内成功表达.%Objective To construct an eukaryotic expression vector of human Par-4 gene with green fluorescent protein gene which iS named pIRES2-EGFP/Par-4 and transfect jt into K562 cell line. Methods Using pDNR/Par-4 plasmid as a template.the full length Par-4 cDNA was amplified by PCR and subsequently cloned into T-A vector.Then subcloned into pIRES2-EGFP vector.After identified by digestion of restriefive endonucleases.pIRES2-EGFP/Par-4 was further confirmed by sequencing.Then it was transfected into K562 cells with Superfect reagents.The total proteins were isolated and Par-4 was detected by Western blotting. Results The exact sequences of pIRES2-EGFP/Par-4 vector were confirmed by digestion of restrictive endonucleases and sequencing.After transfection,the expressions of green fluorescent protein were present.The protein expression of Par-4 has been detected in transfected ceils hv Western blotting.Conclusion The vector pIRES2-EGFP/Par-4 has been constructed and could Successfully express Par-4 gene in K562 cells.

  8. Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

    International Nuclear Information System (INIS)

    The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 x 10-8 to 2.0 x 10-6 M with a detection limit of 1.6 x 10-8 M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications

  9. Circadian RNA expression elicited by 3’-UTR IRAlu-paraspeckle associated elements

    Science.gov (United States)

    Torres, Manon; Becquet, Denis; Blanchard, Marie-Pierre; Guillen, Séverine; Boyer, Bénédicte; Moreno, Mathias; Franc, Jean-Louis; François-Bellan, Anne-Marie

    2016-01-01

    Paraspeckles are nuclear bodies form around the long non-coding RNA, Neat1, and RNA-binding proteins. While their role is not fully understood, they are believed to control gene expression at a post-transcriptional level by means of the nuclear retention of mRNA containing in their 3’-UTR inverted repeats of Alu sequences (IRAlu). In this study, we found that, in pituitary cells, all components of paraspeckles including four major proteins and Neat1 displayed a circadian expression pattern. Furthermore the insertion of IRAlu at the 3’-UTR of the EGFP cDNA led to a rhythmic circadian nuclear retention of the egfp mRNA that was lost when paraspeckles were disrupted whereas insertion of a single antisense Alu had only a weak effect. Using real-time video-microscopy, these IRAlu were further shown to drive a circadian expression of EGFP protein. This study shows that paraspeckles, thanks to their circadian expression, control circadian gene expression at a post-transcriptional level. DOI: http://dx.doi.org/10.7554/eLife.14837.001 PMID:27441387

  10. Preparation and characterization of a stable BHK-21 cell line constitutively expressing the Schmallenberg virus nucleocapsid protein.

    Science.gov (United States)

    Zhang, Yongning; Wu, Shaoqiang; Song, Shanshan; Lv, Jizhou; Feng, Chunyan; Lin, Xiangmei

    2015-08-01

    Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that predominantly infects livestock such as cattle, sheep, and goats. Its nucleocapsid (N) protein is an ideal target antigen for SBV diagnosis. In this study, a stable BHK-21 cell line, BHK-21-EGFP-SBV-N, constitutively expressing the SBV N protein was obtained using a lentivector-mediated gene transfer system combined with puromycin selection. To facilitate the purification of recombinant SBV N protein, the coding sequence for a hexa-histidine tag was introduced into the C-terminus of the SBV N gene during construction of the recombinant lentivirus vector pLV-EGFP-SBV-N. The BHK-21-EGFP-SBV-N cell line was demonstrated to spontaneously emit strong enhanced green fluorescent protein (EGFP) signals that exhibited a discrete punctate distribution throughout the cytoplasm. SBV N mRNA and protein expression in this cell line were detected by real-time RT-PCR and western blot, respectively. The expressed recombinant SBV N protein carried an N-terminal EGFP tag, and was successfully purified using Ni-NTA agarose by means of its C-terminal His tag. The purified SBV N protein could be recognized by SBV antisera and an anti-SBV monoclonal antibody (mAb) 2C8 in an indirect enzyme-linked immunosorbent assay and western blot analyses. Indirect immunofluorescence assays further demonstrated that the stable cell line reacts with SBV antisera and mAb 2C8. These results suggest that the generated cell line has the potential to be used in the serological diagnosis of SBV. PMID:26013296

  11. Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Grass carp reovirus (GCRV), a disaster agent to aquatic animals, belongs to Genus Aquareovirus of family Reoviridea. Sequence analysis revealed GCRV genome segment 8 (s8) was 1296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa. To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter, the recombinant baculovirus, which contained the GCRVs8 and eGFP (enhanced green fluorescence protein)genes, was constructed by using the Bac-to-Bac insect expression system. In this study, the whole GCRVs8 and eGFP genes, amplified by PCR, were constructed into a pFastBacDual vector under polyhedron (PH) and p10 promoters, respectively. The constructed dual recombinant plasmid (pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid (AcGCRVs8/eGFP) by transposition. Finally, the recombinant bacluovirus (vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells. The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection, and gradually enhanced and extended around 5days culture in P1(Passage1) stock. The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus (BV) stock. Additionally, PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus. Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.

  12. Expression of porcine CFL2b gene in C2C12 cells and its effect on the expression of MyHC

    OpenAIRE

    Zhao, Wei; ZENG Rui-Xia; Su, Yu-Hong; BA Cai-Feng; SU Rong-Jian; Song, Hui-Juan

    2008-01-01

    Porcine CFL2b gene is expressed mainly in skeletal muscle, it affects muscle development and myofibrillar formation. In order to study the relationship between CFL2b gene and muscle fiber trait, stable C2C12 cell clones groups expressing porcine CFL2b gene were obtained by directed cloning and gene transfection technology. The expression of pEGFP-N1–CFL2b were detected using GFP fluorescence and Western Blotting. The expression level of myosin heavy chain (MyHC) isoforms (2x.2b and slow) in C...

  13. The stealth episome: suppression of gene expression on the excised genomic island PPHGI-1 from Pseudomonas syringae pv. phaseolicola.

    Directory of Open Access Journals (Sweden)

    Scott A C Godfrey

    2011-03-01

    Full Text Available Pseudomonas syringae pv. phaseolicola is the causative agent of halo blight in the common bean, Phaseolus vulgaris. P. syringae pv. phaseolicola race 4 strain 1302A contains the avirulence gene avrPphB (syn. hopAR1, which resides on PPHGI-1, a 106 kb genomic island. Loss of PPHGI-1 from P. syringae pv. phaseolicola 1302A following exposure to the hypersensitive resistance response (HR leads to the evolution of strains with altered virulence. Here we have used fluorescent protein reporter systems to gain insight into the mobility of PPHGI-1. Confocal imaging of dual-labelled P. syringae pv. phaseolicola 1302A strain, F532 (dsRFP in chromosome and eGFP in PPHGI-1, revealed loss of PPHGI-1::eGFP encoded fluorescence during plant infection and when grown in vitro on extracted leaf apoplastic fluids. Fluorescence-activated cell sorting (FACS of fluorescent and non-fluorescent PPHGI-1::eGFP F532 populations showed that cells lost fluorescence not only when the GI was deleted, but also when it had excised and was present as a circular episome. In addition to reduced expression of eGFP, quantitative PCR on sub-populations separated by FACS showed that transcription of other genes on PPHGI-1 (avrPphB and xerC was also greatly reduced in F532 cells harbouring the excised PPHGI-1::eGFP episome. Our results show how virulence determinants located on mobile pathogenicity islands may be hidden from detection by host surveillance systems through the suppression of gene expression in the episomal state.

  14. Mitochondrial physiology and gene expression analyses reveal metabolic and translational dysregulation in oocyte-induced somatic nuclear reprogramming.

    Directory of Open Access Journals (Sweden)

    Telma C Esteves

    Full Text Available While reprogramming a foreign nucleus after somatic cell nuclear transfer (SCNT, the enucleated oocyte (ooplasm must signal that biomass and cellular requirements changed compared to the nucleus donor cell. Using cells expressing nuclear-encoded but mitochondria-targeted EGFP, a strategy was developed to directly distinguish maternal and embryonic products, testing ooplasm demands on transcriptional and post-transcriptional activity during reprogramming. Specifically, we compared transcript and protein levels for EGFP and other products in pre-implantation SCNT embryos, side-by-side to fertilized controls (embryos produced from the same oocyte pool, by intracytoplasmic injection of sperm containing the EGFP transgene. We observed that while EGFP transcript abundance is not different, protein levels are significantly lower in SCNT compared to fertilized blastocysts. This was not observed for Gapdh and Actb, whose protein reflected mRNA. This transcript-protein relationship indicates that the somatic nucleus can keep up with ooplasm transcript demands, whilst transcription and translation mismatch occurs after SCNT for certain mRNAs. We further detected metabolic disturbances after SCNT, suggesting a place among forces regulating post-transcriptional changes during reprogramming. Our observations ascribe oocyte-induced reprogramming with previously unsuspected regulatory dimensions, in that presence of functional proteins may no longer be inferred from mRNA, but rather depend on post-transcriptional regulation possibly modulated through metabolism.

  15. Characterization of gene expression regulated by human OTK18 using Drosophila melanogaster as a model system for innate immunity

    Indian Academy of Sciences (India)

    Cole R. Spresser; Sarah E. Marshall; Kimberly A. Carlson

    2008-08-01

    OTK18 is a human transcriptional suppressor implicated in the regulation of human immunodeficiency virus type-one infection of mononuclear phagocytes. It is ubiquitously expressed in all normal tissues, but its normal homeostatic function is yet to be characterized. One hypothesis is that OTK18 aids in the regulation of the innate immune system. To test this hypothesis, cDNA microarray analysis was performed on the total RNA extracted from Drosophila melanogaster embryonic Schneider 2 (S2) cells transfected with either pEGFP-OTK18 (enhanced green fluorescent protein) or empty vector controls (pEGFP-N3) for 6, 12 and 24 h. cDNA microarray analysis revealed differential expression of genes known to be important in regulation of Drosophila innate immunity. The expression levels of two genes, Metchnikowin and CG16708 were verified by quantitative real-time reverse transcription PCR. These results suggest a role for OTK18 in innate immunity.

  16. Tissue-specific expression of ferritin H regulates cellular iron homoeostasis in vivo.

    Science.gov (United States)

    Wilkinson, John; Di, Xiumin; Schönig, Kai; Buss, Joan L; Kock, Nancy D; Cline, J Mark; Saunders, Thomas L; Bujard, Hermann; Torti, Suzy V; Torti, Frank M

    2006-05-01

    Ferritin is a ubiquitously distributed iron-binding protein. Cell culture studies have demonstrated that ferritin plays a role in maintenance of iron homoeostasis and in the protection against cytokine- and oxidant-induced stress. To test whether FerH (ferritin H) can regulate tissue iron homoeostasis in vivo, we prepared transgenic mice that conditionally express FerH and EGFP (enhanced green fluorescent protein) from a bicistronic tetracycline-inducible promoter. Two transgenic models were explored. In the first, the FerH and EGFP transgenes were controlled by the tTA(CMV) (Tet-OFF) (where tTA and CMV are tet transactivator protein and cytomegalovirus respectively). In skeletal muscle of mice bearing the FerH/EGFP and tTA(CMV) transgenes, FerH expression was increased 6.0+/-1.1-fold (mean+/-S.D.) compared with controls. In the second model, the FerH/EGFP transgenes were controlled by an optimized Tet-ON transactivator, rtTA2(S)-S2(LAP) (where rtTA is reverse tTA and LAP is liver activator protein), resulting in expression predominantly in the kidney and liver. In mice expressing these transgenes, doxycycline induced FerH in the kidney by 14.2+/-4.8-fold (mean+/-S.D.). Notably, increases in ferritin in overexpressers versus control littermates were accompanied by an elevation of IRP (iron regulatory protein) activity of 2.3+/-0.9-fold (mean+/-S.D.), concurrent with a 4.5+/-2.1-fold (mean+/-S.D.) increase in transferrin receptor, indicating that overexpression of FerH is sufficient to elicit a phenotype of iron depletion. These results demonstrate that FerH not only responds to changes in tissue iron (its classic role), but can actively regulate overall tissue iron balance. PMID:16448386

  17. Small Protein B upregulates sensor kinase bvgS expression in Aeromonas veronii

    Directory of Open Access Journals (Sweden)

    Zhu eLiu

    2015-06-01

    Full Text Available Earlier studies reveal that Small Protein B (SmpB, a class of well-conserved tmRNA-binding proteins, is essential for the trans-translation process, which functions as a system for translation surveillance and ribosome rescue. Here, we report a previously unrecognized mechanism by which SmpB alone positively regulates the expression of a sensor kinase, BvgS, in Aeromonas veronii. A reporter plasmid was constructed in which the promoter of bvgS was used to control the expression of the enhanced green fluorescent protein (eGFP gene. When the reporter plasmid was co-transformed with a SmpB expression construct into E. coli, the relative fluorescence intensity increased about 3-fold. Transformation with a truncated form of smpB gene showed that the C-terminus had little effect, while N-terminus unexpectedly increased eGFP production. Next, a series of SmpB mutants were generated by site-directed mutagenesis. When the mutants SmpB (G11S or SmpB (E32AG was used in the experiment, eGFP expression dropped significantly compared with that of wild type SmpB. Further,purified SmpB was shown to bind the promoter regions of bvgS in the agarose gel retardation assay. Quantitative RT-PCR analysis showed that eGFP transcript levels increased approximately 25-fold in the presence of SmpB. Likewise, bvgS transcripts decreased significantly in smpB knockout A. veronii. Similar to BvgS inhibition, smpB knockout in A. veronii displayed a reduced capability in salt tolerance. Collectively, the data presented here will facilitate a deeper understanding of SmpB-mediated regulatory circuits as a transcriptional factor in A. veronii.

  18. Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals.

    Science.gov (United States)

    Cho, Yeon Sook; Kim, Byung Soo; Sim, Chan Kyu; Kim, Inki; Lee, Myeong Sup

    2016-01-01

    Interleukin-7 (IL-7) is a cytokine essential for T cell homeostasis, and is clinically important. However, the regulatory mechanism of IL-7 gene expression is not well known, and a systematic approach to screen chemicals that regulate IL-7 expression has not yet been developed. In this study, we attempted to develop human reporter cell lines using CRISPR/Cas9-mediated genome editing technology. For this purpose, we designed donor DNA that contains an enhanced green fluorescent protein (eGFP) gene, drug selection cassette, and modified homologous arms which are considered to enhance the translation of the eGFP reporter transcript, and also a highly efficient single-guide RNA with a minimal off-target effect to target the IL-7 start codon region. By applying this system, we established IL-7 eGFP reporter cell lines that could report IL-7 gene transcription based on the eGFP protein signal. Furthermore, we utilized the cells to run a pilot screen campaign for IL-7-upregulating chemicals in a high-throughput format, and identified a chemical that can up-regulate IL-7 gene transcription. Collectively, these results suggest that our IL-7 reporter system can be utilized in large-scale chemical library screening to reveal novel IL-7 regulatory pathways and to identify potential drugs for development of new treatments in immunodeficiency disease. PMID:27589392

  19. Cell-type specific oxytocin gene expression from AAV delivered promoter deletion constructs into the rat supraoptic nucleus in vivo.

    Directory of Open Access Journals (Sweden)

    Raymond L Fields

    Full Text Available The magnocellular neurons (MCNs in the hypothalamus selectively express either oxytocin (OXT or vasopressin (AVP neuropeptide genes, a property that defines their phenotypes. Here we examine the molecular basis of this selectivity in the OXT MCNs by stereotaxic microinjections of adeno-associated virus (AAV vectors that contain various OXT gene promoter deletion constructs using EGFP as the reporter into the rat supraoptic nucleus (SON. Two weeks following injection of the AAVs, immunohistochemical assays of EGFP expression from these constructs were done to determine whether the EGFP reporter co-localizes with either the OXT- or AVP-immunoreactivity in the MCNs. The results show that the key elements in the OT gene promoter that regulate the cell-type specific expression the SON are located -216 to -100 bp upstream of the transcription start site. We hypothesize that within this 116 bp domain a repressor exists that inhibits expression specifically in AVP MCNs, thereby leading to the cell-type specific expression of the OXT gene only in the OXT MCNs.

  20. Construction and Co-expression of Bicistronic Plasmid Encoding Human WEE1 and Stem Cell Factor

    Institute of Scientific and Technical Information of China (English)

    Ping LEI; Wen-Han LI; Wen-Jun LIAO; Bing YU; Hui-Fen ZHU; Jing-Fang SHAO; Guan-Xin SHEN

    2005-01-01

    To protect the hematopoietic stem cells (HSCs) from apoptosis induced by chemotherapy and promote HSC proliferation, bi-functional gene delivery systems are increasingly investigated in gene therapy.In the present study, we constructed a bicistronic vector, pWISG, expressing the anti-apoptotic protein human WEE1 (WEE1Hu) and the fusion protein of the proliferation-stimulating stem cell factor (SCF) and enhanced green fluorescent protein (EGFP) separately with internal ribosome entry site (IRES). We first examined the expression and location of WEE1Hu in Chinese hamster ovary (CHO) cells and showed that WEE1Hu was located in the nucleus, which was confirmed by immunohistochemistry and Western blot. We determined the expression and receptor-binding ability of the SCF-EGFP fusion protein on CD34+ cells,which were proved by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry,respectively. Furthermore, inhibition of cisplatin-induced apoptosis was observed in CD34+ cells transfected with pWISG, which implies that protection for CD34+ cells was achieved via WEE1Hu and SCF-EGFP. Our study suggests that the introduction of two functional genes via bicistronic vector is more powerful and efficient than single gene therapy.

  1. Anti-tumor effect of a recombinant plasmid expressing human interleukin-12: an initial research

    International Nuclear Information System (INIS)

    Objective: To study the anti-tumor effect of a recombinant plasmid expressing human interleukin-12 (pEGFP-CIIL- 12) in vivo and in vitro. Methods: We transduct the recombinant gene (pEGFP-CIIL-12) to liver cancer cell HepG2 in vitro, and detect reproductive activity of the cell using MTT and the activity of expressing vascular endothelial growth factor(VEGF) using semiquantitative PCR. And then, we deliver the gene to rabbit liver tumor tissue intraarterial and combine with chemoembolization to observe the anti- tumor effect to VX2 tumor in vivo. Results: There are no statistical difference compared With control group in activity of reproductive and expressing VEGF in vitro. In vivo, tumor growth rate significantly reduce in gene therapy combined with chemoembolization group. Conclusion: Recombinant gene (pEGFP-ClIL-12) exhibit significant anti-tumor effect in vivo but not in vitro, perhaps the anti-tumor effect is associated with an indirect pathway instead of a direct pathway. (authors)

  2. Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Mandy Y M Lo

    Full Text Available Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

  3. Small animal PET imaging of HSV1-tk gene expression with 124IVDU in liver by the hydrodynamic injection

    International Nuclear Information System (INIS)

    The liver is an important target organ for gene transfer due to its capacity for synthesizing serum protein and its involvement in numerous genetic diseases. High level of foreign gene expression in liver can be achieved by a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), so called hydrodynamic injection. This study is aimed to evaluate liver specific-gene expression of herpes simplex virus type 1 thymidine kinase(HSV1-tk) by hydrodynamic injection and image HSV1-tk expression using 124IVDU-PET. We constructed herpes simplex virus type 1 thymidine kinase (HSV1-tk)-expressing pDNA (pHSV1-tk) modified from pEGFP-N1. Hydrodynamic injection was performed using 40 μg of plasmid (pEGFP/N1 or pHSV1-tk) in 2 ml of 0.85% saline solution for 20∼22g mice in 5 seconds intravenously. At 1 d post-hydrodynamic injection, biodistribution study was performed at 2 h post-injection of radiolabeled IVDU, fluorescence image was obtained using optical imager and small animal PET image was acquired with 124IVDU at 2 h post-injection. After PET imaging, digital whole body autoradiography (DWBA) was performed. Expression of HSV1-tk and EGFP was confirmed by RT-PCR in each liver tissue. In liver of pHSV1-tk and pEGFP/N1 injection groups, 123IVDU uptake was 5.65%ID/g and 0.98%ID/g, respectively. 123IVDU uptake in liver of pHSV1-tk injection group showed 5.7-fold higher than that of pEGFP/N1 injection group (p124IVDU uptake was selectively localized in liver of pHSV1-tk injection group and also checked in DWBA, but showed minimal uptake in liver of pEGFP/N1 injection mice. Hydrodynamic injection was effective to liver-specific delivery of plasmid DNA. Small animal PET image of 124IVDU could be used in the evaluation of noninvasive reporter gene imaging in liver

  4. 携带ePNP基因的减毒伤寒沙门菌的构建及目的基因胰腺癌细胞中的表达%Construction of attenuated Salmonella typhimurium containing PNP gene and expression in pancreatic cancer cells

    Institute of Scientific and Technical Information of China (English)

    林勇; 刘强; 陈卫昌

    2012-01-01

    Objective To develop attenuated Salmonella typhimurium SL3261 harboring recombi-nant expressing vector of pEGFP-Cl-PNP and to detect the expression in pancreatic cancer cells. Methods PNP gene was amplified by PCR cloned into a recombinant eukaryotic expressing plasmid pEGFP-Cl-ePNP and then identified by enzyme digesting,PCR,and sequencing analysis. pEGFP-Cl-PNP vector was electrotransfered to attenuated Salmonella typhimurium SL3261. Then the stability of the morphology and suface antigen of attenuated Salmonella typhimunium containing plasmid were detected. Meanwhile the reconstructed SL3261 was cocultured with BxPc-3 cells to detect their invasion. Fluorescent microscope observasion and RT-PCR was applied to observe GFP and PNP mRNA in the host cells. Results It was proved that PNP gene segments were inserted into pEGFP-Cl correctly and transferred into attenuated Salmonella typhimunium successfully. Conclusion Attenuated Salmonella typhimunium SL3261 containing recombined eukaryotic expressing plasmid pEGFP-Cl-PNP was successfully established and correctly expressed in pancreatic cancer cells.%目的 构建含有pEGFP-C1-PNP质粒的减毒鼠伤寒沙门菌SL3261菌株,检测其在胰腺癌细胞BxPc-3中的表达.方法 PCR扩增得到PNP基因,构建真核表达载体pEGFP-C1-PNP,并进行酶切、PCR和测序鉴定.利用电转化法将重组质粒转入减毒鼠伤寒沙门菌SL3261,进行形态学和表面抗原稳定性检测.含质粒SL3261菌株与BxPc-3细胞体外共培养,利用荧光显微镜观察和RT-PCR检测绿色荧光蛋白的表达和PNP基因的转录.结果 目的基因正确连接pEGFP-C1中,并成功转入减毒鼠伤寒沙门菌,感染细胞亦检测到目的基因的表达.结论 成功构建了能携带ePNP基因真核表达质粒的SL3261菌株,且该基因能在胰腺癌细胞中正确表达.

  5. A p38 substrate-specific MK2-EGFP translocation assay for identification and validation of new p38 inhibitors in living cells: a comprising alternative for acquisition of cellular p38 inhibition data.

    Directory of Open Access Journals (Sweden)

    Roman Anton

    Full Text Available The fundamental role of p38 mitogen-activated protein kinases (MAPKs in inflammation underlines their importance as therapeutic targets for various inflammatory medical conditions, including infectious, vascular, neurobiological and autoimmune disease. Although decades of research have yielded several p38 inhibitors, most clinical trials have failed, due to lack of selectivity and efficacy in vivo. This underlines the continuous need to screen for novel structures and chemotypes of p38 inhibitors. Here we report an optimized MK2-EGFP translocation assay in a semi-automated image based High Content Analysis (HCA system to screen a combinatorial library of 3362 proprietary compounds with extensive variations of chemotypes. By determining the levels of redistribution of MK2-EGFP upon activation of the Rac/p38 pathway in combination with compound treatment, new candidates were identified, which modulate p38 activity in living cells. Based on integrated analysis of TNFα release from human whole blood, biochemical kinase activity assays and JNK3 selectivity testing, we show that this cell based assay reveals a high overlap and predictability for cellular efficacy, selectivity and potency of tested compounds. As a result we disclose a new comprehensive short-list of subtype inhibitors which are functional in the low nanomolar range and might provide the basis for further lead-optimization. In accordance to previous reports, we demonstrate that the MK2-EGFP translocation assay is a suitable primary screening approach for p38-MAPK drug development and provide an attractive labor- and cost saving alternative to other cell based methods including determination of cytokine release from hPBMCs or whole blood.

  6. A p38 Substrate-Specific MK2-EGFP Translocation Assay for Identification and Validation of New p38 Inhibitors in Living Cells: A Comprising Alternative for Acquisition of Cellular p38 Inhibition Data

    Science.gov (United States)

    Anton, Roman; Bauer, Silke M.; Keck, Peter R. W. E. F.; Laufer, Stefan; Rothbauer, Ulrich

    2014-01-01

    The fundamental role of p38 mitogen-activated protein kinases (MAPKs) in inflammation underlines their importance as therapeutic targets for various inflammatory medical conditions, including infectious, vascular, neurobiological and autoimmune disease. Although decades of research have yielded several p38 inhibitors, most clinical trials have failed, due to lack of selectivity and efficacy in vivo. This underlines the continuous need to screen for novel structures and chemotypes of p38 inhibitors. Here we report an optimized MK2-EGFP translocation assay in a semi-automated image based High Content Analysis (HCA) system to screen a combinatorial library of 3362 proprietary compounds with extensive variations of chemotypes. By determining the levels of redistribution of MK2-EGFP upon activation of the Rac/p38 pathway in combination with compound treatment, new candidates were identified, which modulate p38 activity in living cells. Based on integrated analysis of TNFα release from human whole blood, biochemical kinase activity assays and JNK3 selectivity testing, we show that this cell based assay reveals a high overlap and predictability for cellular efficacy, selectivity and potency of tested compounds. As a result we disclose a new comprehensive short-list of subtype inhibitors which are functional in the low nanomolar range and might provide the basis for further lead-optimization. In accordance to previous reports, we demonstrate that the MK2-EGFP translocation assay is a suitable primary screening approach for p38-MAPK drug development and provide an attractive labor- and cost saving alternative to other cell based methods including determination of cytokine release from hPBMCs or whole blood. PMID:24743242

  7. ZNF217 expression correlates with the biological behavior of human ovarian cancer cells

    Institute of Scientific and Technical Information of China (English)

    Lilin Hang; Min Zhang; Fanliang Meng; Mei Zhong; Jing Li 

    2014-01-01

    Objective:The aim of the study was to investigate the correlation of zinc-finger protein 217 (ZNF217) gene ex-pression with the biological behavior of human ovarian cancer HO-8910 cel s. Methods:The expression of ZNF217 in ovarian carcinoma cel lines was detected by RT-PCR and Western blot, respectively. The biological behaviors of the transfectants were investigated by MTT, in vitro Boyden chamber and in vivo invasion assay, respectively. Results:RT-PCR and Western blotting revealed that transfection of ZNF217 into the HO-8910 cel s significantly increased their proliferation along with mark-edly enhanced in vitro and in vivo invasion and metastatic abilities. MTT assay showed that the proliferation ability of pEGFP-N1-ZNF217/HO-8910 cel s was significantly higher than that of pEGFP-N1/HO-8910 cel s and HO-8910 cel s (P<0.001). The Boyden chamber assay showed that the numbers of migrating pEGFP-N1-ZNF217/HO-8910, pEGFP-N1/HO-8910 and HO-8910 cel s were (141.25 ± 13.91) cel s/200 × field, (82.50 ± 11.73) cel s/200 × field and (81.75 ± 12.12) cel s/200 × field, respectively, with a significant dif erence between them (F=29.274, P<0.001). The nude mouse experiment showed that the in vivo tumor formation ability of pEGFP-N1-ZNF217/HO-8910 cel s was significantly higher than that of pEGFP-N1/HO-8910 cel s (P<0.001). Conclusion:Based on these clinical and laboratory observations, we conclude that ZNF217 may contribute to ovarian cancer invasion and metastasis, and associated with worse clinical outcomes. We evaluated ZNF217’s role as a biomarker of ovarian carcinogenesis and tumor progression in patient samples and explored possible molecular mechanisms in promoting tumor growth and invasion.

  8. Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression

    Science.gov (United States)

    Yang, Dongshan; Song, Jun; Zhang, Jifeng; Xu, Jie; Zhu, Tianqing; Wang, Zhong; Lai, Liangxue; Chen, Y. Eugene

    2016-01-01

    The laboratory rabbit has been a valuable model system for human disease studies. To make the rabbit model more amendable to targeted gene knockin and stable gene over-expression, we identified a rabbit orthologue of the mouse Rosa26 locus through genomic sequence homology analysis. Real-time PCR and 5′ RACE and 3′ RACE experiments revealed that this locus encodes two transcript variants of a long noncoding RNA (lncRNA) (rbRosaV1 and rbRosaV2). Both variants are expressed ubiquitously and stably in different tissues. We next targeted the rabbit Rosa26 (rbRosa26) locus using CRISPR/Cas9 and produced two lines of knock-in rabbits (rbRosa26-EGFP, and rbRosa26-Cre-reporter). In both lines, all the founders and their offspring appear healthy and reproduce normally. In F1 generation animals, the rbRosa26-EGFP rabbits express EGFP, and the rbRosa26-Cre-reporter rabbits express tdTomato ubiquitously in all the tissues examined. Furthermore, disruption of rbRosa26 locus does not adversely impact the animal health and reproduction. Therefore, our work establishes rbRosa26 as a safe harbor suitable for nuclease mediated gene targeting. The addition of rbRosa26 to the tool box of transgenic research is expected to allow diverse genetic manipulations, including gain-of function, conditional knock out and lineage-tracing studies in rabbits. PMID:27117226

  9. Single cell derived murine embryonic stem cell clones stably express Rex1-specific green fluorescent protein and their differentiation study

    International Nuclear Information System (INIS)

    Embryonic stem cells (ESCs) often display high rates of apoptosis and spontaneous differentiation in routine culture, thus bring the proliferation of these cells highly inefficient. Moreover, little is known about the factors that are indispensable for sustaining self-renewal. To surmount these issues, we established transgenic mES cell lines expressing the enhanced green fluorescent protein (EGFP) under the control of the Rex1 promoter which is a key regulator of pluripotency in ES cells. In addition, we provided a simplified and improved protocol to derive transgenic mESCs from single cell. Finally, we showed that embryoid body (EB) development was faster than adherent differentiation in terms of differentiation ratio by real-time tracking of the EGFP expression. Therefore, these cell lines can be tracked and selected both in vitro and in vivo and should be invaluable for studying the factors that are indispensable for maintaining pluripotency

  10. Spinal neurons that contain gastrin-releasing peptide seldom express Fos or phosphorylate extracellular signal-regulated kinases in response to intradermal chloroquine

    Science.gov (United States)

    Bell, Andrew M; Gutierrez-Mecinas, Maria; Polgár, Erika; Todd, Andrew J

    2016-01-01

    Background Gastrin-releasing peptide (GRP) is thought to play a role in the itch evoked by intradermal injection of chloroquine. Although some early studies suggested that GRP was expressed in pruriceptive primary afferents, it is now thought that GRP in the spinal cord is derived mainly from a population of excitatory interneurons in lamina II, and it has been suggested that these are involved in the itch pathway. To test this hypothesis, we used the transcription factor Fos and phosphorylation of extracellular signal-regulated kinases (ERK) to look for evidence that interneurons expressing GRP were activated following intradermal injection of chloroquine into the calf, in mice that express enhanced green fluorescent protein (EGFP) in these cells. Results Injection of chloroquine resulted in numerous Fos- or phospho-ERK (pERK) positive cells in the somatotopically appropriate part of the superficial dorsal horn. The proportion of all neurons in this region that showed Fos or pERK was 18% and 21%, respectively. However, among the GRP–EGFP, only 7% were Fos-positive and 3% were pERK-positive. As such, GRP–EGFP cells were significantly less likely than other neurons to express Fos or to phosphorylate ERK. Conclusions Both expression of Fos and phosphorylation of ERK can be used to identify dorsal horn neurons activated by chloroquine injection. However, these results do not support the hypothesis that interneurons expressing GRP are critical components in the itch pathway. PMID:27270268

  11. Construction of the recombinant vector carrying herpes simplex virus thymidine kinase and cytokine genes expressed in cell line Tca8113

    Institute of Scientific and Technical Information of China (English)

    JI Guang-hui; ZOU Jing-zhi; QU Le; YUE Ying; KUAI Jian-ke

    2004-01-01

    Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell lineTca8113. Methods: IL-2 cDNA was obtained by reverse transcription. Hsv-tk, IL-2 and IRES genes were amplified by PCR. The purified amplification products were inserted into pGEM-T-Easy, and transformed into E. coli JM109. The purified recombinant plasmids were identified by restriction endonucleases. The recombinant plasmids were digested and pEGFPN3 were linearized, DNA fragments of Hsv-tk, IRES and IL-2 were ligated into linearized pEGFP-N3, and then transferred into E. coli JM109. The recombinant tk-IL-2 genes were cloned separately and introduced into the expression vector pEGFPN3 containing GFP. The recombinant vectors were identified by their restriction sites through PCR. The plasmids pEGFP-TI was also transfected into Tca8113 cells by calcium phosphate method for the expression of fusion proteins. Fusion genes expressing vector PL(TI)SN was generated by the fusion of HSV-tk, IRES and IL-2 with the use of DNA recombination technology. The recombinant retroviruses were transferred into Tca8113 cells by lipofectamine. The positive clones were obtained after G418 selection and named Tca/TI respectively. Results: The pEGFP-TI pasmid was identified respectively by restriction endonucleases, and their fragment sizes were 1 120 bp and 450 bp. The pEGFP-TI pasmid as templates were amplified respectively by PCR, and their PCR products were 1 120 bp and 450 bp. The pEGFP-TI vectors were used to transfect Tca8113 cell, and the cells with fluorescence accounted for 60 % of the total amount. Conclusion: pFGFP- tk- IRES- IL-2 expressing vector is easy to assess the expression of tk-IRES-IL-2-GFP fusion protein localization in transfected cells. The successful construction of expressing vector containing fusion genes of Hsv-tk, IRES and IL-2 may be

  12. Imaging of Herpes Simplex Virus Type 1 Thymidine Kinase Gene Expression with Radiolabeled 5-(2-iodovinyl)-2'-deoxyuridine (IVDU) in Liver by Hydrodynamic-based Procedure

    International Nuclear Information System (INIS)

    Hydrodynamic-based procedure is a simple and effective gene delivery method to lead a high gene expression in liver tissue. Non-invasive imaging reporter gene system has been used widely with herpes simplex virus type 1 thymidine kinase (HSV1-tk) and its various substrates. In the present study, we investigated to image the expression of HSV1-tk gene with 5-(2-iodovinyl)-2'-deoxyuridine (IVDU) in mouse liver by the hydrodynamicbased procedure. HSV1-tk or enhanced green fluorescence protein (EGFP) encoded plasmid DNA was transferred into the mouse liver by hydrodynamic injection. At 24 h post-injection, RT-PCR, biodistribution, fluorescence imaging, nuclear imaging and digital wholebody autoradiography (DWBA) were performed to confirm transferred gene expression. In RT-PCR assay using mRNA from the mouse liver, specific bands of HSV1-tk and EGFP gene were observed in HSV1-tk and EGFP expressing plasmid injected mouse, respectively. Higher uptake of radiolabeled IVDU was exhibited in liver of HSV1-tk gene transferred mouse by biodistribution study. In fluorescence imaging, the liver showed specific fluorescence signal in EGFP gene transferred mouse. Gamma-camera image and DWBA results showed that radiolabeled IVDU was accumulated in the liver of HSV1-tk gene transferred mouse. In this study, hydrodynamic-based procedure was effective in liver-specific gene delivery and it could be quantified with molecular imaging methods. Therefore, co-expression of HSV1-tk reporter gene and target gene by hydrodynamic-based procedure is expected to be a useful method for the evaluation of the target gene expression level with radiolabeled IVDU

  13. Physiological properties of enkephalin-containing neurons in the spinal dorsal horn visualized by expression of green fluorescent protein in BAC transgenic mice

    Directory of Open Access Journals (Sweden)

    Kofuji Takefumi

    2011-05-01

    Full Text Available Abstract Background Enkephalins are endogenous opiates that are assumed to modulate nociceptive information by mediating synaptic transmission in the central nervous system, including the spinal dorsal horn. Results To develop a new tool for the identification of in vitro enkephalinergic neurons and to analyze enkephalin promoter activity, we generated transgenic mice for a bacterial artificial chromosome (BAC. Enkephalinergic neurons from these mice expressed enhanced green fluorescent protein (eGFP under the control of the preproenkephalin (PPE gene (penk1 promoter. eGFP-positive neurons were distributed throughout the gray matter of the spinal cord, and were primarily observed in laminae I-II and V-VII, in a pattern similar to the distribution pattern of enkephalin-containing neurons. Double immunostaining analysis using anti-enkephalin and anti-eGFP antibodies showed that all eGFP-expressing neurons contained enkephalin. Incubation in the presence of forskolin, an activator of adenylate cyclase, increased the number of eGFP-positive neurons. These results indicate that eGFP expression is controlled by the penk1 promoter, which contains cyclic AMP-responsive elements. Sections obtained from sciatic nerve-ligated mice exhibited increased eGFP-positive neurons on the ipsilateral (nerve-ligated side compared with the contralateral (non-ligated side. These data indicate that PPE expression is affected by peripheral nerve injury. Additionally, single-neuron RT-PCR analysis showed that several eGFP positive-neurons in laminae I-II expressed glutamate decarboxylase 67 mRNA and that some expressed serotonin type 3 receptors. Conclusions These results suggest that eGFP-positive neurons in laminae I-II coexpress enkephalin and γ-aminobutyric acid (GABA, and are activated by forskolin and in conditions of nerve injury. The penk1-eGFP BAC transgenic mouse contributes to the further characterization of enkephalinergic neurons in the transmission and

  14. 脂肪组织特异性表达载体的构建%Construction of Adipose Tissue - specific Expression Vector

    Institute of Scientific and Technical Information of China (English)

    华晓敏; 许登高; 潘庆杰

    2012-01-01

    采用PCR技术克隆了小鼠脂肪组织特异表达的脂肪酸结合蛋白ap2基因增强子和启动子,通过DNA重组技术将该基因增强子和启动子重组于pEGFP - N1真核表达载体上,构建pEGFP - N1 - ap2重组质粒,通过PCR扩增、酶切电泳分析和测序的方法对重组质粒进行鉴定,并转染小鼠前脂肪细胞,通过荧光素酶活性检测特异性表达强度.结果表明,本实验克隆的ap2基因增强子和启动子的碱基组成与GenBank中的ap2基因序列完全一致,通过DNA重组技术将该基因增强子和启动子重组于pEGFP- N1真核表达载体上,成功构建了脂肪组织特异表达的重组质粒.为以后的转基因动物的研究奠定了基础.%The mouse adipose tissue -specific fatty acid binding protein ap2 gene enhancer /promoter was amplified by PCR amplification, and it was recombined into pEGFP - Nl eukaryotic expression vector by recombinant DNA technology, to obtain pEGFP - Nl - ap2 recombinant plasmid, which was identified by PCR amplification, enzyme digestion and DNA sequencing and infected with mouse pre - adipocytes, and its expression was detected by the fluorescence detection of the enzyme activity specific expression strength. The results showed that, cloned gene enhancer and promoter is consistent with the ap2 gene sequences in GenBank. The enhancer / promoter was recombined into pEGFP - Nl eukaryotic expression vector by recombinant DNA technology. The construction of the adipose tissue - specific expression vector was successfully constructed, which can provide a necessary basis for further study.

  15. Localization of DIR1 at the tissue, cellular and subcellular levels during Systemic Acquired Resistance in Arabidopsis using DIR1:GUS and DIR1:EGFP reporters

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    Thilmony Roger

    2011-09-01

    Full Text Available Abstract Background Systemic Acquired Resistance (SAR is an induced resistance response to pathogens, characterized by the translocation of a long-distance signal from induced leaves to distant tissues to prime them for increased resistance to future infection. DEFECTIVE in INDUCED RESISTANCE 1 (DIR1 has been hypothesized to chaperone a small signaling molecule to distant tissues during SAR in Arabidopsis. Results DIR1 promoter:DIR1-GUS/dir1-1 lines were constructed to examine DIR1 expression. DIR1 is expressed in seedlings, flowers and ubiquitously in untreated or mock-inoculated mature leaf cells, including phloem sieve elements and companion cells. Inoculation of leaves with SAR-inducing avirulent or virulent Pseudomonas syringae pv tomato (Pst resulted in Type III Secretion System-dependent suppression of DIR1 expression in leaf cells. Transient expression of fluorescent fusion proteins in tobacco and intercellular washing fluid experiments indicated that DIR1's ER signal sequence targets it for secretion to the cell wall. However, DIR1 expressed without a signal sequence rescued the dir1-1 SAR defect, suggesting that a cytosolic pool of DIR1 is important for the SAR response. Conclusions Although expression of DIR1 decreases during SAR induction, the protein localizes to all living cell types of the vasculature, including companion cells and sieve elements, and therefore DIR1 is well situated to participate in long-distance signaling during SAR.

  16. Simultaneous detection of both GDNF and GFRα1 expression patterns in the mouse central nervous system

    Directory of Open Access Journals (Sweden)

    Clara Ortega-de San Luis

    2016-06-01

    Full Text Available Glial cell line-derived neurotrophic factor (GDNF is proposed as a therapeutic tool in Parkinson’s disease, addiction-related disorders, and neurodegenerative conditions affecting motor neurons. Despite the high amount of work about GDNF therapeutic application, the neuronal circuits requiring GDNF trophic support in the brain and spinal cord are poorly characterized. Here, we defined GDNF and GDNF family receptor-α 1 (GFRα1 expression pattern in the brain and spinal cord of newborn and adult mice. We performed systematic and simultaneous detection of EGFP and LacZ expressing alleles in reporter mice and asked whether modifications of this signaling pathway lead to a significant central nervous system (CNS alteration. GFRα1 was predominantly expressed by neurons but also by an unexpected population of non-neuronal cells. GFRα1 expression pattern was wider in neonatal than in adult CNS and GDNF expression was restricted in comparison with GFRα1 at both developmental time points. The use of confocal microscopy to imaging X-gal deposits and EGFP allowed us to identify regions containing cells that expressed both proteins and to discriminate between auto and non-autotrophic signaling. We also suggested long-range GDNF-GFRα1 circuits taking advantage of the ability of the EGFP genetically encoded reporter to label long distance projecting axons. The complete elimination of either the ligand or the receptor during development did not produce major abnormalities, suggesting a preponderant role for GDNF signaling during adulthood. In the spinal cord, our results pointed to local modulatory interneurons as the main target of GDNF produced by Clarke’s column cells. Our work increases the understanding on how GDNF signals in the CNS and establish a crucial framework for posterior studies addressing either the biological role of GDNF or the optimization of trophic factor-based therapies.

  17. Characterization of dopamine D1 and D2 receptor-expressing neurons in the mouse hippocampus.

    Science.gov (United States)

    Gangarossa, Giuseppe; Longueville, Sophie; De Bundel, Dimitri; Perroy, Julie; Hervé, Denis; Girault, Jean-Antoine; Valjent, Emmanuel

    2012-12-01

    The hippocampal formation is part of an anatomical system critically involved in learning and memory. Increasing evidence suggests that dopamine plays an important role in learning and memory as well as in several forms of synaptic plasticity. However, the precise identification of neuronal populations expressing D1 or D2 dopamine receptors within the hippocampus is still lacking. To clarify this issue, we used BAC transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter of dopamine D1 or D2 receptors. In Drd1a-EGFP mice, sparse GFP-expressing neurons were detected among glutamatergic projecting neurons of the granular layer of the dentate gyrus and GABAergic interneurons located in the hilus. A dense immunofluorescence was observed in the outer and medial part of the molecular layer of the dentate gyrus as well as in the inner part of the molecular layer of CA1 corresponding to the terminals of pyramidal neurons of the entorhinal cortex defining the perforant and the temporo-ammonic pathway respectively. Finally, scattered D1 receptor-expressing neurons were also identified as GABAergic interneurons in the CA3/CA1 fields of the hippocampus. In Drd2-EGFP transgenic mice, GFP was exclusively detected in the glutamatergic mossy cells located in the polymorphic layer of the dentate gyrus. This pattern was confirmed in Drd2-Cre mice crossed with NLS-LacZ-Tau(mGFP) :LoxP and RCE:LoxP reporter lines. Our results demonstrate that D1 and D2 receptor-expressing neurons are strictly segregated in the mouse hippocampus. By clarifying the identity of D1 and D2 receptor-expressing neurons in the hippocampus, this study establishes a basis for future investigations aiming at elucidating their roles in the hippocampal network. PMID:22777829

  18. Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms

    Energy Technology Data Exchange (ETDEWEB)

    Ford, Nicole R.; Hecht, Karen A.; Hu, Dehong; Orr, Galya; Xiong, Yijia; Squier, Thomas; Rorrer, Gregory L.; Roesijadi, Guritno

    2016-01-08

    The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins incorporating a tetracysteine tag for site-directed labeling with biarsenical affinity probes and either EGFP or single chain antibody to test colocalization of probes with the EGFP-tagged recombinant protein or binding of biosilica-immobilized antibodies to large and small molecule antigens, respectively. Site-directed labeling with the biarsenical probes demonstrated colocalization with EGFP-encoded proteins in nascent and mature biosilica, supporting their use in studying biosilica maturation. Isolated biosilica transformed with a single chain antibody against either the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT) effectively bound the respective antigens. A marked increase in fluorescence lifetime of the TNT surrogate Alexa Fluor 555-trinitrobenzene reflected the high binding specificity of the transformed isolated biosilica. These results demonstrated the potential use of biosilica-immobilized single chain antibodies as binders for large and small molecule antigens in sensing and therapeutics.

  19. Tissue-specific and neural activity-regulated expression of human BDNF gene in BAC transgenic mice

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    Palm Kaia

    2009-06-01

    Full Text Available Abstract Background Brain-derived neurotrophic factor (BDNF is a small secreted protein that has important roles in the developing and adult nervous system. Altered expression or changes in the regulation of the BDNF gene have been implicated in a variety of human nervous system disorders. Although regulation of the rodent BDNF gene has been extensively investigated, in vivo studies regarding the human BDNF gene are largely limited to postmortem analysis. Bacterial artificial chromosome (BAC transgenic mice harboring the human BDNF gene and its regulatory flanking sequences constitute a useful tool for studying human BDNF gene regulation and for identification of therapeutic compounds modulating BDNF expression. Results In this study we have generated and analyzed BAC transgenic mice carrying 168 kb of the human BDNF locus modified such that BDNF coding sequence was replaced with the sequence of a fusion protein consisting of N-terminal BDNF and the enhanced green fluorescent protein (EGFP. The human BDNF-BAC construct containing all BDNF 5' exons preceded by different promoters recapitulated the expression of endogenous BDNF mRNA in the brain and several non-neural tissues of transgenic mice. All different 5' exon-specific BDNF-EGFP alternative transcripts were expressed from the transgenic human BDNF-BAC construct, resembling the expression of endogenous BDNF. Furthermore, BDNF-EGFP mRNA was induced upon treatment with kainic acid in a promotor-specific manner, similarly to that of the endogenous mouse BDNF mRNA. Conclusion Genomic region covering 67 kb of human BDNF gene, 84 kb of upstream and 17 kb of downstream sequences is sufficient to drive tissue-specific and kainic acid-induced expression of the reporter gene in transgenic mice. The pattern of expression of the transgene is highly similar to BDNF gene expression in mouse and human. This is the first study to show that human BDNF gene is regulated by neural activity.

  20. Establishment of Stable High Expression Cell Line with Green Fluorescent Protein and Resistance Genes

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shengtao; LIU Wenli; HE Peigen; GONG Feili; YANG Dongliang

    2006-01-01

    In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.

  1. Regulation of gene expression in mammalian cells using the lac repressor

    International Nuclear Information System (INIS)

    The study describes the construction of a one step inducible lac repressor/operator mammalian expression system within the context of DNA mediated vaccination (naked DNA). It is novel in that it contains, on a single vector (pSO1), all the cis and trans controlling elements necessary to manipulate (switch on/off) and control the expression of a reporter gene in a mammalian cell. The enhanced green fluorescent protein (EGFP) gene was cloned downstream of the cytomegalovirus immediate-early enhancer promoter (PCMV IE) and a lac operator (LacO), which was in turn regulated by the expression of the lac repressor under the control of a second PCMV IE. The number of cells fluorescing were greatly increased following isopropyl B-D thiogalactoside (IPTG) induction. In the repressed state, fewer pSO1 transfected cells expressed the EGFP, and the fluorescence intensity was also lower than that observed for the induced pSO1 transfected cells. Observation by microscopy was quantified by FACScan analysis on the different populations of cells. There was always a significant difference between the induced and repressed pSO1 transfected cells in terms of the percentage of the population of cells fluorescing and the intensity of the fluorescence. Of the repressed pSO1 transfected HeLa cells, only about 10% showed fluorescence at 507 nm wavelength. However, of the induced pSO1 transfected HeLa cells, 68% showed fluorescence at 24 h, 51% at 48 h and 46% at 72 h post-transfection. To demonstrate this system's ability to be manipulated, two experiments were conducted in parallel. Almost 40% of the pSO1 transfected HeLa cells (24 h repressed, followed by 24 h induced) responded to the induction and expressed EGFP. A similar result was obtained for pSO1 transfected HeLa cells (48 h repressed, followed by 24 h induced); 36% of the cells responded and expressed EGFP. Alternatively, there was a decrease in the number of EGFP expressing cells when pSO1 transfected HeLa cells were induced for

  2. Regeneration of alveolar type I and II cells from Scgb1a1-expressing cells following severe pulmonary damage induced by bleomycin and influenza.

    Directory of Open Access Journals (Sweden)

    Dahai Zheng

    Full Text Available The lung comprises an extensive surface of epithelia constantly exposed to environmental insults. Maintaining the integrity of the alveolar epithelia is critical for lung function and gaseous exchange. However, following severe pulmonary damage, what progenitor cells give rise to alveolar type I and II cells during the regeneration of alveolar epithelia has not been fully determined. In this study, we have investigated this issue by using transgenic mice in which Scgb1a1-expressing cells and their progeny can be genetically labeled with EGFP. We show that following severe alveolar damage induced either by bleomycin or by infection with influenza virus, the majority of the newly generated alveolar type II cells in the damaged parenchyma were labeled with EGFP. A large proportion of EGFP-expressing type I cells were also observed among the type II cells. These findings strongly suggest that Scgb1a1-expressing cells, most likely Clara cells, are a major cell type that gives rise to alveolar type I and II cells during the regeneration of alveolar epithelia in response to severe pulmonary damage in mice.

  3. Egr-1 promoter regulating effect on granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression induced by doxorubicin and ionizing radiation

    International Nuclear Information System (INIS)

    Objective: To explore the regulating effects of Egr-1 promoter activated by ionizing radiation (IR) and doxorubicin (ADM) on the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) genes. Methods: The human GM-CSF cDNA and enhanced green fluorescent protein (EGFP) cDNA were linked together with IRES(internal ribosome entry site) and then inserted into the expression vector pCIneo under control of the Egr-1 promoter(Egr-EG). The vector was transferred into human bone marrow stromal cell line HFCL by liposome transfection. And the cells were exposure to ADM and IR. The activity of EGFP in HFCL/EG cells were detected by FACS. The effect of N-acetylcysteine on the expression of EGFP following exposure to ADM and IR was examined. The amounts of GM-CSF in HFCL/EG after chemotherapy or radiation were measured with ELISA. The effects of GM-CSF in HFCL/EG cultural supernatants on expansion of CFU-GM derived from cord blood were also studied. RT-PCR analysis for the expression of GM-CSF mRNA in HFCL/EG after exposure to ADM or IR. Results: The percentage of EGFP+ HFCL/EG cells exposed to ADM and IR was increased compared with non-treatment group (1.2 % and 15.2 % vs 18.2 %, t = 5.11, P < 0.01). The levels of secreted GM-CSF in HFCL/EG cells exposed to ADM and IR was increased (P < 0.01), but no difference between ADM group and IR group (P 0.05). The expression of EGFP by HFCL/EG treated with ADM and IR was significantly decreased by N-acetylcysteine. The effects of GM-CSF in HFCL/EG cultural supernatants on expansion of CFU-GM in ADM group and IR group were significantly higher than that in HFCL group and non-treatment group. However, The CFU-GM count of IR group was higher than that of ADM group. The expression of GM-CSF mRNA in HFCL/EG cells exposed to ADM and IR was significantly increased(t = 4.37, P < 0.01). Conclusions: GM-CSF gene expression regulated by Egr-1 promoter induced by ADM and IR could help the recovery from hematopoietic injury

  4. Construction and detection of expression vectors of microRNA-9a in BmN cells

    Institute of Scientific and Technical Information of China (English)

    Yong HUANG; Quan ZOU; Sheng-peng WANG; Shun-ming TANG; Guo-zheng ZHANG; Xing-jia SHEN

    2011-01-01

    MicroRNAs (miRNAs) are small endogenous RNAs molecules,approximately 21-23 nucleotides in length,which regulate gene expression by base-pairing with 3' untranslated regions (UTRs) of target mRNAs.However,the functions of only a few miRNAs in organisms are known.Recently,the expression vector of artificial miRNA has become a promising tool for gene function studies.Here,a method for easy and rapid construction of eukaryotic miRNA expression vector was described.The cytoplasmic actin 3 (A3) promoter and flanked sequences of miRNA-9a (miR-9a)precursor were amplified from genomic DNA of the silkworm (Bombyx mori) and was inserted into pCDNA3.0 vector to construct a recombinant plasmid.The enhanced green fluorescent protein (EGFP) gene was used as reporter gene.The Bombyx mori N (BmN) cells were transfected with recombinant miR-9a expression plasmid and were harvested 48 h post transfection.Total RNAs of BmN cells transfected with recombinant vectors were extracted and the expression of miR-9a was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot.Tests showed that the recombinant miR-9a vector was successfully constructed and the expression of miR-9a with EGFP was detected.=miRNA-9a (miR-9a),EGFP gene,Bombyx mori N (BmN) Cells,Expression vector

  5. Construction of recombinant adenoviral vector co-expressing interleukin-7 and enhanced green fluorescent protein%IL-7和EGFP双基因共表达重组腺病毒载体的构建

    Institute of Scientific and Technical Information of China (English)

    宁昌; 余长林; 李建军; 胡锴勋

    2011-01-01

    Objective:To construct the adenoviral vector co - expressing interleukin - 7( IL -7 ) and enhanced green fluorescent protein( EGFP),to lay a experimental foundation for further study on the infection into stem cell. Methods: The target gene IL -7 was cloned into the shuttle plasmid expressed the report gene EGFP. Then the re-combinant shuttle plasmid was transformed into Dh5a bacteria to recombine with backbone vector pAdxsi. Next,the plasmid pAd - EGFP - mIL7 was amplified in H293 cells and purifired, then the viral titer was determined. Results: The recombinanted shuttle plasmid pShuttle - EGFP - mIL7 digested with restriction endonucleases was confirmed by two products which length were respectively about 0. 5kb and 5. 1kb; the recombinanted plasmid pAdxsi - EGFP -mIL7 digested with restriction endonucleases was confirmed by seven products which length were respectively about 14K,11.8K,3. Lkb,2.66kb,2.47K,1.45K and 0.6K; recombinant adenoviral amplifired with titer of 2×l010pfu/ ml. Conclusion: The recombinant adenoviral vector pAdxsi - EGFP - mIL7 was successfully constructed.%目的:构建白细胞介素7(IL-7)和增强型绿色荧光蛋白(EGFP)共表达的重组腺病毒载体,为进一步感染干细胞奠定基础.方法:将目的基因IL-7克隆到含有报告基因EGFP的穿梭质粒中,然后再将构建的重组穿梭质粒转移至pAdxsi载体中,构建重组腺病毒载体质粒,继而在H293细胞中扩增,纯化后测定病毒滴度.结果:pShuttle-EGFP-mIL7重组穿梭质粒经酶切鉴定得到0.5kb和5.1kb 2条带;pAdxsi-EGFP-mIL7重组腺病毒载体质粒经酶切鉴定得到14K、11.8K、3.1kb、2.66kb、2.47K、1.45K、0.6K 7条带;TCID50法测定纯化后的病毒滴度为2×1010pfu/ml.结论:pAdxsi-EGFP-mIL7重组腺病毒载体构建成功.

  6. Cloning, Expression and Characterization of Zebra Fish Ferroportin in Hek 293T Cell Line

    Directory of Open Access Journals (Sweden)

    A Memarnejadian

    2012-01-01

    Full Text Available Background: Ferroportin (Fpn, a regulator of iron homeostasis is a conserved membrane protein that exports iron across the enterocytes, macrophages and hepatocytes into the blood circulation. Fpn has also critical influence on survival of microorganisms whose growth is dependent upon iron, thus preparation of Fpn is needed to study the role of iron in immunity and pathogenesis of micoorganisms.Methods: To prepare and characterize a recombinant ferroportin, total RNA was extracted from Indian zebrafish duodenum, and used to synthesize cDNA by RT-PCR. PCR product was first cloned in Topo TA vector and then subcloned into the GFP expression vector pEGFP-N1. The final resulted plasmid (pEGFP-ZFpn was used for expression of Fpn-EGFP protein in Hek 293T cells.Results: The expression was confirmed by appearance of fluorescence in Hek 293 T cells. Recombinant Fpn was further characterized by submission of its predicted amino acid sequences to the TMHMM V2.0 prediction server (hidden Markov model, NetOGlyc 3.1 and NetNGlyc 3.1 servers. The obtained Fpn from indian zebrafish also contained eight transmembrane domains with N- and C-termini inside the cytoplasm and harboured 78 O-glycosylated amino acids.Conclusion: The recombinant Fpn from Indian zebra fish was successfully expressed in Hek 293 cell line. Although the discrepancy in two amino acids was observed in our produced Fpn and resulted in an additional O-glycosylation site, but had no effect on the topology of the protein compared to other Fpn described by other researchers. Therefore this construct can be used in future iron studies.

  7. Fluorescent reporter signals, EGFP and DsRed, encoded in HIV-1 facilitate the detection of productively infected cells and cell-associated viral replication levels

    Directory of Open Access Journals (Sweden)

    Kazutaka eTerahara

    2012-01-01

    Full Text Available Flow cytometric analysis is a reliable and convenient method for investigating molecules at the single cell level. Previously, recombinant human immunodeficiency virus type 1 (HIV-1 strains were constructed that express a fluorescent reporter, either enhanced green fluorescent protein or DsRed, which allow the monitoring of HIV-1-infected cells by flow cytometry. The present study further investigated the potential of these recombinant viruses in terms of whether the HIV-1 fluorescent reporters would be helpful in evaluating viral replication based on fluorescence intensity. When primary CD4+ T cells were infected with recombinant viruses, the fluorescent reporter intensity measured by flow cytometry was associated with the level of CD4 downmodulation and Gag p24 expression in infected cells. Interestingly, some HIV-1-infected cells, in which CD4 was only moderately downmodulated, were reporter-positive but Gag p24-negative. Furthermore, when the activation status of primary CD4+ T cells was modulated by T cell receptor-mediated stimulation, we confirmed the preferential viral production upon strong stimulation and showed that the intensity of the fluorescent reporter within a proportion of HIV-1-infected cells was correlated with the viral replication level. These findings indicate that a fluorescent reporter encoded within HIV-1 is useful for the sensitive detection of productively-infected cells at different stages of infection and for evaluating cell-associated viral replication at the single cell level.

  8. Expression liver-directed genes by employing synthetic transcriptional control units

    Institute of Scientific and Technical Information of China (English)

    Marie-Luise Lemken; Wolfgang A. Wybranietz; Ulrike Schmidt; Florian Graepler; Sorin Armeanu; Michael Bitzer; Ulrich M. Lauer

    2005-01-01

    AIM: To generate and characterize the synthetic transcriptional control units for transcriptional targeting of the liver,thereby compensating for the lack of specificity of currently available gene therapeutic vector systems.METHODS: Synthetic transcriptional control unit constructs were generated and analyzed for transcriptional activities in different cell types by FACS quantification, semi-quantitative RT-PCR, and Western blotting. RESULTS: A new bifunctionally-enhanced green fluorescent protein (EGFP)/neor fusion gene cassette was generated,and could flexibly be used both for transcript quantification and for selection of stable cell clones. Then, numerous synthetic transcriptional control units consisting of a minimal promoter linked to "naturally" derived composite enhancer elements from liver-specific expressed genes or binding sites of liver-specific transcription factors were inserted upstream of this reporter cassette. Following liposome-mediated transfection, EGFP reporter protein quantification by FACS analysis identified constructs encoding multimerized composite elements of the apolipoprotein B100 (ApoB) promoter or the ornithin transcarbamoylase (OTC) enhancer to exhibit maximum transcriptional activities in liver originating cell lines, but only background levels in non-liver originating cell lines. In contrast, constructs encoding only singular binding sites of liver-specific transcription factors, namely hepatocyte nuclear factor (HNF)1, HNF3, HNF4, HNF5, or CAAT/enhancer binding protein (C/EBP) only achieved background levels of EGFP expression. Finally, both semi-quantitative RT-PCR and Western blotting analysis of Hep3B cells demonstrated maximum transcriptional activities for a multimeric 4xApoB cassette construct, which fully complied with the data obtained by initial FACS analysis.CONCLUSION: Synthetic transcriptional control unit constructs not only exhibit a superb degree of structural compactness, but also provide new means for liver

  9. Effects of XPD and PPARγ on Proliferation of Human Hepatoma G2 Cells and Expression of ERG%XPD和PPARγ对HepG2增殖及ERG表达的影响

    Institute of Scientific and Technical Information of China (English)

    何月; 罗文; 张吉翔

    2012-01-01

    目的 探讨人着色性干皮病基因D(xeroderma pigmentosum group D,XPD)和PPARγ对HepG2增殖及ERG表达的影响.方法 用脂质体转染法瞬时转染重组质粒pEGFP-N2/XPD和空载质粒pEGFP-N2入HepG2细胞中,转染后给予10 μmol/L的GW9662(PPARγ抑制剂)孵育48 h.实验分为6组:空白对照组、脂质体组、pEGFP-N2组、pEGFP-N2/XPD组、pEGFP-N2/XPD + GW9662组及GW9662组.用RT-PCR和Western印迹检测XPD、PPARγ和ERG表达的变化;MTT法观察细胞增殖活力;流式细胞仪检测细胞凋亡.结果 RT-PCR和Western印迹检测示:重组质粒pEGFP-N2/XPD的转染使得XPD表达增高(P均 <0.001);XPD表达增高使得ERG表达降低,同时使得PPARγ表达增高,GW9662能抑制XPD这一作用.MTT结果显示:XPD表达增高抑制了细胞增殖活力,GW9662能抑制XPD降低细胞活力的作用(P 均<0.001).流式细胞仪结果显示:pEGFP-N2/XPD组较未转染组的HepG2细胞凋亡指数明显增加,GW9662能抑制XPD这一作用(P均 <0.001).结论 XPD可通过PPARγ途径抑制肝癌细胞增殖和促进凋亡的; XPD是通过PPARγ途径下调ERG的表达.%Objective To investigate the effects of xeroderma pigmentosum group D (XPD) on the proliferation of human hepatoma G2 cells ( HepG2) and the expression of ERG. Methods The HepG2 cells was transfected with pEGFP-N2/XPD and pEGFP-N2 (the vacant vector plasmid) by Lipofectamine 2000, and they were incubated with 10 μmol/L GW9662 (a PPARγ inhibitor) for 48 h. Six groups were set up in the study, including blank control group, Lipofectamine group, pEGFP-N2 group, pEGFP-N2/XPD group, pEGFP-N2/XPD + GW9662 group and GW9662 group. By using RT-PCR and Western blotting, the expression levels of XPD, ERG, PPARγ and cdk7 were detected. The apoptosis rate was measured by flow cytometry, and the cell viability detected by MTT. Results The mRNA and protein expression levels of XPD were remarkably increased after pEGFP-N2/XPD transfection (P <0. 001) . Over-expression of XPD up

  10. Pattern of CXCR7 Gene Expression in Mouse Brain Under Normal and Inflammatory Conditions.

    Science.gov (United States)

    Banisadr, Ghazal; Podojil, Joseph R; Miller, Stephen D; Miller, Richard J

    2016-03-01

    The chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 acting via its G-protein coupled receptor (GPCR) CXCR4 has been implicated in neurogenesis, neuromodulation, brain inflammation, HIV-1 encephalopathy and tumor growth. CXCR7 was identified as an alternate receptor for SDF-1/CXCL12. Characterization of CXCR7-deficient mice demonstrated a role for CXCR7 in fetal endothelial biology, cardiac development, and B-cell localization. Despite its ligand binding properties, CXCR7 does not seem to signal like a conventional GPCR. It has been suggested that CXCR7 may not function alone but in combination with CXCR4. Here, we investigated the regional localization of CXCR7 receptors in adult mouse brain using CXCR7-EGFP transgenic mice. We found that the receptors were expressed in various brain regions including olfactory bulb, cerebral cortex, hippocampus, subventricular zone (SVZ), hypothalamus and cerebellum. Extensive CXCR7 expression was associated with cerebral blood vessels. Using cell type specific markers, CXCR7 expression was found in neurons, astrocytes and oligodendrocyte progenitors. GAD-expressing neurons exhibited CXCR7 expression in the hippocampus. Expression of CXCR7 in the dentate gyrus included cells that expressed nestin, GFAP and cells that appeared to be immature granule cells. In mice with Experimental Autoimmune Encephalomyelitis (EAE), CXCR7 was expressed by migrating oligodendrocyte progenitors in the SVZ. We then compared the distribution of SDF-1/CXCL12 and CXCR7 using bitransgenic mice expressing both CXCR7-EGFP and SDF-1-mRFP. Enhanced expression of SDF-1/CXCL12 and CXCR7 was observed in the corpus callosum, SVZ and cerebellum. Overall, the expression of CXCR7 in normal and pathological nervous system suggests CXCR4-independent functions of SDF-1/CXCL12 mediated through its interaction with CXCR7. PMID:25997895

  11. Construction and identification of recombination expression vector Ksp-Cadherin-Gpx1-Klk1

    Institute of Scientific and Technical Information of China (English)

    解立怡; 薛武军; 项和立; 麻孙凯

    2008-01-01

    Objective To construct and identify the Gpx1-Klk1 vector which contains kidney-specific promoter (Ksp-cadherin). Methods Through PCR amplification, the human Gpx1, Klk1, and Ksp-cadherin cDNA were obtained by taking Gpx1 cDNA, Klk1 cDNA, and Ksp-cadherin BAC as templates. After being testified, the PCR products were inserted into the expressive vector pIRES-EGFP step-by-step to produce a recombinant vector Ksp-cadherin-Gpx1-Klk1. This vector was examined by restriction enzyme digestion and sequence analysis...

  12. Effects of L1-ORF2 fragments on green fluorescent protein gene expression

    OpenAIRE

    Xiu-Fang Wang; Xia Jin; Xiaoyan Wang; Jing Liu; Jingjing Feng; QinQing Yang; Wenli Mu; Xiaojuan Shi; Zhanjun Lu

    2009-01-01

    The retrotransposon known as long interspersed nuclear element-1 (L1) is 6 kb long, although most L1s in mammalian and other eukaryotic cells are truncated. L1 contains two open reading frames, ORF1 and ORF2, that code for an RNA-binding protein and a protein with endonuclease and reverse transcriptase activities, respectively. In this work, we examined the effects of full length L1-ORF2 and ORF2 fragments on green fluorescent protein gene (GFP) expression when inserted into the pEGFP-C1 vect...

  13. c-Jun氨基末端激酶1反义真核表达载体及其蛋白缺陷细胞株的构建与鉴定%Construction and identification of antisense c-Jun N-terminal kinase 1 eukaryotic fluorescent expressing plasmids and JNK1+ human embryo lung fibroblasts cell line

    Institute of Scientific and Technical Information of China (English)

    徐辉; 何晓庆; 陈瑞; 尹仕伟; 彭雷; 王国强; 李爱萍; 周建伟; 刘起展

    2008-01-01

    目的 构建反义JNK1荧光真核细胞表达载体,建立JNK1蛋白缺陷人胚肺成纤维细胞(HELF)株.方法 用Trizol试剂抽提HELF细胞中总RNA,以反转录PCR扩增JNK1目的 片断,双酶切,纯化PCR产物后,反向插入pEGFP-C1绿色荧光质粒,构建反义pEGFP-C1-asJNK1真核表达载体;大量抽提质粒并转染至HELF细胞中.24 h后使用G418筛选,挑选单克隆细胞扩大培养,经荧光显微成像和蛋白免疫印迹鉴定.结果 pEGFP-C1-asJNK1表达载体DNA测序结果与预期目的 片断序列一致,且JNK1蛋白表达水平明显抑制.结论 反义pEGFP-C1-asJNK1真核表达载体构建成功,JNK1蛋白质缺陷HELF细胞株成功建立.%Objective To construct antisense c-Jun N-terminal kinase 1 (JNK1) eukaryotic fluorescent expressing vector and JNK1+ human embryo lung fibroblasts cell line. Methods Trizol reagent was used to extract total RNA in HELF. The proper primers of JNK1 were chosen and synthesized. RT-PCR and gene recombinant techniques were used to construct the fragment of JNK1. After purification, the PCR products were cut, and JNK1 were inserted reversely into eukaryotic fluorescent expressing vector pEGFP-C1. Enzyme-cutting and DNA auto-sequencing were used to prove the successful construction of JNK1 eukaryotic expressing vector. Then plasmids were extracted and transfected into HELF cells and screen by G418 24 h later. Monoclone was chosen and cultured. Fluorescent imaging and Western blot were used to identify the JNK+HELF cell line. Results Sequence analysis of pEGFP-C1-as JNK1 plasmids was same as expected. The expression level of JNK1 was inhibited markedly. Conclusion Construction of antisensc JNK1 eukaryotic fluorescent expressing vectors and JNK + HELF cell line is successful.

  14. Expression of human carcinoembryonic antigen-related cell adhesion molecule 6 and alveolar progenitor cells in normal and injured lungs of transgenic mice.

    Science.gov (United States)

    Lin, Shin-E; Barrette, Anne Marie; Chapin, Cheryl; Gonzales, Linda W; Gonzalez, Robert F; Dobbs, Leland G; Ballard, Philip L

    2015-12-01

    Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is expressed in the epithelium of various primate tissues, including lung airway and alveoli. In human lung, CEACAM6 is developmentally and hormonally regulated, protects surfactant function, has anti-apoptotic activity and is dysregulated in cancers. We hypothesized that alveolar CEACAM6 expression increases in lung injury and promotes cell proliferation during repair. Studies were performed in CEABAC transgenic mice-containing human CEACAM genes. The level of CEACAM6 in adult CEABAC lung was comparable to that in human infants; expression occurred in epithelium of airways and of some alveoli but rarely co-localized with markers of type I or type II cells. Ten days after bleomycin instillation, both the number of CEACAM6(+) cells and immunostaining intensity were elevated in injured lung areas, and there was increased co-localization with type I and II cell markers. To specifically address type II cells, we crossed CEABAC mice with animals expressing EGFP driven by the SP-C promoter. After bleomycin injury, partially flattened, elongated epithelial cells were observed that expressed type I cell markers and were primarily either EGFP(+) or CEACAM6(+). In cell cycle studies, mitosis was greater in CEACAM6(+) non-type II cells versus CEACAM6(+)/EGFP(+) cells. CEACAM6 epithelial expression was also increased after hyperoxic exposure and LPS instillation, suggesting a generalized response to acute lung injuries. We conclude that CEACAM6 expression is comparable in human lung and the CEABAC mouse. CEACAM6 in this model appears to be a marker of a progenitor cell population that contributes to alveolar epithelial cell replenishment after lung injury. PMID:26702074

  15. Anti-sense RNA Inhibits the Expression of Synaptotagmin Ⅱ in RBL-2H3 and Enhances the Exocytosis of Lysosomes in RBL-2H3

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    The expression of synaptotagmin Ⅱ (Syt2) in RBL-2H3 (RBL) and its role during exocytosis of RBL was investigated. The expression of Syt2 in RBL was detected by western blot and Syt2 gene was amplified by PCR. The anti-sense full length Syt2 cDNA expression vector was constructed with pEGFP-N1 and transfected into RBL by electroporation, and stable transfectants were selected by using G418. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. The results showed that Syt2 was expressed in RBL.The anti-sense expression vector pEGFP-N1-Syt2-AS was constructed and the sequence of insertion was completely consistent with rat Syt2 (accession number in GeneBank: NM012665). The stable transfectants (RBL-Syt2-AS) were obtained. Western blot showed that RBL-Syt2-AS expressed a lower level of Syt2 (8 % and 10 % of control cells), indicating that the expression of Syt2 in RBLSyt2-AS was markedly down-regulated by anti-RNA. Compared with control, the release of cathepsin D by RBL-Syt2-AS was increased. It was concluded that Syt2 expressed in RBL and could inhibit exocytosis of lysosomes in RBL.

  16. TLR4 signaling induced TLR2 expression in the process of mimic cerebral ischemia/reperfusion in vitro

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Both TLR4 and TLR2 participated in the mediation of the inflammatory injury in the process of partial cerebral ischemia/reperfusion.However,it still remains unclear whether a crosstalk exists between TLR2 and TLR4 in ischemic cerebral damage.In the present study,we investigated the effect of TLR4 signaling on TLR2 expression during mimic cerebral I/R in vitro.BV-2 cells were cultured and treated with ischemia/reperfusion,then transfected with the plasmid pEGFP-H1/TLR4-siRNA,the plasmid pEGFP-H1/control sequence-siRNA and the blank plasmid,respectively.Interestingly,the expression of TLR2 and TLR4 mRNA and protein,NF-κB p65 mRNA and supernatant TNF-α level were significantly higher in ischemia/reperfusion treated cells than those lack of ischemia/reperfusion treatment,and as compared with those in ischemia/reperfusion treated cells without transfection,no significant differences about the above mentioned gene and protein expression were found in the blank plasmid tranfected cells and the plasmid pEGFP-H1/control sequence-siRNA transfected cells respectively,while the expression levels in the plasmid pEGFP-H1/TLR4-siRNA transfected cells were significantly lower.Additionally,in order to determine the effects of pyrrolidinediethyldithiocarbamate (PDTC),an NF-κB inhibitor,on the TLR4-induced TLR2 expression in BV-2 cells treated with ischemia/reperfusion,it was found that TLR4 and TLR2 mRNA expressions in PDTC pretreated cells were significantly lower in comparison with normal saline pretreated cells and non-pretreated cells.The data suggested that TLR2 activation,signaled by TLR4 and regulated by NF-κB,might be directly involved play an important role in ischemia/reperfusion induced brain damage.

  17. Construction and Identification of α-MHC Promoter-driven CREG Eukaryotic Expressive Plasmid%心肌α肌球蛋白重链启动子驱动的CREG蛋白表达载体的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    栾波; 李杰; 段岩; 张娜; 霍煜; 梁卓; 闫承慧; 韩雅玲

    2011-01-01

    目的:构建心肌特异性α-肌球蛋白重链(α-myosin heavy chain,α-MHC)启动子启动E1A基因阻遏子(cellular repressor of E1A-stimulated genes,CREG)和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)融合的真核表达载体.绿色荧光蛋白作为报告基因,方便在心肌细胞中直接观察CREG蛋白的表达,为心肌特异性转CREG基因动物模型制备提供载体.方法:用BamH I和EcoR I双酶切pcDNA3.1 myc-His/hCREG质粒得到CREG基因,亚克隆入增强绿色荧光蛋白表达质粒pEGFP-N1中,构建pCREG-EGFP-Nl;根据Genebank中公布的α-MHC基因的启动子序列,人工合成pUC57-α-MHC启动子基因序列,经AseI和NheI双酶切得到启动子α-MHC,亚克隆入pCREG-EGFP-N1中替代原CMV启动子,构建pα-MHC-CREG-EGFP-N1,测序鉴定.用脂质体法将该质粒转染体外培养的小鼠原代心肌细胞,荧光显微镜下观测绿色荧光蛋白的表达;Western blot检测CREG蛋白的表达.结果:成功构建pα-MHC-CREG-EGFP-N1质粒,酶切及测序结果正确;成功转染入原代培养小鼠心肌细胞,在荧光显微镜下可见绿色荧光蛋白的表达,Western blot检测到CREG蛋白的表达.结论:重组质粒pα-MHC-CREG-EGFP-N1体外转染入原代培养小鼠心肌细胞后,目的基因能够在心肌细胞中有效表达,检测方法简便可靠,为下一步建立心肌细胞特异性表达CREG的过表达转基因小鼠、深入探讨CREG在心肌疾病发生中的生物学功能研究奠定了基础.%Objective: To construct a eukaryotic expressive plasmid for cellular repressor of ElA-stimulated genes (CREG) driven by a cardiac specific promoter α-MHC and reported by green fluorescent protein, and then to observe its expression in mouse primary cardiomyocytes in vitro. Methods: CREG fragment was released from pcDNA3.1 myc-His/hCREG plasmid by BamH I and EcoR I digestion enzymes, and then was subcloned into pEGFP-Nl plasmid to construct pCREG-EGFP-Nl plasmid. The α -MHC

  18. Bioinformatics analysis and expression of a novel protein ROP48 in Toxoplasma gondii.

    Science.gov (United States)

    Zhou, Jian; Wang, Lin; Zhou, Aihua; Lu, Gang; Li, Qihang; Wang, Zhilin; Zhu, Meiyan; Zhou, Huaiyu; Cong, Hua; He, Shenyi

    2016-06-01

    Toxoplasma gondii is an obligate intracellular apicomplexan parasite, and can infect warmblooded animals and humans all over the world. In the past years, ROP family genes encoding particular proteins of T. gondii had made a great contribution to toxoplasmosis. In this study, we used multiple bioinformatics approaches to predict the physical and chemical characteristics, transmembrane domain, epitope, and topological structure of the rhoptry protein 48 (ROP48). The results indicated that ROP48 protein was mainly located in the membrane and had several positive linear-B cell epitopes and Th-cell epitopes, which suggested that ROP48 is a potential DNA vaccine candidate against toxoplasmosis. Then the PCR product amplified from the ROP48 cDNA was inserted into a pEASY-T1 vector to build a recombinant cloning plasmid. After sequencing, ROP48 was subcloned into a eukaryotic expression plasmid pEGFP-C1 to obtain pEGFP-C1-ROP48 (pROP48). After identification by PCR and restriction enzyme digestion, the recombinant plasmid pROP48 was transfected into HEK 293-T cell and identified by RT-PCR. The results showed that the eukaryotic expression plasmid pROP48 was constructed and transfected to the cells of HEK 293-T successfully. Western blotting showed that the expressed proteins can be recognized by anti-STAg mouse sera. PMID:27078655

  19. Construction of Porcine CCK pDNA and Its Expression in COS-7 Cells

    Institute of Scientific and Technical Information of China (English)

    BAI Jigang; L(U) Yi; BAI Qiaoling

    2007-01-01

    CCK correlates with the generation and progression of pancreatic cancer. The research aims to construct eukaryotic expression plasmid pIRES2-EGFP/CCK (CCK pDNA) and transiently express it in COS-7 cells. Total RNA was extracted from porcine intestinal mucosa. RT-PCR was used to amplify the aimed segments CCKcDNA which was then digested with EcoR1 and BamH1 and inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct CCK pDNA. The constructed plasmid was transfected into COS-7 cells by lepofectamine TM2000-mediated transfer method.The expression of CCK in transfected COS-7 cells was detected 24, 48 and 72 h post-transfection with fluorescence microscopy and the expression level of CCK mRNA in transfected COS-7 cells was assayed by using RT-PCR. The results showed CCK pDNA was successfully constructed and expressed transiently in COS-7 cells. Green fluorescent protein could be detected in the COS-7 cells transfected with porcine CCK pDNA 24 h post-transfection. At 48th h post-transfection, the number of positive cells was increased significantly and much brighter green fluorescence could be detected.And 72 h post-transfection, the green fluorescence of positive cells became even stronger, while no green fluorescence was detected in the control group. The expression of CCK mRNA in the cells was detectable by using RT-PCR. In COS-7 cells transfected with CCK pDNA a high level of porcine CCK mRNA was detected while no expression of porcine CCKmRNA was found in the cells transfected with null plasmid. It was concluded CCK pDNA was expressed successfully in COS-7 cells,which lays a foundation for further research on the relationship between CCK and tumor.

  20. Chronic levodopa treatment alters expression and function of dopamine D3 receptor in the MPTP/p mouse model of Parkinson's disease.

    Science.gov (United States)

    Cote, Samantha R; Kuzhikandathil, Eldo V

    2015-01-12

    Chronic treatment with levodopa or antipsychotics results in manifestation of side-effects such as dyskinesia which correlates with changes in expression and function of receptors and signaling proteins. Previous studies have suggested a role for the dopamine D3 receptor in Parkinson's disease (PD) and tardive dyskinesia. Yet the expression and signaling function of D3 receptor in these disorders is not well understood. Here we tested the hypothesis that chronic levodopa treatment alters both expression and function of D3 receptors in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine plus probenecid (MPTP/p) mouse model of PD. drd3-EGFP reporter mice were injected biweekly with saline or MPTP and probenecid for a 5-week period. During the last two weeks of the 5-week period, the mice were administered saline or levodopa twice daily. Locomotor activity was measured during the treatment period. D3 receptor expression was determined by western blot analysis. D3 receptor signaling function was determined at tissue and single cell level by measuring the activation of D3 receptor-mitogen activated protein kinase (MAPK) pathway. The drd3-EGFP mice administered MPTP/p exhibited akinesia/bradykinesia. Expression of D3 receptor protein in the dorsal striatum specifically increased in the MPTP/p-treated mice administered levodopa. In the dorsal striatum of levodopa and MPTP/p-treated drd3-EGFP mice, administration of a D3 receptor-selective dose of agonist, PD128907, failed to activate D3 receptor-MAPK signaling. These results suggest that MPTP-induced lesion and chronic levodopa treatment alters D3 receptor expression and function in the dorsal striatum which could contribute to the development of dyskinesias and other motor side-effects. PMID:25445374

  1. Immunogenicity and efficacy of a recombinant adenovirus expressing hemagglutinin from the H5N1 subtype of swine influenza virus in mice.

    Science.gov (United States)

    Wu, Yunpu; Qiao, Chuanling; Yang, Huanliang; Chen, Yan; Xin, Xiaoguang; Chen, Hualan

    2014-04-01

    The H5N1 influenza viruses infect a range of avian species and have recently been isolated from humans and pigs. In this study we generated a replication-defective recombinant adenovirus (rAd-H5HA-EGFP) expressing the hemagglutinin (HA) gene of H5N1 A/Swine/Fujian/1/2001 (SW/FJ/1/01) and evaluated its immunogenicity and protective efficacy in BALB/c mice. The recombinant virus induced high levels of hemagglutination inhibition (HI) antibody at a median tissue culture infective dose of 10(8) or 10(7). Compared with mice in the control groups, the mice vaccinated with rAd-H5HA-EGFP did not show apparent weight loss after challenge with either the homologous SW/FJ/1/01 or the heterologous H5N1 A/Chicken/Hunan/77/2005 (CK/HuN/77/05). Replication of the challenge virus was partially or completely inhibited, and viruses were detected at significantly lower numbers in the organs of the vaccinated mice, all of which survived the challenge with CK/HuN/77/05, whereas most of the control mice did not. These results indicate that rAd-H5HA-EGFP can provide effective immune protection from highly pathogenic H5N1 viruses in mice and is therefore a promising new candidate vaccine against H5N1 influenza in animals. PMID:24688173

  2. Ezrin基因敲低及过表达对脑胶质瘤细胞U87迁移的影响%Effect of Ezrin gene knockdown and over-expression on invasion of glioma U87 cells

    Institute of Scientific and Technical Information of China (English)

    刘乃杰; 秦治刚; 孙利波; 金星一; 叶保国; 张金男; 朱庆三

    2013-01-01

    Objective To study the relationship between Ezrin gene and infiltrative growth of glioma through Ezrin gene knockdown and over-expression. Methods According to Ezrin gene sequence in GenBank, primers were designed using Prime Primer 5. 0 software, with which the gene fragment encoding CDS region of Ezrin gene was amplified from U87 cells and cloned into expression vector pEGFP-1. U87 cells were transfected with the constructed recombinant plasmid pEGFP-C 1/Ezrin as well as plasmids shRNA-EZrin-2 and pEGFP-C 1 in mediation of LipofectimineTM 2000 respectively, then deter-mined for expression of Ezrin protein by Western blot, and for migration by scarification test. Results The homologies of mRNAs of cloned Ezrin gene were 99% to those of homo sapiens ezrin (EZR) and transcript variant 1 reported in Gen-Bank. The homologies of amino acids encoding by the cloned gene was 99% to that of ezrin [Homo sapiens] (Sequence ID: ref-NP_003370. 2-, Length:586), with variations of S66P, K258R, P265L and K577R, while the opening read frame was correct. The relative expression level (1. 17) of Ezrin protein in U87 cells transfected with recombinant plasmid pEGFP-C1 /Ezrin was higher those transfected with shRNA-Ezrin-2 (0. 47) and pEGFP-C1 (0. 82). Scarification test showed migration of a small quantity of U87 cells transfected with shRNA-Ezrin-2 to the wound. However, in pEGFP-C1/ Ezrin transfection group, the wound was almost filled with cells. Conclusion Ezrin gene knockdown blocked , while the over-expression promoted the migration of U87 cells, indicating that Ezrin gene involved in the invasive growth of U87 cells.%目的 分析Ezrin基因敲低及过表达对脑胶质瘤细胞U87迁移的影响,以探讨脑胶质瘤浸润性生长的机理.方法 从U87细胞中扩增Ezrin基因CDS区片段,克隆至表达载体pEGFP-C1中,构建Ezrin基因表达质粒pEGFP-C1/Ezrin.将pEGFP-C1/Ezrin、Ezrin基因shRNA质粒shRNA-Ezrin-2和pEGFP-C1以脂质体LipofectimineTM 2000

  3. Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression

    International Nuclear Information System (INIS)

    Human exposure to genotoxic agents in the environment and everyday life represents a serious health threat. Fast and reliable assessment of genotoxicity of chemicals is of main importance in the fields of new chemicals and drug development as well as in environmental monitoring. The tumor suppressor gene p21, the major downstream target gene of activated p53 which is responsible for cell cycle arrest following DNA damage, has been shown to be specifically up-regulated by genotoxic carcinogens. The aim of our study was to develop a human cell-based biosensor system for simple and fast detection of genotoxic agents. Metabolically active HepG2 human hepatoma cells were transfected with plasmid encoding Enhanced Green Fluorescent Protein (EGFP) under the control of the p21 promoter (p21HepG2GFP). DNA damage was induced by genotoxic agents with known mechanisms of action. The increase in fluorescence intensity, due to p21 mediated EGFP expression, was measured with a fluorescence microplate reader. The viability of treated cells was determined by the colorimetric MTS assay. The directly acting alkylating agent methylmethane sulphonate (MMS) showed significant increase in EGFP production after 48 h at 20 μg/mL. The indirectly acting carcinogen benzo(a)pyren (BaP) and the cross-linking agent cisplatin (CisPt) induced a dose- dependent increase in EGFP fluorescence, which was already significant at concentrations 0.13 μg/mL and 0.41 μg/mL, respectively. Vinblastine (VLB), a spindle poison that does not induce direct DNA damage, induced only a small increase in EGFP fluorescence intensity after 24 h at the lowest concentration (0.1 μg/mL), while exposure to higher concentrations was associated with significantly reduced cell viability. The results of our study demonstrated that this novel assay based on the stably transformed cell line p21HepG2GFP can be used as a fast and simple biosensor system for detection of genetic damage caused by chemical agents

  4. 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori

    Science.gov (United States)

    Wang, Yuancheng; Wang, Feng; Wang, Riyuan; Zhao, Ping; Xia, Qingyou

    2015-01-01

    Fundamental and applied studies of silkworms have entered the functional genomics era. Here, we report a multi-gene expression system (MGES) based on 2A self-cleaving peptide (2A), which regulates the simultaneous expression and cleavage of multiple gene targets in the silk gland of transgenic silkworms. First, a glycine-serine-glycine spacer (GSG) was found to significantly improve the cleavage efficiency of 2A. Then, the cleavage efficiency of six types of 2As with GSG was analyzed. The shortest porcine teschovirus-1 2A (P2A-GSG) exhibited the highest cleavage efficiency in all insect cell lines that we tested. Next, P2A-GSG successfully cleaved the artificial human serum albumin (66 kDa) linked with human acidic fibroblast growth factor (20.2 kDa) fusion genes and vitellogenin receptor fragment (196 kD) of silkworm linked with EGFP fusion genes, importantly, vitellogenin receptor protein was secreted to the outside of cells. Furthermore, P2A-GSG successfully mediated the simultaneous expression and cleavage of a DsRed and EGFP fusion gene in silk glands and caused secretion into the cocoon of transgenic silkworms using our sericin1 expression system. We predicted that the MGES would be an efficient tool for gene function research and innovative research on various functional silk materials in medicine, cosmetics, and other biomedical areas. PMID:26537835

  5. Inhibition of Bcl-2 expression by a novel tumor-specific RNA interference system increases chemosensitivity to 5-fluorouracil in Hela cells

    Institute of Scientific and Technical Information of China (English)

    Sheng-lin HUANG; Yi WU; Hai YU; Ping ZHANG; Xing-qian ZHANG; Lei YING; Han-fang ZHAO

    2006-01-01

    Aim: RNA interference (RNAi) has been proposed as a potential treatment for cancer, but the lack of cellular targets limits its use in cancer gene therapy. No current technology has achieved direct tumor-specific gene silencing using RNAi.In the present study we attempt to develop a tumor-specific RNAi system using the human telomerase reverse transcriptase (hTERT) promoter; furthermore, we analyzed its inhibitive effect on Bcl-2 expression. Methods: The vectors containing a small hairpin RNA (shRNA) to target exogenous reporters [firefly luciferase and enhanced green fluorescent protein (EGFP)] and endogenous gene (Bcl-2)were constructed. Luciferase expression was determined by dual luciferase assay.Reverse transcription-polymerase chain reaction (RT-PCR), fluorescence microscopy and fluorescence-activated cell sorting (FACS) were used to measure EGFP expression. Inhibition of Bcl-2 was evaluated by RT-PCR and Western blotting.Cell proliferation and viability were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. FACS was used to analyze the cell cycle distribution profile. Results: We showed that with the hTERT promoter directly driving shRNA transcription, expression of the exogenous reporters (LUC and EGFP) in tumor cells, but not normal cells, was specifically inhibited in vitro. The hTERT promoter-driven shRNA also depressed the expression of Bcl-2. Inhibition of Bcl-2 did not affect cell proliferation, but increased the chemosensitivity of HeLa cells to 5-fluorouracil. Conclusion: The present study describes an efficient RNAi system for gene silencing that is specific to tumor cells using the hTERT promoter. Suppression of Bcl-2 by using this system sensitized HeLa cells to 5-fluorouracil. This system may be useful for RNAi therapy.

  6. RNA干扰技术抑制SGIV ICP18绿色荧光融合蛋白表达的研究%The Inhibition of Expression of SGIV ICP18-GFP in FHM Cell by RNA Interferencing

    Institute of Scientific and Technical Information of China (English)

    夏立群; 张红莲; 梁海鹰; 秦启伟

    2011-01-01

    Singapore grouper iridovirus (SGIV) is a major pathogen resulting in heavy economic losses to grouper aquaculture. In this study, recombant eukaryotic vector pEGFP-ICP18 which inserted with SGIV ICP18 gene was transfected into Fathead minnow (FHM) cells, and ICP18-GFP fusion protein was successfully expressed in FHM cells with a finely punctate cytoplasmic pattern. Candidate siRNA targeting SGIV ICP18 gene (siRNA-ICP18) was designed and chemically synthesized. To investigate the inhibition effect of siRNA-ICP18, pEGFP-ICP18 and siRNA were co-transfected into FHM cells, and the green fluorescence was observed by fluorescence microscope after transfection. The green fluorescence in FHM cells co-transfected with pEGFP-ICP18 and siRNA-ICP18 were 60% ~ 80% fewer than that of negative control, which show the siRNA-ICP18 can effectively silence the extrinsic SGIV ICP18 gene in FHM cells during 24 ~48 h after transfection.%将含有新加坡石斑鱼虹彩病毒(Singapore gmuper iridovirus,SGIV)ICP18基因的真核表达载体pEGFP-ICPl8转染到胖头鲤细胞(Fathead minnow cells,FHM)中进行融合表达,用荧光显微镜观察到ICP18-GFP融合蛋白呈点状分布于FHM细胞的细胞质中.根据SGIV ICPl8的序列,设计并体外化学合成了特异性干扰SGIVICP18的siRNA(siRNA-ICP18),与pEGFP-ICP18共转染到FHM细胞中,通过荧光显微镜观察不同时间点的荧光强度变化.与序列非特异性siRNA(siRNA-negative)阴性对照相比,转染后24-48 h,共转染siRNA-ICP18和pEGFP-ICP18的实验细胞中发荧光的细胞数量较阴性对照少60%-80%左右,说明体外化学合成的siRNA-ICP18可有效抑制FHM细胞中外源导入SGIV ICP18基因的表达.

  7. Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy.

    Science.gov (United States)

    Duwé, Sam; De Zitter, Elke; Gielen, Vincent; Moeyaert, Benjamien; Vandenberg, Wim; Grotjohann, Tim; Clays, Koen; Jakobs, Stefan; Van Meervelt, Luc; Dedecker, Peter

    2015-10-27

    "Smart fluorophores", such as reversibly switchable fluorescent proteins, are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available, and many display reduced biological performance compared to more classical variants. We present the development of robustly photoswitchable variants of enhanced green fluorescent protein (EGFP), named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37 °C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multimodal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of ∼70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the "on"- and "off"-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded "smart fluorophores" can be readily optimized for biological performance and provides a practical strategy for developing maturation- and stability-enhanced photochromic fluorescent proteins. PMID:26308583

  8. Construction, characterization and expression of full length cDNA clone of sheep YAP1 gene.

    Science.gov (United States)

    Sun, Wei; Li, Da; Su, Rui; Musa, Hassan H; Chen, Ling; Zhou, Hong

    2014-02-01

    RT-PCR, 5'RACE, 3'RACE were used to clone sheep full length cDNA sequence of YAP1 (Yes-associated protein 1), eukaryotic expression plasmid and a mutant that cannot be phosphorylated at Ser42 was successfully constructed. The amino acid sequence analysis revealed that sheep YAP1 gene encoded water-soluble protein and its relative molecular weight and isoelectric point was 44,079.0 Da and 4.91, respectively. Sub-cellular localization of YAP1 was in the nucleus, it is hydrophilic non-transmembrane and non-secreted protein. YAP1 protein contained 33 phosphorylation sites, seven glycosylation sites and two WW domains. The secondary structure of YAP1 was mainly composed of random coil, while the tertiary structure of domain area showed a forniciform helix structure. YAP1 gene was expressed in different tissues, the highest expression was in kidney and the lowest was in hypothalamus. The CDS of sheep YAP1was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, cloned into pMD19-T simple vector by T/A ligation. YAP1 coding region was further sub-cloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR, restriction enzyme and sequencing were used to confirm the recombinant plasmid. The sheep full-length YAP1 cDNA sequence is 1712 in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine by PCR, restriction digestion and sequencing. The result showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, this will help for further studies on the YAP1 protein expression and its biological activities. PMID:24381103

  9. Inhibition of HIV-1 Integrase gene expression by 10-23 DNAzyme

    Indian Academy of Sciences (India)

    Nirpendra Singh; Atul Ranjan; Souvik Sur; Ramesh Chandra; Vibha Tandon

    2012-07-01

    HIV Integrase (IN) is an enzyme that is responsible for the integration of the proviral genome into the human genome, and this integration step is the first step of the virus hijacking the human cell machinery for its propagation and replication. 10-23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. We have designed three novel DNAzymes, DIN54, DIN116, and DIN152, against HIV-1 Integrase gene using Mfold software and evaluated them for target site cleavage activity on the in vitro transcribed mRNA. All DNAzymes were tested for its inhibition of expression of HIV Integrase protein in the transiently transfected cell lines. DIN116 and DIN152 inhibited IN-EGFP expression by 80% and 70% respectively.

  10. Construction of a bicistronic recombinant adenoviral vector for human interleukin-10 and enhanced green fluorescent protein expression in bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    LIN Jian-qing; LIN Cai-zhu; LIN Xian-zhong; ZENG Kai; GAO You-guang

    2012-01-01

    Background Human interleukin-10 (hlL-10) is a cytokine synthesis inhibitory factor,which is involved in various immune responses.The purpose of this study was to construct an adenoviral vector carrying the hlL-10 gene for expression of biologically active hlL-10 in rat bone marrow mesenchymal stem cells (rMSCs).Methods A pSNAV2.0-hlL10 plasmid was used as a template to obtain a hlL-10 cDNA fragment that was subcloned by restriction enzyme digestion and ligation into a pDC316-IRES-EGFP-lacZ alpha plasmid carrying an enhanced green fluorescent protein (EGFP) marker gene.The pDC316-hlL-10-IRES-EGFP plasmid was linearized by Pmel digestion and used to transfect HEK293 packaging cells using the adenovirus packaging system AdMax.Virus particles were amplified by repeatedly infecting HEK293 cells with the seed virus and then purified by ion exchange.After the number of virus particles and titer was determined,rMSCs were infected with the adenoviral vector.The infection rate was determined by fluorescence microscopy and flow cytometry,and hlL-10 protein expression in rMSCs was measured by Western blotting.Results The virus particle concentration,OD260/280 value and virus titer of the amplified and purified recombinant adenovirus were 3.2×1011 VP/ml,approximately 2.0,and 1.1×1010 TCID50/ml,respectively.Bright green fluorescence was observed by fluorescence microscopy and flow cytometry in the recombinant adenovirus-infected rMSCs.GFP expression was considered the multiplicity of infection (MOI) and was time-dependent.The infection rate was 92.9% at 100 MOI.Conclusions A bicistrenic recombinant adenoviral vector for hlL-10 and EGFP gene expression were successfully constructed.The infection rate of rMSCs by the adenovirus was high (92.9% at 100 MOI) and the target gene hlL-10 was highly expressed in cells.The present study provides an experimental basis for further research of immunosuppressive therapy using hlL-10.The expression level of hlL-10 protein as detected by

  11. Establishment and Identification of Chinese Hamster Ovary Cell Lines with Stable Expression of Soluble CD40 Ligands

    Directory of Open Access Journals (Sweden)

    JIANG Hua-wei

    2014-09-01

    Full Text Available Objective: To establish the Chinese Hamster Ovary (CHO cell lines with stable expression of soluble CD40 ligands (sCD40L. Methods: Recombinant plasmid pIRES2-EGFP-sCD40L, enzyme digestion and sequencing identification were obtained by cloning sCD40L coding sequences into eukaryotic expression vector pIRES2-EGFP from carrier pDC316-sCD40 containing sCD40L. CHO cells were transfected by electroporation, followed by screening of resistant clones with G418, after which monoclones were obtained by limited dilution assay and multiply cultured. Flow cytometer and reverted fluorescence microscope were applied to observe the expression of green fluorescent protein, while sCD40L expression was detected by polymerase chain reaction (PCR, reverse transcription-polymerase chain reaction (RT-PCR and enzyme-linked immunosorbent assay (ELISA from aspects of deoxyribose nucleic acid (DNA, messenger ribonucleic acid (mRNA and protein, respectively. CHO-sCD40L was cultured together with MDA-MB-231 cells to compare the expression changes of surface molecule fatty acid synthase (Fas by flow cytometer and observe the apoptosis of MDA-MB-231 cells after Fas activated antibodies (CH-11 were added 24 h later. Results: Plasmid pIRES2-EGFP-sCD40L was successfully established, and cell lines with stable expression of sCD40L were obtained with cloned culture after CHO cell transfection, which was named as B11. Flow cytometer and reverted fluorescence microscope showed >90% expression of green fluorescent protein, while PCR, RT-PCR and ELISA suggested integration of sCD40L genes into cell genome DNA, transcription of sCD40L mRNA and sCD40L protein expression being (4.5±2.1 ng/mL in the supernatant of cell culture, respectively. After co-culture of B11 and MDA-MB-231 cells, the surface Fas expression of MDA-MB-231 cells was increased from (3±1.02 % to (34.8±8.75%, while the apoptosis rate 24 h after addition of CH11 from (5.4±1.32% to (20.7±5.24%, and the differences

  12. Transfection and expression of exogenous gene in laying hens oviduct in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    GAO Bo; SUN Huai-chang; SONG Cheng-yi; WANG Zhi-yue; CHEN Qin; SONG Hong-qin

    2005-01-01

    To examine whether or not the regulatory sequence of chicken ovalbumin gene can drive transgene expression specifically in hen oviduct, the authors constructed an oviduct-specific expression vector (pOV), containing 3.0 kilobases (kb) of the 5'-flanking sequence and 3.0 kb of the 3'-flanking sequence of the chicken ovalbumin gene. Jellyfish green fluorescence protein (EGFP) reporter gene and bacterial LacZ reporter gene were respectively inserted into the downstream of the 5'-regulatory region.The recombinants were named as pOVEGFP and pOVLacZ. Two transfer systems, in vitro and in vivo, were used to verify the function of the vector. In vitro, the plasmid DNA pOVEGFP and pEGFP-N1 were transfected respectively by the polyethyleneimine procedure into the primary chicken oviduct epithelium (PCOE) and fibroblasts cells isolated from laying hens. In vivo, the recombinant vector pOVLacZ was injected into egg-laying hens via wing vein and the tissues were collected for RT-PCR analysis.The results showed that expression of pEGFP-N1 was achieved at low level in oviduct epithelial cells and at high level in fibroblasts, but that the recombinant vector was not expressed in both cells. RT-PCR analysis showed that the LacZ gene was transcribed in the oviduct, but not in the heart, liver, kidney and spleen of the injected hens. Accordingly, the ?-galactosidase activity was only detected in the oviduct magnum (116.7 mU/ml) and eggs (16.47 mU/ml). These results indicated that the cloned regulation regions of chicken ovalbumin gene could drive exogenous gene expression specifically in the oviducts of hens. In vivo gene injection via wing vein may serve as a rapid production system of recombinant proteins in chicken eggs. In addition, the cultured primary oviduct cells from laying hens were not efficient temporary expression systems for analyzing the function of regulating elements of ovalbumin gene.

  13. Identification of the subgenomic promoter of the coat protein gene of cucumber fruit mottle mosaic virus and development of a heterologous expression vector.

    Science.gov (United States)

    Rhee, Sun-Ju; Jang, Yoon Jeong; Lee, Gung Pyo

    2016-06-01

    Heterologous gene expression using plant virus vectors enables research on host-virus interactions and the production of useful proteins, but the host range of plant viruses limits the practical applications of such vectors. Here, we aimed to develop a viral vector based on cucumber fruit mottle mosaic virus (CFMMV), a member of the genus Tobamovirus, whose members infect cucurbits. The subgenomic promoter (SGP) in the coat protein (CP) gene, which was used to drive heterologous expression, was mapped by analyzing deletion mutants from a CaMV 35S promoter-driven infectious CFMMV clone. The region from nucleotides (nt) -55 to +160 relative to the start codon of the open reading frame (ORF) of CP was found to be a fully active promoter, and the region from nt -55 to +100 was identified as the active core promoter. Based on these SGPs, we constructed a cloning site in the CFMMV vector and successfully expressed enhanced green fluorescent protein (EGFP) in Nicotiana benthamiana and watermelon (Citrullus lanatus). Co-inoculation with the P19 suppressor increased EGFP expression and viral replication by blocking degradation of the viral genome. Our CFMMV vector will be useful as an expression vector in cucurbits. PMID:26976138

  14. Effects of XPD and P53 on Proliferation of HepG2.2.15 and Expression of Hepatitis B Virus X Protein%XPD和P53对HepG2.2.15增殖及乙型肝炎病毒X蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    丁浩; 张吉翔

    2012-01-01

    Objective: To investigate effects of xeroderma pigmentosum D (XPD) and P53 on the proliferation of HepC2.2.1S, human hepatoma cells and the expressions of hepatitis B virus X protein (HBx), Bcl-2 and Bax. Methods: Re-combinant plasmid pEGFP-N2/XPD and vacant vector plasmid pEGFP-N2 were transfected into HepG2.2.15 by liposome. On the next day, these cells were incubated with Pifithrin-a (P53 inhibitor) at a 20 (xmol/L concentration for 24 h. Cells were divided into five groups, blank control group, pEGFP-N2 group, pEGFP-N2/XPD group, pEGFP-N2/XPD + Pifithrin-a group and Pifithrin-a group. The expressions of XPD, HBx, Bcl-2 and Bax were detected by RT-PCR and Western blotting. The cell proliferation was detected by MTT. The cell cycles were examined with flow cytometry. Results: (1) The expression of XPD increased by transfection of pEGFP-N2/XPD (P < 0.001). The increased XPD expression down-regulated the expressions of HBx and Bcl-2, and up-regulated the expression of Bax, while Pifithrin-a abolished the above effects of XPD(P < 0.01, respectively). (2) MTT results showed that the increased XPD expression inhibited the cell proliferation. This inhibition induced by XPD can be inhibited by Pifithrin-a (P < 0.001). (3) Flow cytometry results showed that the up-regulated XPD expression increased the cell number of Gi phase and decreased that of S phase, while the effect of XPD on cell cycle was abolished by Pifithrin-a (P < 0.001, respectively). Conclusion: XPD can suppress the proliferation of hepatoma carcinoma cells, down-regulate the expression of HBx and Bcl-2, and up-regulate the expression of Bax through P53 pathway. There may be mutual influences among XPD, P53 and HBx that co-regulate hepatocarcmogenes.s.%目的:探讨人着色性干皮病基因D(XPD)和P53对人肝癌细胞株HepG2.2.15增殖及乙型肝炎病毒X蛋白(HBx)、Bcl-2和Bax基因表达的影响.方法:用脂质体转染法将重组质粒pEGFP-N2/XPD和空载质粒pEGFP-N2转染HepG2.2.15

  15. Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI

    Directory of Open Access Journals (Sweden)

    Margison Geoffrey P

    2006-03-01

    Full Text Available Abstract Background A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES sequence of encephalomyocarditis virus (EMCV and/or foot-and-mouth disease virus (FMDV cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP, O6-methylguanine-DNA-methyltransferase (MGMT, and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. Results All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector. Conclusion The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.

  16. Modified gateway system for double shRNA expression and Cre/lox based gene expression

    Directory of Open Access Journals (Sweden)

    Leung Lisa

    2011-03-01

    Full Text Available Abstract Background The growing need for functional studies of genes has set the stage for the development of versatile tools for genetic manipulations. Results Aiming to provide tools for high throughput analysis of gene functions, we have developed a modified short hairpin RNA (shRNA and gene expression system based on Gateway Technology. The system contains a series of entry and destination vectors that enables easy transfer of shRNA or cDNA into lentiviral expression systems with a variety of selection or marker genes (i.e. puromycin, hygromycin, green fluorescent protein-EGFP, yellow fluorescent protein-YFP and red fluorescent protein-dsRed2. Our shRNA entry vector pENTR.hU6.hH1 containing two tandem human shRNA expression promoters, H1 and U6, was capable of co-expressing two shRNA sequences simultaneously. The entry vector for gene overexpression, pENTR.CMV.ON was constructed to contain CMV promoter with a multiple cloning site flanked by loxP sites allowing for subsequent Cre/lox recombination. Both shRNA and cDNA expression vectors also contained attL sites necessary for recombination with attR sites in our destination expression vectors. As proof of principle we demonstrate the functionality and efficiency of this system by testing expression of several cDNA and shRNA sequences in a number of cell lines. Conclusion Our system is a valuable addition to already existing library of Gateway based vectors and can be an essential tool for many aspects of gene functional studies.

  17. Potent and specific inhibition of SARS-CoV antigen expression by RNA interference

    Institute of Scientific and Technical Information of China (English)

    TAO Peng; ZHANG Jun; TANG Ni; ZHANG Bing-qiang; HE Tong-chuan; HUANG Ai-long

    2005-01-01

    Background Severe acute respiratory syndrome (SARS) is an infectious disease caused by SARS-CoV. There are no effective antiviral drugs for SARS although the epidemic of SARS was controlled. The aim of this study was to develop an RNAi (RNA interference) approach that specifically targeted the N gene sequence of severe acute respiratory syndrome associated coronavirus (SARS-CoV) by synthesizing short hairpin RNA (shRNA) in vivo, and to assess the inhibitory effect of this shRNA on SARS-CoV N antigen expression. Methods The eukaryotic expression plasmid pEGFP-C1-N, containing SARS-CoV N gene, was co-transfected into 293 cells with either the RNAi plasmid pshRNA-N or unrelated control plasmid pshRNA-HBV-C4. At 24, 48 and 72 hours post transfection, the green fluorescence was observed through a fluorescence microscope. The RNA levels of SARS-CoV N were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression of Green Fluorescent Protein (GFP) and protein N were detected using Western blot.Results The vector, pshRNA-N expressing shRNA which targeted the N gene of SARS-CoV, was successfully constructed. The introduction of RNAi plasmid efficiently and specifically inhibited the synthesis of protein N. RT-PCR showed that RNAs of N gene were clearly reduced when the pEGFP-C1-N was cotransfected with pshRNA-N, whereas the control vector did not exhibit inhibitory effect on N gene transcription.Conclusions Our results demonstrate that RNAi mediated silencing of SARS-CoV gene could effectively inhibit expression of SARS-CoV antigen, hence RNAi based strategy should be further explored as a more efficacious antiviral therapy of SARS-CoV infection.

  18. Enhanced gene expression promoted by hybrid magnetic/cationic block copolymer micelles.

    Science.gov (United States)

    Haladjova, E; Rangelov, S; Tsvetanov, Ch B; Posheva, V; Peycheva, E; Maximova, V; Momekova, D; Mountrichas, G; Pispas, S; Bakandritsos, A

    2014-07-15

    We report on novel gene delivery vector systems based on hybrid polymer-magnetic micelles. The hybrid micelles were prepared by codissolution of hydrophobically surface modified iron oxide and amphiphilic polystyrene-b-poly(quaternized 2-vinylpyridine) block copolymer (PS-b-P2QVP) in organic solvent. After extensive dialysis against water, micelles with positively charged hydrophilic corona of PQVP and hydrophobic PS core were prepared, in which magnetic nanoparticles were randomly distributed. The hybrid micelles were used to form complexes with linear (salmon sperm, 2000 bp, corresponding to M(w) of 1.32 × 10(6) Da) and plasmid (pEGFP-N1, 4730 bp, corresponding to M(w) of 3.12 × 10(6) Da) DNA. The resulting magnetopolyplexes of phosphate:amine (P/N) ratios in the 0.05-20 range were characterized by light scattering, ζ-potential measurements, and transmission electron microscopy as well as cytotoxicity and gel retardation assays. The investigated systems displayed a narrow size distribution, particle dimensions below 360 nm, whereas their ζ-potential values varied from positive to negative depending of the P/N ratio. The resulting vector nanosystems exhibited low toxicity. They were able to introduce pEGFP-N1 molecules into the cells. The application of a magnetic field markedly boosted the transgene expression efficiency of the magnetopolyplexes, which was even superior to those of commercial transfectants such as Lipofectamine and dendritic polyethylenimine. PMID:24945823

  19. Abundant GFP expression and LTP in hippocampal acute slices by in vivo injection of sindbis virus.

    Science.gov (United States)

    D'Apuzzo, M; Mandolesi, G; Reis, G; Schuman, E M

    2001-08-01

    Virus-mediated gene transfer into neurons is a powerful tool for the analysis of neuronal structure and function. Recombinant sindbis virus has been previously used to study protein function in hippocampal neuron cultures as well as in hippocampal organotypic slice cultures. Nevertheless, some concern still exists about the physiological relevance of these cultured preparations. Acute hippocampal slices are a widely used preparation for the study of synaptic transmission, but currently recombinant gene delivery is usually achieved only through time-consuming transgenic techniques. In this study, we show that a subregion of the CA1 area in acute hippocampal slices can be specifically altered to express a gene of interest. A sindbis virus vector carrying an enhanced green fluorescent protein (EGFP) reporter was injected in vivo into the hippocampus of adult rats. After 18 h, rats were killed, and acute hippocampal slices, infected in the CA1 field, were analyzed morphologically and electrophysiologically. Infected slices showed healthy and stable electrophysiological responses as well as long-term potentiation. In addition, infected pyramidal cells were readily recognized in living slices by two-photon imaging. Specifically, the introduction of an EGFP-Actin fusion protein greatly enhanced the detection of fine processes and dendritic spines. We propose this technique as an efficient tool for studying gene function in adult hippocampal neurons. PMID:11495971

  20. Transgenic Expression of a Single Transcription Factor Pdx1 Induces Transdifferentiation of Pancreatic Acinar Cells to Endocrine Cells in Adult Mice.

    Science.gov (United States)

    Miyazaki, Satsuki; Tashiro, Fumi; Miyazaki, Jun-Ichi

    2016-01-01

    A promising approach to new diabetes therapies is to generate β cells from other differentiated pancreatic cells in vivo. Because the acinar cells represent the most abundant cell type in the pancreas, an attractive possibility is to reprogram acinar cells into β cells. The transcription factor Pdx1 (Pancreas/duodenum homeobox protein 1) is essential for pancreatic development and cell lineage determination. Our objective is to examine whether exogenous expression of Pdx1 in acinar cells of adult mice might induce reprogramming of acinar cells into β cells. We established a transgenic mouse line in which Pdx1 and EGFP (enhanced green fluorescent protein) could be inducibly expressed in the acinar cells. After induction of Pdx1, we followed the acinar cells for their expression of exocrine and endocrine markers using cell-lineage tracing with EGFP. The acinar cell-specific expression of Pdx1 in adult mice reprogrammed the acinar cells as endocrine precursor cells, which migrated into the pancreatic islets and differentiated into insulin-, somatostatin-, or PP (pancreatic polypeptide)-producing endocrine cells, but not into glucagon-producing cells. When the mice undergoing such pancreatic reprogramming were treated with streptozotocin (STZ), the newly generated insulin-producing cells were able to ameliorate STZ-induced diabetes. This paradigm of in vivo reprogramming indicates that acinar cells hold promise as a source for new islet cells in regenerative therapies for diabetes. PMID:27526291

  1. Transgenic Expression of a Single Transcription Factor Pdx1 Induces Transdifferentiation of Pancreatic Acinar Cells to Endocrine Cells in Adult Mice

    Science.gov (United States)

    Miyazaki, Satsuki; Tashiro, Fumi; Miyazaki, Jun-ichi

    2016-01-01

    A promising approach to new diabetes therapies is to generate β cells from other differentiated pancreatic cells in vivo. Because the acinar cells represent the most abundant cell type in the pancreas, an attractive possibility is to reprogram acinar cells into β cells. The transcription factor Pdx1 (Pancreas/duodenum homeobox protein 1) is essential for pancreatic development and cell lineage determination. Our objective is to examine whether exogenous expression of Pdx1 in acinar cells of adult mice might induce reprogramming of acinar cells into β cells. We established a transgenic mouse line in which Pdx1 and EGFP (enhanced green fluorescent protein) could be inducibly expressed in the acinar cells. After induction of Pdx1, we followed the acinar cells for their expression of exocrine and endocrine markers using cell-lineage tracing with EGFP. The acinar cell-specific expression of Pdx1 in adult mice reprogrammed the acinar cells as endocrine precursor cells, which migrated into the pancreatic islets and differentiated into insulin-, somatostatin-, or PP (pancreatic polypeptide)-producing endocrine cells, but not into glucagon-producing cells. When the mice undergoing such pancreatic reprogramming were treated with streptozotocin (STZ), the newly generated insulin-producing cells were able to ameliorate STZ-induced diabetes. This paradigm of in vivo reprogramming indicates that acinar cells hold promise as a source for new islet cells in regenerative therapies for diabetes. PMID:27526291

  2. Feline Neural Progenitor Cells II: Use of Novel Plasmid Vector and Hybrid Promoter to Drive Expression of Glial Cell Line-Derived Neurotrophic Factor Transgene

    Directory of Open Access Journals (Sweden)

    X. Joann You

    2012-01-01

    Full Text Available Sustained transgene expression is required for the success of cell transplant-based gene therapy. Most widely used are lentiviral-based vectors which integrate into the host genome and thereby maintain sustained transgene expression. This requires integration into the nuclear genome, and potential risks include activation of oncogenes and inactivation of tumor suppressor genes. Plasmids have been used; however lack of sustained expression presents an additional challenge. Here we used the pCAG-PyF101-eGFP plasmid to deliver the human GDNF gene to cat neural progenitor cells (cNPCs. This vector consists of a CAGG composite promoter linked to the polyoma virus mutant enhancer PyF101. Expression of an episomal eGFP reporter and GDNF transgene were stably maintained by the cells, even following induction of differentiation. These genetically modified cells appear suitable for use in allogeneic models of cell-based delivery of GDNF in the cat and may find veterinary applications should such strategies prove clinically beneficial.

  3. Regulation of ADAM12 cell-surface expression by protein kinase C epsilon

    DEFF Research Database (Denmark)

    Sundberg, Christina; Thodeti, Charles Kumar; Kveiborg, Marie;

    2004-01-01

    constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell......The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin......-immunoprecipitated from membrane-enriched fractions of PMA-treated cells, 3) RD cells transfected with EGFP-tagged, myristoylated PKCepsilon expressed more ADAM12 at the cell surface than did non-transfected cells, and 4) RD cells transfected with a kinase-inactive PKCepsilon mutant did not exhibit ADAM12 cell...

  4. Endoplasmic reticulum stress response in an INS-1 pancreatic β-cell line with inducible expression of a folding-deficient proinsulin

    Directory of Open Access Journals (Sweden)

    Lai Elida

    2010-07-01

    Full Text Available Abstract Background Cells respond to endoplasmic reticulum stress (ER stress by activating the unfolded protein response. To study the ER stress response in pancreatic β-cells we developed a model system that allows for pathophysiological ER stress based on the Akita mouse. This mouse strain expresses a mutant insulin 2 gene (C96Y, which prevents normal proinsulin folding causing ER stress and eventual β-cell apoptosis. A double-stable pancreatic β-cell line (pTet-ON INS-1 with inducible expression of insulin 2 (C96Y fused to EGFP was generated to study the ER stress response. Results Expression of Ins 2 (C96Y-EGFP resulted in activation of the ER stress pathways (PERK, IRE1 and ATF6 and caused dilation of the ER. To identify gene expression changes resulting from mutant insulin expression we performed microarray expression profiling and real time PCR experiments. We observed an induction of various ER chaperone, co-chaperone and ER-associated degradation genes after 24 h and an increase in pro-apoptotic genes (Chop and Trib3 following 48 h of mutant insulin expression. The latter changes occurred at a time when general apoptosis was detected in the cell population, although the relative amount of cell death was low. Inhibiting the proteasome or depleting Herp protein expression increased mutant insulin levels and enhanced cell apoptosis, indicating that ER-associated degradation is maintaining cell survival. Conclusions The inducible mutant insulin expressing cell model has allowed for the identification of the ER stress response in β-cells and the repertoire of genes/proteins induced is unique to this cell type. ER-associated degradation is essential in maintaining cell survival in cells expressing mutant insulin. This cell model will be useful for the molecular characterization of ER stress-induced genes.

  5. Upregulation of human telomerase reverse transcriptase mRNA expression by in vitro transfection of hepatitis B virus X gene into human hepatocarcinoma and cholangiocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Zhen-Liang Qu; Sheng-Quan Zou; Nai-Qiang Cui; Xian-Zhong Wu; Ming-Fang Qin; Di Kong; Zhen-Li Zhou

    2005-01-01

    AIM: To study the changes of human telomerase reverse transcriptase (hTERT) mRNA expression in human hepatocarcinoma cell lines (HepG2) and cholangiocarcinoma cell lines (QBC939) after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis.METHODS: HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein (EGFP) coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency.Cells were harvested and total RNA was extracted using TRIzol() reagent. The expression of hTERT mRNA in HepG2and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting.RESULTS: Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector.Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939cells only when transfected with HBx gene.CONCLUSION: HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection.

  6. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent

    2010-06-01

    Full Text Available Abstract Background Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro. Results In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP regeneration systems: creatine phosphate (CP, phosphoenolpyruvate (PEP, and 3-phosphoglyceric acid (3-PGA. The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture. Conclusions Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma

  7. Construction, expression and activity detection of the recombinant plasmids of acid-sensing ion channels genes%酸敏感离子通道基因重组质粒的构建、表达及其活性测定

    Institute of Scientific and Technical Information of China (English)

    苏敬敬; 董强

    2009-01-01

    To construct the recombinant plasmids containing the full length cDNA of mouse ASICi a and ASIC2a genes and to express ASICI a and ASIC2a fusion proteins with biological activity, the full length eDNA of mouse ASICI a and ASIC2a genes were obtained by RT-PCR and cloned into the eukaryotic expression vector pEGFP-N3 respectively in the present study. Then, recombinant plasmids pEGFP-ASIC1a and pEGFP-ASIC2a were constructed. After identified by DNA sequencing,the recombinant plasmids were transfected into Chinese hamster ovary (CHO) cells respectively with lipofectamine reagent. The distribution of the fusion protein expression in the cells was detected by fluorescence microscope and Western-blot after transfection. After the celts wth recombinant plasmids were acidified, cell viability and LDH release were determined to evaluate the biological activity of fusion protein. Our results showed that the full length cDNA of mouse ASIC1a and ASIC2a genes had been cloned into the vector pEGFP-N3 and transfected into CHO cells. Obvious green fluorescence was de-tected around CHO cell membrane with recombinant plasmids. Western blot showed that ASIC1a and ASIC2a fusion proteins were respec-tively expressed at about 90 kD and 88 kD. Compared with pH7.4-treated cells, cell viability was reduced and LDH release increased sig-nificantly in the acidified ceils with recombinant plasmids, which could be inhibited by ASICs blockers. The present results suggest that the recombinant plasmids containing the full length cDNA of mouse pEGFP-AS1C1a and pEGFP-ASIC2a genes were successfully construc-ted and expressed with biological activity in CHO cells.%为了构建含有小鼠酸敏感离子通道(ASIC1a、ASIC2a)基因全长eDNA的重组质粒,并表达有生物学活性的ASIC1a和ASIC2a融合蛋白,本研究通过逆转录聚合酶链反应(RT-PCR)分别获得小鼠ASIC1a和ASIC2a全长cDNA,并将其克隆人真核表达载体pEGFP-N3中,构建含小鼠全长ASIC1a和ASIC2a基因的重组质粒pEGFP

  8. Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Mehdi Sharifi Tabar

    2015-10-01

    Full Text Available Objective: Genetic modification of human embryonic stem cells (hESCs is critical for their extensive use as a fundamental tool for cell therapy and basic research. Despite the fact that various methods such as lipofection and electroporation have been applied to transfer the gene of interest (GOI into the target cell line, however, there are few reports that compare all parameters, which influence transfection efficiency. In this study, we examine all parameters that affect the efficiency of electroporation and lipofection for transient and long-term gene expression in three different cell lines to introduce the best method and determinant factor. Materials and Methods: In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for transient expression of green fluorescent protein in two genetically different hESC lines, Royan H5 (XX and Royan H6 (XY, as well as human foreskin fibroblasts (hFF. For long-term EGFP expression VASA and OLIG2 promoters (germ cell and motoneuron specific genes, respectively, were isolated and subsequently cloned into a pBluMAR5 plasmid backbone to drive EGFP expression. Flow cytometry analysis was performed two days after transfection to determine transient expression efficiency. Differentiation of drug resistant hESC colonies toward primordial germ cells (PGCs was conducted to confirm stable integration of the transgene. Results: Transient and stable expression suggested a variable potential for different cell lines against transfection. Analysis of parameters that influenced gene transformation efficiency revealed that the vector concentrations from 20-60 μg and the density of the subjected cells (5×105 and 1×106 cells were not as effective as the genetic background and voltage rate. The present data indicated that in contrast to the circular form, the linearized vector generated more distinctive drug resistant colonies. Conclusion

  9. Expression of transgenes targeted to the Gt(ROSA26Sor locus is orientation dependent.

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    Douglas Strathdee

    Full Text Available BACKGROUND: Targeting transgenes to a chosen location in the genome has a number of advantages. A single copy of the DNA construct can be inserted by targeting into regions of chromatin that allow the desired developmental and tissue-specific expression of the transgene. METHODOLOGY: In order to develop a reliable system for reproducibly expressing transgenes it was decided to insert constructs at the Gt(ROSA26Sor locus. A cytomegalovirus (CMV promoter was used to drive expression of the Tetracycline (tet transcriptional activator, rtTA2(s-M2, and test the effectiveness of using the ROSA26 locus to allow transgene expression. The tet operator construct was inserted into one allele of ROSA26 and a tet responder construct controlling expression of EGFP was inserted into the other allele. CONCLUSIONS: Expression of the targeted transgenes was shown to be affected by both the presence of selectable marker cassettes and by the orientation of the transgenes with respect to the endogenous ROSA26 promoter. These results suggest that transcriptional interference from the endogenous gene promoter or from promoters in the selectable marker cassettes may be affecting transgene expression at the locus. Additionally we have been able to determine the optimal orientation for transgene expression at the ROSA26 locus.

  10. Expression of GFP in tumor cells and fluorescent examination by confocal microscope

    Science.gov (United States)

    Jin, Ying; Xing, Da; Xu, Chaoyang

    2002-04-01

    The green fluorescent protein (GFP), from the bioluminescent jellyfish Aequorea victoria, yields a bright green fluorescence when expressed in either eukaryotic or prokaryotic cells and illuminated by blue or UV light. The characteristic properties of GFP make this protein a good candidate for use as a molecular reporter to monitor patterns of protein localization, gene expression, and intracellular protein trafficking in living cells. In this study, the plasmid EGFP encoding GFP was used to transfect SWO cells (a cancer cell line of nerve gelatinous tissue) mediated by liposome: (1) The plasmid EGFP-C1, purchased from Clontech Co., propagated in suitable E. coli strain (JM 109), was extracted by Concert High Purity Plasmid Miniprep (Gibco). (2) SWO was cultured in RPMI 1640 (10% FCS and 25 mM HEPES), 37 degree(s)C, 5% CO2. Cancer cells were transfected in 6-cm tissue culture dishes by Lipofectin Reagent (Gibco) for 6-12 hr using 2 ug DNA. (3) Then, infected cells were collected in medium containing 800 ug/ml G418, and the resistant clones were harvested and subcloned in fresh culture medium maintaining 800 ug/ml G418. (4) The cells were examined by using Nikon fluorescent microscope (E600) and Bio-Rad confocal microscope (MRC 600). (5) Next step, the cancer cells, stably expressing GFP after in vivo transduction, were implanted by surgical orthotopic implantation (SOI) in nude mice. Tracking of these cancer cells will become more sensitive and rapid than the traditional procedure of histopathological examination or immunohistochemistry. This method demonstrates external, noninvasive, whole-body, real-time fluorescence optical imaging of internally growing tumors and metastases in transplanted animals.

  11. Establishment of A Malignant Pleural Effusion Mouse Model with Lewis Lung 
Carcinoma Cell Lines Expressing Enhanced Green Fluorescent Protein

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    Xingqun MA

    2012-06-01

    Full Text Available Background and objective Malignant pleural effusion (MPE is a poor prognosis factor in patients with advanced lung cancer. The aim of this study is to establish a mouse model of MPE using Lewis lung carcinoma (LLC cell lines expressing enhanced green fluorescent protein (EGFP. Methods The mouse model was created by injecting LLC-EGFP cells directly into the pleural cavity of mice that were sacrificed periodically. The dynamic growth and metastasis of tumor cells were screened using in vivo fluorescence imaging. The remaining mice were subjected to transverse computed tomography (CT imaging periodically to analyze the formation rate of pleural effusion. The survival rate and tumor metastasis were also observed. Pleural fluid was gently aspirated using a 1 mL syringe and its volume was measured. When two or more mice bore pleural effusion at the same time, we calculated the average volume. The correlation of pleural effusion with the integrated optical density (IOD were analyzed. Results Four days after the inoculation of LLC-EGFP cells, green fluorescence was observed by opening the chest wall. The tumor formation rate was 100%, and the IOD gradually increased after inoculation. The metastasis sites were mediastinal, and the hilar lymph nodes were contralateral pleural as well as pericardial. The metastasis rates were 87%, 73% and 20%, respectively. The CT scan revealed that the formation rates of pleural effusion on days 7, 14 and 21 were 13%, 46% and 53%, respectively. The average volume of pleural effusion increased obviously on day 10 and peaked on day 16 with a value of 0.5 mL. The mean survival time of nude mice was 28.8 days. The volume of pleural effusion and IOD were significantly correlated (r=0.91, P<0.000,1. Conclusion A mouse model of lung cancer malignant pleural effusion was successfully established by injecting LLC lines expressing EGFP into the pleural cavity under a microscope. The model can enable dynamic observations of the

  12. High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.

    Science.gov (United States)

    Zhang, Ningning; Gonzalez, Maria; Savary, Brett; Xu, Jianfeng

    2016-03-01

    Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (∼7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures. PMID:26627201

  13. Recombinant protein expression by targeting pre-selected chromosomal loci

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    Krömer Wolfgang

    2009-12-01

    Full Text Available Abstract Background Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs. Results We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs were targeted by Flp recombinase mediated cassette exchange (RMCE. The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context

  14. Tobamoviral movement protein transiently expressed in a single epidermal cell functions beyond multiple plasmodesmata and spreads multicellularly in an infection-coupled manner.

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    Tamai, A; Meshi, T

    2001-02-01

    Cell-to-cell movement of a plant virus requires expression of the movement protein (MP). It has not been fully elucidated, however, how the MP functions in primary infected cells. With the use of a microprojectile bombardment-mediated DNA infection system for Tomato mosaic virus (ToMV), we found that the cotransfected ToMV MP gene exerts its effects in the initially infected cells and in their surrounding cells to achieve multicellular spread of movement-defective ToMV. Five other tobamoviral MPs examined also transcomplemented the movement-defective phenotype of ToMV, but the Cucumber mosaic virus 3a MP did not. Together with the cell-to-cell movement of the mutant virus, a fusion between the MP and an enhanced green fluorescent protein variant (EGFP) expressed in trans was distributed multicellularly and localized primarily in plasmodesmata between infected cells. In contrast, in noninfected sites the MP-EGFP fusion accumulated predominantly inside the bombarded cells as irregularly shaped aggregates, and only a minute amount of the fusion was found in plasmodesmata. Thus, the behavior of ToMV MP is greatly modulated in the presence of a replicating virus and it is highly likely that the MP spreads in the infection sites, coordinating with the cell-to-cell movement of the viral genome. PMID:11204775

  15. Functional Characterization of the Mannitol Promoter of Pseudomonas fluorescens DSM 50106 and Its Application for a Mannitol-Inducible Expression System for Pseudomonas putida KT2440.

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    Jana Hoffmann

    Full Text Available A new pBBR1MCS-2-derived vector containing the Pseudomonas fluorescens DSM10506 mannitol promoter PmtlE and mtlR encoding its AraC/XylS type transcriptional activator was constructed and optimized for low basal expression. Mannitol, arabitol, and glucitol-inducible gene expression was demonstrated with Pseudomonas putida and eGFP as reporter gene. The new vector was applied for functional characterization of PmtlE. Identification of the DNA binding site of MtlR was achieved by in vivo eGFP measurement with PmtlE wild type and mutants thereof. Moreover, purified MtlR was applied for detailed in vitro investigations using electrophoretic mobility shift assays and DNaseI footprinting experiments. The obtained data suggest that MtlR binds to PmtlE as a dimer. The proposed DNA binding site of MtlR is AGTGC-N5-AGTAT-N7-AGTGC-N5-AGGAT. The transcription activation mechanism includes two binding sites with different binding affinities, a strong upstream binding site and a weaker downstream binding site. The presence of the weak downstream binding site was shown to be necessary to sustain mannitol-inducibility of PmtlE. Two possible functions of mannitol are discussed; the effector might stabilize binding of the second monomer to the downstream half site or promote transcription activation by inducing a conformational change of the regulator that influences the contact to the RNA polymerase.

  16. Generation and characterization of transgenic mice expressing mitochondrial targeted red fluorescent protein selectively in neurons: modeling mitochondriopathy in excitotoxicity and amyotrophic lateral sclerosis

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    Wang Yi

    2011-11-01

    Full Text Available Abstract Background Mitochondria have roles or appear to have roles in the pathogenesis of several chronic age-related and acute neurological disorders, including Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis, Parkinson's disease, and cerebral ischemia, and could be critical targets for development of rational mechanism-based, disease-modifying therapeutics for treating these disorders effectively. A deeper understanding of neural tissue mitochondria pathobiologies as definitive mediators of neural injury, disease, and cell death merits further study, and the development of additional tools to study neural mitochondria will help achieve this unmet need. Results We created transgenic mice that express the coral (Discosoma sp. red fluorescent protein DsRed2 specifically in mitochondria of neurons using a construct engineered with a Thy1 promoter, specific for neuron expression, to drive expression of a fusion protein of DsRed2 with a mitochondrial targeting sequence. The biochemical and histological characterization of these mice shows the expression of mitochondrial-targeted DsRed2 to be specific for mitochondria and concentrated in distinct CNS regions, including cerebral cortex, hippocampus, thalamus, brainstem, and spinal cord. Red fluorescent mitochondria were visualized in cerebral cortical and hippocampal pyramidal neurons, ventrobasal thalamic neurons, subthalamic neurons, and spinal motor neurons. For the purpose of proof of principle application, these mice were used in excitotoxicity paradigms and double transgenic mice were generated by crossing Thy1-mitoDsRed2 mice with transgenic mice expressing enhanced-GFP (eGFP under the control of the Hlxb9 promoter that drives eGFP expression specifically in motor neurons and by crossing Thy1-mitoDsRed2 mice to amyotrophic lateral sclerosis (ALS mice expressing human mutant superoxide dismutase-1. Conclusions These novel transgenic mice will be a useful tool for better understanding

  17. Construction of eukaryotic expression vector and expression of stage-specific gene T314 from newborn larvae of Trichinella spiralis%旋毛虫新生幼虫期特异性T314基因真核表达质粒的构建及表达

    Institute of Scientific and Technical Information of China (English)

    于建立; 白雪; 吴秀萍; 刘明远

    2012-01-01

    目的 构建旋毛虫(Trichinella spirais)新生幼虫期特异性T314基因真核表达质粒,并在BHK细胞中进行表达.方法 从旋毛虫新生幼虫RNA中通过RT-PCR技术扩增无信号肽T314全长基因,定向克隆至真核表达载体pEGFP-N1中,构建重组真核表达质粒T314-pEGFP-N1,脂质体法转染BHK细胞,荧光显微镜观察EGFP的表达,Western blot检测T314融合蛋白的表达.结果 重组真核表达质粒T314-pEGFP-N1经双酶切及测序证实构建正确;转染的BHK细胞48 h转染效率最高;表达的T314融合蛋白可与旋毛虫感染的猪血清发生特异性反应.结论 已成功构建了T314基因重组真核表达质粒T314-pEGFP-N1,并在BHK细胞中融合表达,为进一步研究旋毛虫包囊形成机制奠定了基础.%Objective To construct a eukaryotic expression vector for stage-specific gene T314 from newborn larvae of Trichinella spiralis and express in BHK cells. Methods Signal peptide-free full-length T314 gene was amplified from RNA of newborn larvae of T. Spiralis by RT-PCR and cloned into eukaryotic expression vector Pegfp-Nl. BHK cells were transfected with the constructed recombinant plasmid T314-Pegfp-Nl in mediation of Hposome, then observed for expression of EGFP by fluorescent microscopy, and for that of T314 fusion protein by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid T314-Pegfp-Nl was constructed correctly. The transfection efficacy of BHK cells reached the maximum 48 h after transfection. The expressed T314 fusion protein showed specific reaction with porcine serum infected with T. Spiralis. Conclusion The recombinant eukaryotic expression vector T314-Pegfp-Nl for T314 gene was successfully constructed, and fusion protein was expressed in BHK cells, which laid a foundation of further study on mechanism of nurse cell formation of T. Spiralis.

  18. 人雌激素受体β绿色荧光蛋白真核表达载体的构建与表达%Construction of ERβ green fluorescent protein eukaryotic expression vector and its expression

    Institute of Scientific and Technical Information of China (English)

    李慧; 安莲效; 郭静; 顾月清

    2011-01-01

    目的 构建人雌激素受体β(EBβ)绿色荧光蛋白(GFP)真核表达载体,并稳定转染乳腺癌MCF-7细胞.方法 通过双酶切将pCMV5-hERβ中的人ERβ cDNA克隆到pEGFP-C3中,构建并鉴定重组质粒pEGFP-hERβ,然后转染乳腺癌MCF-7细胞,采用G418(500 μg/mL)筛选出稳定转染的细胞系,在荧光显微镜下观察融合蛋白质在真核细胞内的表达分布,并采用逆转录-聚合酶链反应(RT-PCR)检测ERβ的mRNA转录水平.结果 成功构建重组表达载体pEGFP-hERβ,有效转染并筛选出稳定转染ERβ基因的MCF-7细胞系,外源性ERβ基因在MCF-7细胞中获得了有效转录,并在细胞质和细胞核中均广泛分布.结论 建立了稳定表达ERβ的MCF-7细胞系,为筛选ERβ调节荆和进一步研究ERβ在乳腺癌中的作用及其机制奠定基础.%Purpose To construct a estrogen receptor β (ERβ) green fluorescent protein eukaryotic expression vector and to transfect stably MCF-7 breast cancer cells.Methods The cDNA of human ERβgene was cloned into pEGFP-C3 from pCMV5-hERβ by double digestion to create recombinant plasmid pEGFP-hERβ.The recombinant plasmid was identified and transfected into MCF-7 cells.Then stable transfectants were selected by G418.The expression and localization of ERβ in MCF-7 cells were assayed by fluorescence microscopy.The transcription of ERβ in the stably transfected cells was also examined by RT-PCR.Results The ERβ green fluorescent protein eukaryotic expressio vector was successfully constructed and effectively transfected into MCF-7 cells.Then the stably ERβ-transfected MCF-7 cell line was screened.The transcription of exogenous ERβ in transfected cells was confirmed, and the ERβ was abroad distributed in the cytoplasm and nucleus.Conclusion The stably ERβ-transfected MCF-7 cell line was established.It has provided an important basis for screening ERβ modulators and furtherly investigating the involvement of ERβ in breast cancer as well as its

  19. Regulation of endothelial-specific transgene expression by the LacI repressor protein in vivo.

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    Susan K Morton

    Full Text Available Genetically modified mice have played an important part in elucidating gene function in vivo. However, conclusions from transgenic studies may be compromised by complications arising from the site of transgene integration into the genome and, in inducible systems, the non-innocuous nature of inducer molecules. The aim of the present study was to use the vascular system to validate a technique based on the bacterial lac operon system, in which transgene expression can be repressed and de-repressed by an innocuous lactose analogue, IPTG. We have modified an endothelium specific promoter (TIE2 with synthetic LacO sequences and made transgenic mouse lines with this modified promoter driving expression of mutant forms of connexin40 and an independently translated reporter, EGFP. We show that tissue specificity of this modified promoter is retained in the vasculature of transgenic mice in spite of the presence of LacO sequences, and that transgene expression is uniform throughout the endothelium of a range of adult systemic and cerebral arteries and arterioles. Moreover, transgene expression can be consistently down-regulated by crossing the transgenic mice with mice expressing an inhibitor protein LacI(R, and in one transgenic line, transgene expression could be de-repressed rapidly by the innocuous inducer, IPTG. We conclude that the modified bacterial lac operon system can be used successfully to validate transgenic phenotypes through a simple breeding schedule with mice homozygous for the LacI(R protein.

  20. Cloning and expression of human papilloma virus type 6b-L1 gene

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    Lie-hua DENG

    2011-03-01

    Full Text Available Objective To investigate the expression of green fluorescent protein plasmid of human papilloma virus 6b L1 gene(HPV6bL1 in eukaryotic cells.Methods The L1 gene of PQE40-HPV6bL1 was amplified by PCR,purified by restriction enzyme digestion,and then connected to eukaryotic expression plasmid PEGFP-C1.The recombinant expression vector was then transformed into E.coli DH5a,which was identified by BamH Ⅰ and Hand Ⅲ digestion and the positive vector was selected.The recombinant plasmid PEGFP-HPV6bL1 was transfected into COS-7 cells by liposomal transfection technique and the expression of fusion protein was observed under fluorescence microscope.The generation of HPV6bL1 mRNA was detected by RT-PCR.Results Identification of PEGFP-HPV6bL1 by enzyme digestion and sequencing showed that the length,direction and inserted location of target,which was inserted into the recombinant,was correct and the expression of EGFP in transfected cell was observed.Conclusions A new type of green fluorescent HPV6bL1 eukaryotic expression system has been established.It may provide a research foundation for the study of the protein.

  1. Serial bone marrow transplantation reveals in vivo expression of the pCLPG retroviral vector

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    Fratini Paula

    2010-01-01

    Full Text Available Abstract Background Gene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied. Our group has developed a retroviral vector where transgene expression is controlled by p53 with the intention of harnessing the dynamic and inducible nature of this tumor suppressor and transcription factor. We present here a test of in vivo expression provided by the p53-responsive vector, pCLPG. For this, we used a model of serial transplantation of transduced bone marrow cells. Results We observed, by flow cytometry, that the eGFP transgene was expressed at higher levels when the pCLPG vector was used as compared to the parental pCL retrovirus, where expression is directed by the native MoMLV LTR. Expression from the pCLPG vector was longer lasting, but did decay along with each sequential transplant. The detection of eGFP-positive cells containing either vector was successful only in the bone marrow compartment and was not observed in peripheral blood, spleen or thymus. Conclusions These findings indicate that the p53-responsive pCLPG retrovirus did offer expression in vivo and at a level that surpassed the non-modified, parental pCL vector. Our results indicate that the pCLPG platform may provide some advantages when applied in the hematopoietic system.

  2. Minicircle DNA Provides Enhanced and Prolonged Transgene Expression Following Airway Gene Transfer.

    Science.gov (United States)

    Munye, Mustafa M; Tagalakis, Aristides D; Barnes, Josephine L; Brown, Rachel E; McAnulty, Robin J; Howe, Steven J; Hart, Stephen L

    2016-01-01

    Gene therapy for cystic fibrosis using non-viral, plasmid-based formulations has been the subject of intensive research for over two decades but a clinically viable product has yet to materialise in large part due to inefficient transgene expression. Minicircle DNA give enhanced and more persistent transgene expression compared to plasmid DNA in a number of organ systems but has not been assessed in the lung. In this study we compared minicircle DNA with plasmid DNA in transfections of airway epithelial cells. In vitro, luciferase gene expression from minicircles was 5-10-fold higher than with plasmid DNA. In eGFP transfections in vitro both the mean fluorescence intensity and percentage of cells transfected was 2-4-fold higher with minicircle DNA. Administration of equimolar amounts of DNA to mouse lungs resulted in a reduced inflammatory response and more persistent transgene expression, with luciferase activity persisting for 2 weeks from minicircle DNA compared to plasmid formulations. Transfection of equal mass amounts of DNA in mouse lungs resulted in a 6-fold increase in transgene expression in addition to more persistent transgene expression. Our findings have clear implications for gene therapy of airway disorders where plasmid DNA transfections have so far proven inefficient in clinical trials. PMID:26975732

  3. Identification and Characterization of a Rat Novel Gene RSEP4 Expressed Specifically in Central Nervous System

    Institute of Scientific and Technical Information of China (English)

    Xi-Dao WANG; Ling-Wei KONG; Zhi-Qin XIE; Yu-Qiu ZHANG; Zhi-Xin LIN; Zhi-Qi ZHAO; Lei YU; Nai-He JING

    2004-01-01

    The low-abundantly expressed genes composed the majorities of the mRNAs expressed in the central nervous system (CNS), and were thought to be important for the normal brain functions. Through differential screening a low-abundance cDNA sublibrary with mRNA from neuropathic pain of chronic constriction injury (CCI) model, we have identified a novel rat gene, rat spinal-cord expression protein 4 gene (RSEP4). The total length ofRSEP4 cDNA is 2006 bp, with a 501 nucleotide open reading frame (ORF) that encodes a 167 amino acid polypeptide. Northern blot revealed that RSEP4 was expressed specifically in the CNS. In situ hybridization showed that the mRNA of RSEP4 was strongly expressed in the CA1, CA2, CA3 and DG regions of hippocampus, the Purkinje cells of cerebellum, and the small sensory neurons of dorsal horn and large motor neurons of ventral horn of spinal cord. Over-expression of RSEP4-EGFP fusion protein in the human embryonic kidney 293T cells showed that RSEP4 protein was mainly localized in the cell cytoplasm. These results suggest that RSEP4 may play some roles in the CNS.

  4. Promoter-dependent expression of the fungal transporter HcPT1.1 under Pi shortage and its spatial localization in ectomycorrhiza.

    Science.gov (United States)

    Garcia, Kevin; Haider, Muhammad Zulqurnain; Delteil, Amandine; Corratgé-Faillie, Claire; Conéjero, Geneviève; Tatry, Marie-Violaine; Becquer, Adeline; Amenc, Laurie; Sentenac, Hervé; Plassard, Claude; Zimmermann, Sabine

    2013-01-01

    Mycorrhizal exchange of nutrients between fungi and host plants involves a specialization and polarization of the fungal plasma membrane adapted for the uptake from the soil and for secretion of nutrient ions towards root cells. In addition to the current progress in identification of membrane transport systems of both symbiotic partners, data concerning the transcriptional and translational regulation of these proteins are needed to elucidate their role for symbiotic functions. To answer whether the formerly described Pi-dependent expression of the phosphate transporter HcPT1.1 from Hebeloma cylindrosporum is the result of its promoter activity, we introduced promoter-EGFP fusion constructs in the fungus by Agrotransformation. Indeed, HcPT1.1 expression in pure fungal cultures quantified and visualized by EGFP under control of the HcPT1.1 promoter was dependent on external Pi concentrations, low Pi stimulating the expression. Furthermore, to study expression and localization of the phosphate transporter HcPT1.1 in symbiotic conditions, presence of transcripts and proteins was analyzed by the in situ hybridization technique as well as by immunostaining of proteins. In ectomycorrhiza, expression of the phosphate transporter was clearly enhanced by Pi-shortage indicating its role in Pi nutrition in the symbiotic association. Transcripts were detected in external hyphae and in the hyphal mantle, proteins in addition also within the Hartig net. Exploiting the transformable fungus H. cylindrosporum, Pi-dependent expression of the fungal transporter HcPT1.1 as result from its promoter activity as well as transcript and protein localization in ectomycorrhizal symbiosis are shown. PMID:23850603

  5. A simple, highly efficient method for heterologous expression in mammalian primary neurons using cationic lipid-mediated mRNA transfection

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    Damian J Williams

    2010-11-01

    Full Text Available Expression of heterologous proteins in adult mammalian neurons is a valuable technique for the study of neuronal function. The postmitotic nature of mature neurons prevents effective DNA transfection using simple, cationic lipid-based methods. Adequate heterologous protein expression is often only achievable using complex techniques that, in many cases, are associated with substantial toxicity. Here, a simple method for high efficiency transfection of mammalian primary neurons using in vitro-transcribed mRNA and the cationic lipid transfection reagent Lipofectamine 2000 is described. Optimal transfection conditions were established in adult mouse dissociated dorsal root ganglion (DRG neurons using a 96-well based luciferase activity assay. Using these conditions, a transfection efficiency of 25% was achieved in DRG neurons transfected with EGFP mRNA. High transfection efficiencies were also obtained in dissociated rat superior cervical ganglion (SCG neurons and mouse cortical and hippocampal cultures. Endogenous Ca2+ currents in EGFP mRNA-transfected SCG neurons were not significantly different from untransfected neurons, which suggested that this technique is well suited for heterologous expression in patch clamp recording experiments. Functional expression of a cannabinoid receptor (CB1R, a G protein inwardly-rectifying K+ channel (GIRK4 and a dominant-negative G protein α-subunit mutant (GoA G203T indicate that the levels of heterologous protein expression attainable using mRNA transfection are suitable for most functional protein studies. This study demonstrates that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research.

  6. Expression of porcine CFL2b gene in C2C12 cells and its effect on the expression of MyHC

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    ZHAO Wei

    2008-12-01

    Full Text Available Porcine CFL2b gene is expressed mainly in skeletal muscle, it affects muscle development and myofibrillar formation. In order to study the relationship between CFL2b gene and muscle fiber trait, stable C2C12 cell clones groups expressing porcine CFL2b gene were obtained by directed cloning and gene transfection technology. The expression of pEGFP-N1–CFL2b were detected using GFP fluorescence and Western Blotting. The expression level of myosin heavy chain (MyHC isoforms (2x.2b and slow in C2C12 were determined using Real-time PCR. Result showed that MyHC 2x gene and MyHC 2b genes were obviously up-regulated, MyHC1/slow gene not significant. The research indicated porcine CFL2b gene correlated to muscle fiber trait. And it was associated with pork quality. Porcine CFL2b gene can possible be regarded as a candidate gene for pork quality [Acta Zoologica Sinica 54(6: 1014–1019, 2008].

  7. Clone and expression of human transferrin receptor gene: a marker gene for magnetic resonance imaging

    International Nuclear Information System (INIS)

    Objective: To clone human transferrin receptor (hTfR) gene and construct expression vector producing recombination protein. Methods: Human transferrin receptor gene cDNA was amplified by RT-PCR from human embryonic liver and lung tissue. Recombinant pcDNA3-hTfR and pEGFP-Cl-hTfR plasmids were constructed and confirmed by DNA sequencing. These plasmids were stably transfected into the HEK293 cells. The protein expression in vitro was confirmed by Western Blot. The efficiency of expression and the location of hTfR were also investigated by fluorescence microscopy and confocal fluorescence microscopy. Results: The full length cDNA of hTfR gene (2332 bp) was cloned and sequenced. The hTfR (190 000) was overexpressed in transfected HEK293 cells by Western blot analysis. Fluorescence micrographs displayed that the hTfR was expressed at high level and located predominantly in the cell surface. Conclusions: Human transferrin receptor (hTfR) gene has been successfully cloned and obtained high-level expression in HEK293 cells, and the recombination protein of hTfR distributed predominantly in the cell membrane. (authors)

  8. 'Fluorescent Cell Chip' for immunotoxicity testing: Development of the c-fos expression reporter cell lines

    International Nuclear Information System (INIS)

    The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes-macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals

  9. Inducible expression of photoacoustic reporter gene tyrosinase in cells using a single plasmid

    Science.gov (United States)

    Paproski, Robert J.; Zemp, Roger J.

    2012-02-01

    We have previously demonstrated that tyrosinase is a reporter gene for photoacoustic imaging since tyrosinase is the rate-limiting step in the synthesis of melanin, a pigment capable of producing strong photoacoustic signals. We previously created a cell line capable of inducible tyrosinase expression (important due to toxicity of melanin) by stably transfecting tyrosinase in MCF-7 Tet-OnR cell line (Clontech) which expresses a doxycycline-controlled transactivator. Unfortunately, Clontech provides few Tet-On Advanced cell lines making it difficult to have inducible tyrosinase expression in cell lines not provided by Clontech. In order to simplify the creation of cell lines with inducible expression of tyrosinase, we created a single plasmid that encodes both the transactivator as well as tyrosinase. PCR was used to amplify both the transactivator and tyrosinase from the Tet-OnR Advanced and pTRE-Tight-TYR plasmids, respectively. Both PCR products were cloned into the pEGFP-N1 plasmid and the newly created plasmid was transfected into ZR-75-1, MCF-7, and MIA PaCa-1 cells using lipofectamine. After several days, brown melanin was only observed in cells incubated with doxycycline, suggesting that the newly created single plasmid allowed inducible tyrosinase expression in many different cells lines.

  10. Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation

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    Moen Ingrid

    2012-01-01

    Full Text Available Abstract Background The tumor microenvironment is pivotal in tumor progression. Thus, we aimed to develop a mammary tumor model to elucidate molecular characteristics in the stroma versus the tumor cell compartment by global gene expression. Secondly, since tumor hypoxia influences several aspects of tumor pathophysiology, we hypothesized that hyperoxia might have an inhibitory effect on tumor growth per se. Finally, we aimed to identify differences in gene expression and key molecular mechanisms, both in the native state and following treatment. Methods 4T1 dsRed breast cancer cells were injected into eGFP expressing NOD/SCID mice. Group 1 was exposed to 3 intermittent HBO treatments (Day 1, 4 and 7, Group 2 to 7 daily HBO treatments (both 2.5bar, 100% O2, à 90 min, whereas the controls were exposed to a normal atmosphere. Tumor growth, histology, vascularisation, cell proliferation, cell death and metastasis were assessed. Fluorescence-activated cell sorting was used to separate tumor cells from stromal cells prior to gene expression analysis. Results The purity of sorted cells was verified by fluorescence microscopy. Gene expression profiling demonstrated that highly expressed genes in the untreated tumor stroma included constituents of the extracellular matrix and matrix metalloproteinases. Tumor growth was significantly inhibited by HBO, and the MAPK pathway was found to be significantly reduced. Immunohistochemistry indicated a significantly reduced microvessel density after intermittent HBO, whereas daily HBO did not show a similar effect. The anti-angiogenic response was reflected in the expression trends of angiogenic factors. Conclusions The present in vivo mammary tumor model enabled us to separate tumor and stromal cells, and demonstrated that the two compartments are characterized by distinct gene expressions, both in the native state and following HBO treatments. Furthermore, hyperoxia induced a significant tumor growth

  11. Gene expression in tumor cells and stroma in dsRed 4T1 tumors in eGFP-expressing mice with and without enhanced oxygenation

    International Nuclear Information System (INIS)

    The tumor microenvironment is pivotal in tumor progression. Thus, we aimed to develop a mammary tumor model to elucidate molecular characteristics in the stroma versus the tumor cell compartment by global gene expression. Secondly, since tumor hypoxia influences several aspects of tumor pathophysiology, we hypothesized that hyperoxia might have an inhibitory effect on tumor growth per se. Finally, we aimed to identify differences in gene expression and key molecular mechanisms, both in the native state and following treatment. 4T1 dsRed breast cancer cells were injected into eGFP expressing NOD/SCID mice. Group 1 was exposed to 3 intermittent HBO treatments (Day 1, 4 and 7), Group 2 to 7 daily HBO treatments (both 2.5bar, 100% O2, à 90 min), whereas the controls were exposed to a normal atmosphere. Tumor growth, histology, vascularisation, cell proliferation, cell death and metastasis were assessed. Fluorescence-activated cell sorting was used to separate tumor cells from stromal cells prior to gene expression analysis. The purity of sorted cells was verified by fluorescence microscopy. Gene expression profiling demonstrated that highly expressed genes in the untreated tumor stroma included constituents of the extracellular matrix and matrix metalloproteinases. Tumor growth was significantly inhibited by HBO, and the MAPK pathway was found to be significantly reduced. Immunohistochemistry indicated a significantly reduced microvessel density after intermittent HBO, whereas daily HBO did not show a similar effect. The anti-angiogenic response was reflected in the expression trends of angiogenic factors. The present in vivo mammary tumor model enabled us to separate tumor and stromal cells, and demonstrated that the two compartments are characterized by distinct gene expressions, both in the native state and following HBO treatments. Furthermore, hyperoxia induced a significant tumor growth-inhibitory effect, with significant down-regulation of the MAPK pathway. An

  12. An Entry/Gateway® cloning system for general expression of genes with molecular tags in Drosophila melanogaster

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    Eyer Katie

    2009-01-01

    Full Text Available Abstract Background Tagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. However, over-expression of fusion proteins sometimes leads to the unwanted lethality or developmental defects. Therefore, vectors that can express tagged proteins at physiological levels are desirable tools for studying dosage-sensitive proteins. We developed a set of Entry/Gateway® vectors for expressing fluorescent fusion proteins in Drosophila melanogaster. The vectors were used to generate fluorescent CP190 which is a component of the gypsy chromatin insulator. We used the fluorescent CP190 to study the dynamic movement of related chromatin insulators in living cells. Results The Entry/Gateway® system is a timesaving technique for quickly generating expression constructs of tagged fusion proteins. We described in this study an Entry/Gateway® based system, which includes six P-element destination vectors (P-DEST for expressing tagged proteins (eGFP, mRFP, or myc in Drosophila melanogaster and a TA-based cloning vector for generating entry clones from unstable DNA sequences. We used the P-DEST vectors to express fluorecent CP190 at tolerable levels. Expression of CP190 using the UAS/Gal4 system, instead, led to either lethality or underdeveloped tissues. The expressed eGFP- or mRFP-tagged CP190 proteins are fully functional and rescued the lethality of the homozygous CP190 mutation. We visualized a wide range of CP190 distribution patterns in living cell nuclei, from thousands of tiny particles to less than ten giant ones, which likely reflects diverse organization of higher-order chromatin structures. We also visualized the fusion of multiple smaller insulator bodies into larger aggregates in living cells, which is likely reflective of the dynamic activities of reorganization of chromatin in living nuclei. Conclusion We have developed an efficient cloning system for expressing dosage-sensitive proteins in

  13. Easy expression of the C-terminal heavy chain domain of botulinum neurotoxin serotype A as a vaccine candidate using a bi-cistronic baculovirus system.

    Science.gov (United States)

    Villaflores, Oliver B; Hsei, Chein-Ming; Teng, Chao-Yi; Chen, Ying-Ju; Wey, Jiunn-Jye; Tsui, Pei-Yi; Shyu, Rong-Hwa; Tung, Kuo-Lun; Yeh, Jui-Ming; Chiao, Der-Jiang; Wu, Tzong-Yuan

    2013-04-01

    Clostridial botulinum neurotoxin (BoNT) is one of the most toxic proteins causing the food borne disease, botulism. In previous studies, recombinant BoNT production by Escherichia coli and yeast Pichia pastoris has been hampered by high AT content and codon bias in the gene encoding BoNT and required a synthetic gene to resolve this intrinsic bottleneck. This paper reports the simultaneous expression of the C-terminal heavy chain domain of BoNT (rBoNT/A-HC-6h) and enhanced green fluorescent protein (EGFP) using a bi-cistronic baculovirus-insect cell expression system. The expression of EGFP facilitated the monitoring of viral infection, virus titer determination, and isolation of the recombinant virus. Protein fusion with hexa-His-tag and one-step immobilized metal-ion affinity chromatography (IMAC) purification produced a homogenous, stable, and immunologically active 55-kDa rBoNT/A-HC-6h (about 3mg/L) with >90% purity. Furthermore, measured levels of serum titers were 8-folds for mice vaccinated with the purified rBoNT/A-HC-6h (2μg) than for mice administered with botulinum toxoid after initial immunization. Challenge experiment with botulinum A toxin demonstrated the immunoprotective activity of purified rBoNT/A-HC-6h providing the mice full protection against 10(2) LD50 botulinum A toxin with a dose as low as 0.2μg. This study provided supportive evidence for the use of a bi-cistronic baculovirus-Sf21 insect cell expression system in the facile expression of an immunogenically active rBoNT/A-HC. PMID:23313783

  14. Construction and expression of an optimized, novel human immunodeficiency virus type-1 lentiviral vector containing green fluorescent protein

    Institute of Scientific and Technical Information of China (English)

    Xia Li; Xueling Ma; Lijing Zhao; Hang Gao; Hongjuan Wang; Li Du; Juan Wang; Nan Li; Kangding Liu

    2011-01-01

    The human immunodeficiency virus (HIV) lentiviral vector is an ideal vector for gene therapy. In the present study, the wild-type HIV-1 genome was segregated into four plasmids, and an optimized novel HIV-1 lentiviral vector containing green fluorescent protein and vesicular stomatitis virus G pseudo-capsule was constructed. The plasmids were pHR-CMV-EGFP, pCMVΔ8.9, pRSV-Rev, pCMV-VSV-G. The four plasmid system was co-transfected into 293T cells, and green fluorescent protein expression was observed. The present study obtained lentiviral particles by high-speed centrifugation, and the lentiviral particle titer was 4×10TU/mL after centrifugation. Thus, an optimized novel HIV-1 lentiviral vector was successfully constructed.

  15. Effect of overexpression of wild-type p53 on POLD1 expression and malignant cell behavior in human hepatocellular carcinoma cell line SMMC-7721%野生型p53对肝癌细胞POLD1基因表达及细胞恶性表型的影响

    Institute of Scientific and Technical Information of China (English)

    韦长元; 刘起理; 廖柳凤; 徐恒; 谭晓虹

    2011-01-01

    AIM: To investigate the impact of overexpres-sion of wild-type p53 on cell proliferation and malignant phenotype in human hepatocellular carcinoma cell line SMMC-7721 and to explore possible mechanism involved.METHODS: Enhanced green fluorescence protein gene-containing eukaryotic expressionplasmids expressing p53-specific small interfering RNA (shRNA) (p53-siRNA) or wild-type p53 (pEGFP-p53) were constructed and introduced into SMMC-7721 cells by Lipofection-2000-mediated transfection. Meanwhile, the pEGFP-Cl empty vector was also transfected into SMMC-7721 cells. Cell lines stably expressing p53-siRNA, pEGFP-p53 or pEGFP-Cl were screened in medium containing G418. After transfection, the expression of p53 and POLD1 mRNAs was detected by RT-PCR. The changes in malignant cell behavior were determined by cell growth curve determination and colony formation assay.RESULTS: Compared to control SMMC-7721 cells, p53 mRNA expression was increased and POLD1 gene expression was decreased in SMMC-7721 cells transfected with the plasmid carrying wild-type p53 gene, while p53 mRNA expression was reduced and POLD1 mRNA expression was increased in SMMC-7721 cells transfected with the plasmid carrying p53-siRNA. MTT results showed that cell growth rate was faster in SMMC-7721 cells transfected with the plasmid carrying p53-siRNA than in control SMMC-7721 cells, but was slower in SMMC-7721 cells transfected with the plasmid carrying wild-type p53 gene than in control cells. Colony formation assay showed that colony formation rate was lower in SMMC-7721 cells transfected with the plasmid carrying wild-type p53 gene than in control cells (38.1% vs 52.6%, P < 0.05), but was higher in cells tranfected with the plasmid carrying p53-siRNA than in control cells (72.6% vs 52.6%, P < 0.05). High expression of wild-type p53 inhibited POLD1 transcription and cell proliferation, while low expression of wild-type p53 promoted POLD1 transcription and cell proliferation.CONCLUSION: Wild-type p53

  16. Spatial distribution of D1R- and D2R-expressing medium-sized spiny neurons differs along the rostro-caudal axis of the mouse dorsal striatum.

    Science.gov (United States)

    Gangarossa, Giuseppe; Espallergues, Julie; Mailly, Philippe; De Bundel, Dimitri; de Kerchove d'Exaerde, Alban; Hervé, Denis; Girault, Jean-Antoine; Valjent, Emmanuel; Krieger, Patrik

    2013-01-01

    The striatum projection neurons are striatonigral and striatopallidal medium-sized spiny neurons (MSNs) that preferentially express D1 (D1R) and D2 (D2R) dopamine receptors, respectively. It is generally assumed that these neurons are physically intermingled, without cytoarchitectural organization although this has not been tested. To address this question we used BAC transgenic mice expressing enhanced green fluorescence (EGFP) under the control of Drd1a or Drd2 promoter and spatial point pattern statistics. We demonstrate that D1R- and D2R-expressing MSNs are randomly distributed in most of the dorsal striatum, whereas a specific region in the caudal striatum, adjacent to the GPe, lacks neurons expressing markers for indirect pathway neurons. This area comprises almost exclusively D1R-expressing MSNs. These neurons receive excitatory inputs from the primary auditory cortex and the medial geniculate thalamic nucleus and a rich dopamine innervation. This area contains cholinergic and GABAergic interneurons but apparently no D2R/A2aR modulation because no fluorescence was detected in the neuropil of Drd2-EGFP or Drd2-Cre, and Adora-Cre BAC transgenic mice crossed with reporter mice. This striatal area that expresses calbindin D28k, VGluT1 and 2, is poor in μ opiate receptors and preproenkephalin. Altogether, the differences observed in D1R-MSNs, D2R-MSNs, and interneurons densities, as well as the anatomical segregation of D1R- and D2R/A2aR-expressing MSNs suggest that there are regional differences in the organization of the striatum. PMID:23908605

  17. [Construction of PPENK-MIDGE-NLS gene vector and the expression in rat].

    Science.gov (United States)

    Chen, Xi; Xu, Xuemin; Peng, Xijuan; Jiang, Wei; Yao, Linong

    2015-02-01

    Increasing the production and secretion of endogenous opioid peptide by immune cell can significantly induce myocardial protective effects against ischemia-reperfusion injury. Gene therapy is promising to increase endogenous enkephalin (ENK). However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, gene recombination, oncogene activation, the production of antibacterial antibody and changes in physiological gene expression. Minimalistic immunologically defined gene expression (MIDGE) can overcome all the deficients of viral and plasmid vectors. The exon of rat's preproenkephalin (PPENK) gene was amplified by PCR and the fragments were cloned into pEGFP-N1 plasmids. The recombined plasmids were digested with enzymes to obtain a linear vector contained promoter, preproenkephalin gene, RNA stable sequences and oligodesoxy nucleotides (ODNs) added to both ends of the gene vector to protect gene vector from exonuclease degradation. A nuclear localization sequence (NLS) was attached to an ODN to ensure the effective transport to the nucleus and transgene expression. Flow cytometry, laser confocal microscopy and Western blotting demonstrated that PPENK-MIDGE-NLS can transfect leukocyte of rat in vivo, increase the expression of proenkephalin (PENK) in tissue, and the transfection efficiency depends on gene vector's dosage. These results indicate that PPENK-MIDGE-NLS could be an innovative method to protect and treatment of myocardial ischemia-reperfusion injury. PMID:26062347

  18. Ectopic expression of AID in a non-B cell line triggers A:T and G:C point mutations in non-replicating episomal vectors.

    Directory of Open Access Journals (Sweden)

    Tihana Jovanic

    Full Text Available Somatic hypermutation (SHM of immunoglobulin genes is currently viewed as a two step process initiated by the deamination of deoxycytidine (C to deoxyuridine (U, catalysed by the activation induced deaminase (AID. Phase 1 mutations arise from DNA replication across the uracil residue or the abasic site, generated by the uracil-DNA glycosylase, yielding transitions or transversions at G:C pairs. Phase 2 mutations result from the recognition of the U:G mismatch by the Msh2/Msh6 complex (MutS Homologue, followed by the excision of the mismatched nucleotide and the repair, by the low fidelity DNA polymerase eta, of the gap generated by the exonuclease I. These mutations are mainly focused at A:T pairs. Whereas in activated B cells both G:C and A:T pairs are equally targeted, ectopic expression of AID was shown to trigger only G:C mutations on a stably integrated reporter gene. Here we show that when using non-replicative episomal vectors containing a GFP gene, inactivated by the introduction of stop codons at various positions, a high level of EGFP positive cells was obtained after transient expression in Jurkat cells constitutively expressing AID. We show that mutations at G:C and A:T pairs are produced. EGFP positive cells are obtained in the absence of vector replication demonstrating that the mutations are dependent only on the mismatch repair (MMR pathway. This implies that the generation of phase 1 mutations is not a prerequisite for the expression of phase 2 mutations.

  19. Gene expression

    International Nuclear Information System (INIS)

    We prepared probes for isolating functional pieces of the metallothionein locus. The probes enabled a variety of experiments, eventually revealing two mechanisms for metallothionein gene expression, the order of the DNA coding units at the locus, and the location of the gene site in its chromosome. Once the switch regulating metallothionein synthesis was located, it could be joined by recombinant DNA methods to other, unrelated genes, then reintroduced into cells by gene-transfer techniques. The expression of these recombinant genes could then be induced by exposing the cells to Zn2+ or Cd2+. We would thus take advantage of the clearly defined switching properties of the metallothionein gene to manipulate the expression of other, perhaps normally constitutive, genes. Already, despite an incomplete understanding of how the regulatory switch of the metallothionein locus operates, such experiments have been performed successfully

  20. Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells.

    Science.gov (United States)

    Wang, X Y; Zhang, J H; Sun, Q L; Yao, Z Y; Deng, B G; Guo, W Y; Wang, L; Dong, W H; Wang, F; Zhao, C P; Wang, T Y

    2015-01-01

    Preliminary studies have suggested that a characteristic element of the matrix attachment region (MAR) in human interferon-β mediates the adhesion of vectors to Chinese hamster ovary (CHO) cells. In this study, we investigated if vector adhesion increased nerve growth factor (NGF) expression in CHO cells. The MAR characteristic element sequence of human interferon-β was inserted into the multiple-cloning site of the pEGFP-C1 vector. The target NGF gene was inserted upstream of the MAR characteristic element sequence to construct the MAR/NGF expression vector. The recombinant plasmid was transfected into CHO cells and stable monoclonal cells were selected using G418. NGF mRNA and protein expression was detected by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Plasmid reduction experiments were used to determine the state of transfected plasmid in mammalian cells. The insertion of MAR into the vector increased NGF expression levels in CHO cells (1.93- fold) compared to the control. The recombinant plasmid expressing the MAR sequence was digested into a linear space vector. The inserted MAR and NGF sequences were consistent with those inserted into the plasmid before recombination. Therefore, we concluded that the MAR characteristic element mediates vector adhesion to CHO cells and enhances the stability and efficiency of the target gene expression. PMID:26345852

  1. In-vitro Knock Down of VEGFR2 Expression Using Specific siRNA in the Culture Medium

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    Moslem Jafarisani

    2015-09-01

    Full Text Available Introduction:The use of siRNAforsilencinggene expressionis expandingtoday. Knock down ofvascular endothelial growth factor is one of theobjectives ofthistechnology. In this study we aimed to inhibit the expression of receptor type II of VEGF (VEGFR-2 using specific siRNA in the culture medium.Methods: First, according to the target gene sequences, sequence of the specific siRNA were designed; blasted and built. Using RT-PCR, cDNA of HUVEC was synthesized and PCR with specific primers was reproduced. PEFGP-N1 expression vector was cloned with target gene and confirmed. Then Hela cells which has not any expression of the target genes were transfected whit cloned pEGFP-N1 vector using lipofectamin. GFP expression rate in the Hela cellsContaining initial vector and cloned vector in presence and absence of specific siRNA was assessed. Through evaluation of gene inhibition, decreasing of green fluorescence from GFP, RT-PCR was investigated.Results: The results of the two used siRNA, indicate reduced gene expression 56% and 63% in comparison with the control group (P<0.05.Conclusion: Transfection of specific VEGFR-2 siRNA using lipofectamin significantly inhibits expression of receptor. In fact,it cancut thesignal transduction pathwayand preventsthe creation ofnewblood vessels. Therefore, probably it can act as a new treatment factor for prevention or reduction of neovascularization.

  2. Baculovirus-mediated Expression of p35 Confers Resistance to Apoptosis in Human Embryo Kidney 293 cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Baculovirus has many advantages as vectors for gene transfer. We demonstrated that recombinant baculovirus vectors expressing p35 (Ac-CMV-p35) and eGFP (Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently. The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter. MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells. Cell growth curve showed the Ac-CMV-p35 and Ac- CMV-GFP transduced and non-transduced cells had similar proliferation rate, so baculovirus-mediated p35expression had no adverse effect on cell proliferation. In addition, baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D, UV or serum-free media. These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.

  3. Expression,purification and cell penetrativity of fusion protein PDT/GR-ΔLBD

    Directory of Open Access Journals (Sweden)

    Fang ZHANG

    2011-01-01

    Full Text Available Objective To construct the fusion gene expression vector of penetrating peptide(PDT and the glucocorticoid receptor lack of ligand binding domain(GR-ΔLBD,and evaluate the prokaryotic expression,purification and cell penetrativity of fusion protein PDT/GR-ΔLBD.Methods The target gene fragment GR-ΔLBD was obtained from plasmid pEGFP-GR-ΔLBD by double digestion,and sub-cloned into the prokaryotic expression vector pGEX-PDT to construct the fusion gene expression vector pGEX-PDT/GR-ΔLBD.PDT/GR-ΔLBD fusion protein was obtained after the expression vector was transformed into E.coli,followed by sequential induction with IPTG,treatment with glutathione-agarose resin and elution with glutathione.SDS-PAGE was performed to determine the expression of PDT/GR-ΔLBD fusion protein,and it which was diluted into a final concentration of 0,500 and 1000nmol/L,labeled with fluorescein FITC and co-cultivated with TC-1 cells for 2 hours,and the penetrativity was observed by fluorescence microscopy.Results The successfully constructed prokaryotic expression vector pPDT/GR-ΔLBD had the capacity of expressing protein,and it was 78.6kD in molecular weight,which was consistent with the theoretical value(80kD of the fusion protein PDT/GR-ΔLBD.PDT-GR-ΔLBD,penetrating the nuclear membrane in a concentration-dependent manner,was concentrated within nuclei.Conclusion PDT/GR-ΔLBD fusion protein,with good solubility and cell penetrativity,paves the way for further research on its anti-inflammatory effects.

  4. The Comparative and Functional Study between Two Construction Methods of shRNA Expression Vector Targeted LMP1 Gene Encoded by EBV

    Institute of Scientific and Technical Information of China (English)

    Yi-qin WANG; Yu-cheng YANG; Wen-lu ZHANG; Su-ling HONG

    2007-01-01

    To look for a more stable and convenient way of constructing short hairpin RNA expression vectors targeting the latent membrane protein-1(LMP-1) encoded by Epstein-Barr virus(pshLMP1), and to study the inhibition function of pshLMP1 expression vectors in HNE1 cells, we designed the pshLMP1 expression cassette and pshLMP1 expression vectors by both the annealing method and PCR method and then co-transfected with pEGFP-N1-1158 into HNE1 cells to observe the mRNA and protein levels of LMP-1 genes by green fluorescence analysis, RT-PCR and western blot. pshLMP1 expression vectors were successfully obtained by both methods but better cloning efficiency was achieved and fewer deletions and mutations of nucleotides were achieved with the PCR method. Furthermore, the mRNA and protein levels of LMP-1 genes were down-regulated by pshLMP1 expression vectors. According to our research, we found that the PCR method provides a more efficient way to construct pshLMP1 expression vectors which have the ability to inhibit the function of LMP-1 genes expressed in HNE1 cells, and also provides a novel application of RNA interference technology against-EBV.

  5. The construction and expression of AFP-enhanced green fluorescent protein recombinant and the analysis of measurement characteristics%甲胎蛋白-增强型绿色荧光蛋白重组体的构建表达及其测量特性分析

    Institute of Scientific and Technical Information of China (English)

    张健; 葛丹红; 王雪亮

    2014-01-01

    Objective To construct the fusion protein of alpha fetoprotein (AFP )-enhanced green fluorescent protein(eGFP)recombinant,and to analyze the stability and measurement characteristics.Methods The sequence of eGFP was amplified from pcDNA3.0 plasmid and inserted PET28a plasmid to construct expression plasmid PET28a-eGFP.The coding sequence of AFP was synthetized according to the coding sequence of AFP from the National Center for Biotechnology Information (NCBI)database,added and linked to eGFP sequence by a linker sequence to construct expression plasmid PET28a-eGFP-AFP.Recombinant protein eGFP and eGFP-AFP were purified,and the measurement characteristics and stability were analyzed.Results The expression plasmid of recombinant protein eGFP and eGFP-AFP were constructed,and the proteins were purified.Recombinant protein eGFP and eGFP-AFP had the same excitation spectrum and emission spectrum,which were 450 and 509 nm optimally,and its fluorescence could be stable over 1 2 months.eGFP-AFP could be tested for the level of AFP by routine AFP immunoassay.Conclusions Recombinant protein eGFP and eGFP-AFP have the same fluorescence characteristics and could react with routine immunoassay which establishes the foundation for the next research using eGFP-AFP as calibration and quality control materials.%目的:构建增强型绿色荧光蛋白(eGFP)标记的甲胎蛋白(AFP)的融合蛋白,并对其稳定性和测量特性进行分析。方法从pcDNA3.0质粒中扩增eGFP序列,插入质粒PET28a,构建eGFP表达质粒PET28a-eGFP。根据美国国立生物技术信息中心(NCBI)数据库中的AFP编码序列,应用序列合成方法合成AFP编码序列,并加入连接eGFP的铰链序列,与eGFP序列链接,构建表达质粒PET28a-eGFP-AFP。分别表达和纯化重组蛋白eGFP和eGFP-AFP,分析重组蛋白的荧光特性及稳定性。结果成功构建和纯化重组蛋白eGFP和eGFP-AFP,荧光光谱分析显示其激发和发射光

  6. NF-kB activation and its downstream target genes expression after heavy ions exposure

    Science.gov (United States)

    Chishti, Arif Ali; Baumstark-Khan, Christa; Hellweg, Christine; Schmitz, Claudia; Koch, Kristina; Feles, Sebastian

    2016-07-01

    To enable long-term human space flight cellular radiation response to densely ionizing radiation needs to be better understood for developing appropriate countermeasures to mitigate acute effects and late radiation risks for the astronaut. The biological effectiveness of accelerated heavy ions (which constitute the most important radiation type in space) with high linear energy transfer (LET) for effecting DNA damage response pathways as a gateway to cell death or survival is of major concern not only for space missions but also for new regimes of tumor radiotherapy. In the current research study, the contribution of NF-κB in response to space-relevant radiation qualities was determined by a NF-κB reporter cell line (HEK-pNF-κB-d2EGFP/Neo L2). The NF-κB dependent reporter gene expression (d2EGFP) after ionizing radiation (X-rays and heavy ions) exposure was evaluated by flow cytometry. Because of differences in the extent of NF-κB activation after X-irradiation and heavy ions exposure, it was expected that radiation quality (LET) might play an important role in the cellular radiation response. In addition, the biological effectiveness (RBE) of NF-κB activation and reduction of cellular survival was examined for heavy ions having a broad range of LET (˜0.3 - 9674 keV/µm). Furthermore, the effect of LET on NF-κB target gene expression was analyzed by real time reverse transcriptase quantitative PCR (RT-qPCR). In this study it was proven that NF-κB activation and NF-κB dependent gene expression comprises an early step in cellular radiation response. Taken together, this study clearly demonstrates that NF-κB activation and NF-κB-dependent gene expression by heavy ions are highest in the LET range of ˜50-200 keV/μupm. The up-regulated chemokines and cytokines (CXCL1, CXCL2, CXCL10, IL-8 and TNF) might be important for cell-cell communication among hit as well as unhit cells (bystander effect). The results obtained suggest the NF-κB pathway to be a

  7. The Effects of Interfering COX-2 Gene Expression on Malignant Proliferation of Human Lung Adenocarcinoma A2 Cell in vitro

    Directory of Open Access Journals (Sweden)

    Weiying LI

    2009-02-01

    Full Text Available Background and objective COX-2 was highly expressed in many tumor tissues and was involved in the initiation and development of tumors. The RNAi technique is a method to inhibit gene expression economically, quickly, efficiently and specifically. This study used RNAi technique to explore the interfering effect of COX-2 geneexpression and the influence on the malignant proliferation of A2 cells after quenching COX-2 in vitro . Methods Three COX-2 siRNA expression vectors with human U6 promoter were constructed. The COX-2 siRNA vectors and the vacant vector (pEGFP were transfected into A2 cells with lipofectamine respectively. The cell strains transfected were selected. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferationof A2 cells after silencing COX-2 were detected by cell growth curve and clonogenic assay in vitro . Results The three siRNA and U6 promoter cloned into pEGFP plasmid were validated by PCR, restriction endonucleases identification, DNA sequencing and BLAST alignment. The cell strains transfected were coded as A2-3, A2-7, A2-10 and A2-p respectively. Green fluorescence was seen in A2-p cells and not in A2-3, A2-7 and A2-10 cells in 24 h, 48 h and 72 hafter transfected. The results of RT-PCR and Western blot showed the three siRNA expression vectors acted effectively and the expression of COX-2 was inhibited in different extent. In contrast to A2 cells, COX-2 mRNA levels of A2-3, A2-7 and A2-10 cells were reduced 15.6%, 20.4% and 64.2% respectively, and COX-2 protein expressions of them were reduced 23.7%, 36.7% and 60.2% respectively. The results of cell growth curve and clonogenic assay showed the growth of A2-10 cell slowed and the clonal formation rate was reduced. However the growth of A2-3 and A2-7 cells had no obvious changes vs controls (A2 and A2-p. Conclusion Silencing COX-2 gene in vitro by RNAi technique can significantly inhibit the malignant proliferation of A2

  8. 鸡miR-1749生物信息学分析及真核表达载体构建%Bioinformatics Analysis and Eukaryotic Expression Vector Construction of miR-1749 in Chicken

    Institute of Scientific and Technical Information of China (English)

    王顺红; 王善禾; 蒋可人; 李红; 康相涛; 孙桂荣

    2015-01-01

    The purpose of this study was to explore the function of miR-1749 and the effect of rs15860000 ( C>T) polymorphism on mature miR-1749 biogenesis. The effect of rs15860000 on the secondary struc-ture of miR-1749 precursor was calculated by the Mfold web server. miR-1749 expression vectors with dif-ferent alleles were constructed using pcDNA3. 1-EGFP plasmid and transfected into DF1 cells. Then,the expression of mature miR-1749 was detected. The potential targets of miR-1749 were predicted by Tar-getScan and miRDB and further evaluated by DAVID. The result showed that the C>T mutation caused the free energy change of miR-1749 precursor secondary structure. The pcDNA3. 1-EGFP-pre-miR-1749-C and pcDNA3. 1-EGFP-pre-miR-1749-T expression vector were constructed successfully. The expression of miR-1749 in cells transfected with the recombinant vector was significantly higher than cells transfected with the empty vector (PT)不同基因型对成熟miRNA生成的影响,采用Mfold软件预测C>T突变对miR-1749前体二级结构能值的影响;选用pcDNA3.1-EG-FP质粒,构建miR-1749前体不同等位基因的真核表达载体并转染DF1细胞,检测成熟miR-1749的表达;利用 TargetScan 和 miRDB 软件预测 miR -1749的靶基因,并将靶基因的并集在DAVID数据库进行GO功能富集和KEGG信号转导通路分析。 Mfold预测结果显示,miR-1749前体C>T突变影响其前体二级结构自由能值;成功构建pcDNA3.1-EGFP-pre-miR-1749-C和pcDNA3.1-EGFP-pre-miR-1749-T真核表达载体,转染DF1细胞后,不同基因型重组质粒miR-1749的表达量均显著高于空质粒组( P<0.01), T等位基因成熟miR-1749的表达量显著高于C等位基因(P<0.05)。2个软件预测结果显示,miR-1749靶基因的并集有250个基因,这些基因显著富集于Ras蛋白信号转导调控、GTPase介导信号转导调控、细胞稳态及胚后发育的生物学过程和GnRH、MAPK及细胞黏附分子的信号转导通路。

  9. Melittin restores PTEN expression by down-regulating HDAC2 in human hepatocelluar carcinoma HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Hui Zhang

    Full Text Available Melittin is a water-soluble toxic peptide derived from the venom of the bee. Although many studies show the anti-tumor activity of melittin in human cancer including glioma cells, the underlying mechanisms remain elusive. Here the effect of melittin on human hepatocelluar carcinoma HepG2 cell proliferation in vitro and further mechanisms was investigated. We found melittin could inhibit cell proliferation in vitro using Flow cytometry and MTT method. Besides, we discovered that melittin significantly downregulated the expressions of CyclinD1 and CDK4. Results of western Blot and Real-time PCR analysis indicated that melittin was capable to upregulate the expression of PTEN and attenuate histone deacetylase 2 (HDAC2 expression. Further studies demonstrated that knockdown of HDAC2 completely mimicked the effects of melittin on PTEN gene expression. Conversely, it was that the potential utility of melittin on PTEN expression was reversed in cells treated with a recombinant pEGFP-C2-HDAC2 plasmid. In addition, treatment with melittin caused a downregulation of Akt phosphorylation, while overexpression of HDAC2 promoted Akt phosphorylation. These findings suggested that the inhibitory of cell growth by melittin might be led by HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways.

  10. Arginase-1 is expressed exclusively by infiltrating myeloid cells in CNS injury and disease.

    Science.gov (United States)

    Greenhalgh, Andrew D; Passos Dos Santos, Rosmarini; Zarruk, Juan Guillermo; Salmon, Christopher K; Kroner, Antje; David, Samuel

    2016-08-01

    Resident microglia and infiltrating myeloid cells play important roles in the onset, propagation, and resolution of inflammation in central nervous system (CNS) injury and disease. Identifying cell type-specific mechanisms will help to appropriately target interventions for tissue repair. Arginase-1 (Arg-1) is a well characterised modulator of tissue repair and its expression correlates with recovery after CNS injury. Here we assessed the cellular localisation of Arg-1 in two models of CNS damage. Using microglia specific antibodies, P2ry12 and Fc receptor-like S (FCRLS), we show the LysM-EGFP reporter mouse is an excellent model to distinguish infiltrating myeloid cells from resident microglia. We show that Arg-1 is expressed exclusively in infiltrating myeloid cells but not microglia in models of spinal cord injury (SCI) and experimental autoimmune encephalomyelitis (EAE). Our in vitro studies suggest that factors in the CNS environment prevent expression of Arg-1 in microglia in vivo. This work suggests different functional roles for these cells in CNS injury and repair and shows that such repair pathways can be switched on in infiltrating myeloid cells in pro-inflammatory environments. PMID:27126514

  11. Effects of L1-ORF2 fragments on green fluorescent protein gene expression

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    Xiu-Fang Wang

    2009-01-01

    Full Text Available The retrotransposon known as long interspersed nuclear element-1 (L1 is 6 kb long, although most L1s in mammalian and other eukaryotic cells are truncated. L1 contains two open reading frames, ORF1 and ORF2, that code for an RNA-binding protein and a protein with endonuclease and reverse transcriptase activities, respectively. In this work, we examined the effects of full length L1-ORF2 and ORF2 fragments on green fluorescent protein gene (GFP expression when inserted into the pEGFP-C1 vector downstream of GFP. All of the ORF2 fragments in sense orientation inhibited GFP expression more than when in antisense orientation, which suggests that small ORF2 fragments contribute to the distinct inhibitory effects of this ORF on gene expression. These results provide the first evidence that different 280-bp fragments have distinct effects on the termination of gene transcription, and that when inserted in the antisense direction, fragment 280-9 (the 3' end fragment of ORF2 induces premature termination of transcription that is consistent with the effect of ORF2.

  12. Heterogeneity of astrocytes: from development to injury - single cell gene expression.

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    Vendula Rusnakova

    Full Text Available Astrocytes perform control and regulatory functions in the central nervous system; heterogeneity among them is still a matter of debate due to limited knowledge of their gene expression profiles and functional diversity. To unravel astrocyte heterogeneity during postnatal development and after focal cerebral ischemia, we employed single-cell gene expression profiling in acutely isolated cortical GFAP/EGFP-positive cells. Using a microfluidic qPCR platform, we profiled 47 genes encoding glial markers and ion channels/transporters/receptors participating in maintaining K(+ and glutamate homeostasis per cell. Self-organizing maps and principal component analyses revealed three subpopulations within 10-50 days of postnatal development (P10-P50. The first subpopulation, mainly immature glia from P10, was characterized by high transcriptional activity of all studied genes, including polydendrocytic markers. The second subpopulation (mostly from P20 was characterized by low gene transcript levels, while the third subpopulation encompassed mature astrocytes (mainly from P30, P50. Within 14 days after ischemia (D3, D7, D14, additional astrocytic subpopulations were identified: resting glia (mostly from P50 and D3, transcriptionally active early reactive glia (mainly from D7 and permanent reactive glia (solely from D14. Following focal cerebral ischemia, reactive astrocytes underwent pronounced changes in the expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and reactive astrocyte markers.

  13. Expressing functional siRNAs in mammalian cells using convergent transcription

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    Dawes Ian W

    2003-11-01

    Full Text Available Abstract Background The use of small interfering RNAs (siRNAs as genetic inhibitors of gene expression has been shown to be an effective way of studying gene function in mammalian cells. Recently, different DNA vectors for expression of small hairpin RNAs (shRNAs or co-expression of sense and antisense RNAs have been developed that direct siRNA-mediated gene silencing. One expression cassette design that has been used to express long sense and antisense RNAs in non-mammalian cell types is symmetric transcription using convergent promoters. However, convergent transcription as a way to generate functional siRNAs in mammalian cells has not been reported. This vector design permits the generation of expression constructs containing no repeat sequences, but capable of inducing RNA interference (RNAi-mediated gene silencing. Results With the aim of simplifying the construction of RNAi expression vectors, we report on the production and application of a novel convergent promoter cassette capable of expressing sense and antisense RNAs, that form double-stranded RNA, and mediate gene silencing in mammalian cells. We use this cassette to inhibit the expression of both the EGFP transgene and the endogenous TP53 gene. The gene silencing effect is Dicer-dependent and the level of gene inactivation achieved is comparable to that produced with synthetic siRNA. Furthermore, this expression system can be used for both short and long-term control of specific gene expression in mammalian cells. Conclusion The experiments performed in this study demonstrate that convergent transcription can be used in mammalian cells to invoke gene-specific silencing via RNAi. This method provides an alternative to expression of shRNAs and co-expression of sense and antisense RNAs from independent cassettes or a divergent promoter. The main advantage of the present vector design is the potential to produce a functional siRNA expression cassette with no repeat sequences

  14. Efficiency of different recombinant viral vectors in the transduction of bone marrow mesenchymal stem cells a nd exogenous gene expression%不同重组病毒载体转染大鼠骨间充质干细胞的效率及其基因表达

    Institute of Scientific and Technical Information of China (English)

    潘虹; 刘新建; 吴继红; 田毓华; 谢匡成; 陈霞芳; 张圣海; 黄倩; 林志新

    2008-01-01

    够有效感染体外培养的骨髓间充质干细胞,并表达外源基因,感染效率与病毒用量之间存在量效关系.腺相关病毒rAAV1/2和rAAV2体外感染效果不佳.%BACKGROUND: Genetically engineered cells have been used in the replacement therapy and gene therapy. However, how to select proper donor cells, target cells, and corresponding viral vectors is the most difficult in the therapy.OBJECTIVE: To compare the transduction efficiency of recombinant adenovirus Ad5 and AdSF35, adeno-associated virus rAAV1/2 and rAAV2, and lentivirus LV in bone marrow mesenchymal stem cells (BMSCs) and exogenous gene expression level so as to select the vectors, which can efficiently transduce BMSCs.DESIGN, TIME AND SETTING: Gene engineering controlled observation, performed in the Central Laboratory, Shanghai First People's Hospital between October 2006 and March 2007.MATERIALS: Ten Sprague Dawley rats of clean grade were used to prepare BMSCs. All recombinant viral vectors carded enhanced green fluorescent protein (EGFP) report gene. Ad5 was prepared by the Central Laboratory, Shanghai First People's Hospital. Ad5F35 was gifted by professor Qian Qi-jun from the Second Military Medical University of Chinese PLA. rAAV2 and rAAVI/2 were the products of Benyuan Zhengyang Gene Technique Co.,Ltd. LV was gifted by professor Cuo Li-be from Shanghai Institute of Biochemistry and Cytobiology, Chinese Academy of Sciences.METHODS: Rat BMSCs were in vitro isolated and cultured by density gradient centrifugation. BMSCs of passage 4 were inoculated into 24-well plate at lxlO5/well. One day later, ceils adhered to the wall and allocated to 5 groups. Ad5-EGFP [10, 100,1 000 multiplicity of infection (MOI)], Ad5F35-EGFP (10,10, 1 000 MOI), rAAVI/2-EGFP (1×104,1x10× vg), rAAV2-EGFP(1×104, 1x105vg), and LV-EGFP (30 TU) were respectively added into the 5 groups. BMSCs were transduced for 2 days with Ad virus and separately for 6 days with rAAV and LV virus.MAIN OUTCOME MEASURES

  15. Bmo-miR-2758 Targets BmFMBP-1 (Lepidoptera: Bombycidae) and Suppresses Its Expression in BmN Cells.

    Science.gov (United States)

    Wang, Xin; Tang, Shunming; Song, Fei; Chen, Chen; Guo, Xijie; Shen, Xingjia

    2016-01-01

    MicroRNAs (miRNAs) are an abundant family of endogenous noncoding small RNA molecules. They play crucial roles on regulation of life processes both in plants and animals. Fibroin modulator binding protein-1 (FMBP-1) is a silk gland transcription factor of Bombyx mori, which is considered as a trans-activator of fibroin genes. And bioinformatics prediction showed that at the 3' untranslated region (3' UTR) of BmFMBP-1 there were binding sites for three bmo-miRNAs, bmo-miR-2b*, bmo-miR-305, and bmo-miR-2758, separately. In order to validate whether these bmo-miRNAs involved in the regulation of BmFMBP-1 expression, the expression levels of three bmo-miRNAs and BmFMBP-1 in the middle silk gland (MSG) and posterior silk gland (PSG) during the fourth- and fifth-larval stages of B. mori were measured by semi-quantitative reverse transcription polymerase chain reaction. The results revealed that the expression level of bmo-miR-2758 was the highest in the three, and it expressed higher in the PSG than in the MSG with a similar expression pattern as BmFMBP-1, implying that bmo-miR-2758 may involved in regulation of BmFMBP-1. To validate the regulation function of bmo-miR-2758 on BmFMBP-1, recombinant plasmids pcDNA3 [ie1-egfp-pri-bmo-miR-2758-SV40] and pGL3 [A3-luc-FMBP-1 3' UTR-SV40] were constructed and co-transfected in BmN cells. The dual-luciferase reporter assay system was used for assay of transient expression. The results showed that the expression of the luciferase reporter was significantly decreased when pGL3 [A3-luc-FMBP-1 3' UTR-SV40] co-transfected with pcDNA3 [ie1-egfp-pri-bmo-miR-2758-SV40] (P < 0.01). Furthermore, when the artificial antisense RNA of bmo-miR-2758 (inhibitor) was added to the above co-transfection, the expression of the luciferase reporter was recovered significantly (P < 0.01). These results suggest that bmo-miR-2758 represses the expression of BmFMBP-1 in vitro. PMID:27001963

  16. Mapping eGFP Oligomer Mobility in Living Cell Nuclei

    OpenAIRE

    Nicolas Dross; Corentin Spriet; Monika Zwerger; Gabriele Müller; Waldemar Waldeck; Jörg Langowski

    2009-01-01

    Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS) with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific...

  17. Mapping eGFP Oligomer Mobility in Living Cell Nuclei

    Science.gov (United States)

    Zwerger, Monika; Müller, Gabriele; Waldeck, Waldemar; Langowski, Jörg

    2009-01-01

    Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS) with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific artifacts encountered in live cell studies as well as strategies to prevent them, in particular those arising from the choice of the fluorophore used for calibration of the focal volume, as well as temperature and acquisition conditions used for fluorescence fluctuation measurements. After defining the best acquisition conditions, we show for various human cell lines that the mobility of GFP varies significantly within the cell nucleus, but does not correlate with chromatin density. The intranuclear diffusional mobility strongly depends on protein size: in a series of GFP-oligomers, used as free inert fluorescent tracers, the diffusion coefficient decreased from the monomer to the tetramer much more than expected for molecules free in aqueous solution. Still, the entire intranuclear chromatin network is freely accessible for small proteins up to the size of eGFP-tetramers, regardless of the chromatin density or cell line. Even the densest chromatin regions do not exclude free eGFP-monomers or multimers. PMID:19347038

  18. Mapping eGFP oligomer mobility in living cell nuclei.

    Directory of Open Access Journals (Sweden)

    Nicolas Dross

    Full Text Available Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific artifacts encountered in live cell studies as well as strategies to prevent them, in particular those arising from the choice of the fluorophore used for calibration of the focal volume, as well as temperature and acquisition conditions used for fluorescence fluctuation measurements. After defining the best acquisition conditions, we show for various human cell lines that the mobility of GFP varies significantly within the cell nucleus, but does not correlate with chromatin density. The intranuclear diffusional mobility strongly depends on protein size: in a series of GFP-oligomers, used as free inert fluorescent tracers, the diffusion coefficient decreased from the monomer to the tetramer much more than expected for molecules free in aqueous solution. Still, the entire intranuclear chromatin network is freely accessible for small proteins up to the size of eGFP-tetramers, regardless of the chromatin density or cell line. Even the densest chromatin regions do not exclude free eGFP-monomers or multimers.

  19. Characterization and chondrocyte differentiation stage-specific expression of KRAB zinc-finger protein gene ZNF470

    International Nuclear Information System (INIS)

    As part of a study to identify novel transcriptional regulators of chondrogenesis-related gene expression, we have cloned and characterized cDNA for zinc-finger protein 470 (ZNF470), the human ortholog of which encodes a 717 amino acid residue protein containing 17 Cys2His2 zinc-finger domains, as well as KRAB-A and KRAB-B motifs. The cDNA library used to isolate the initial ZNF470 clone was prepared from human bone marrow-derived mesenchymal progenitor cells at an intermediate stage of chondrogenic differentiation. We have determined the intron-exon structure of the human ZNF470 gene, which has been mapped to a zinc-finger cluster in a known imprinted region of human chromosome 19q13.4. ZNF470 is expressed at high levels in human testis and is expressed at low or undetectible levels in other adult tissues. Human ZNF470 expressed in mammalian cells as an EGFP fusion protein localizes predominantly to the nucleus, consistent with a role in transcriptional regulation. ZNF470, analyzed by quantitative real time PCR, was transiently expressed before the maximal expression of COL2A1 during chondrogenic differentiation in vitro. We have also characterized the bovine ortholog of human ZNF470, which encodes a 508 amino acid residue protein having 10 zinc-finger domains. A bovine ZNF470 cDNA clone was used to examine expression of ZNF470 in bovine articular chondrocytes treated with retinoic acid to stimulate dedifferentiation. Bovine ZNF470 expression was undetectable in freshly isolated bovine articular chondrocytes, but was dramatically upregulated in dedifferentiated retinoic acid-treated chondrocytes. These results, in two model systems, suggest a possible role for ZNF470 in the regulation of chondrogenesis-specific gene expression

  20. A self-inactivating retrovector incorporating the IL-2 promoter for activation-induced transgene expression in genetically engineered T-cells

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    Lejeune Laurence

    2006-11-01

    Full Text Available Abstract Background T-cell activation leads to signaling pathways that ultimately result in induction of gene transcription from the interleukin-2 (IL-2 promoter. We hypothesized that the IL-2 promoter or its synthetic derivatives can lead to T-cell specific, activation-induced transgene expression. Our objective was to develop a retroviral vector for stable and activation-induced transgene expression in T-lymphocytes. Results First, we compared the transcriptional potency of the full-length IL-2 promoter with that of a synthetic promoter composed of 3 repeats of the Nuclear Factor of Activated T-Cells (NFAT element following activation of transfected Jurkat T-cells expressing the large SV40 T antigen (Jurkat TAg. Although the NFAT3 promoter resulted in a stronger induction of luciferase reporter expression post stimulation, the basal levels of the IL-2 promoter-driven reporter expression were much lower indicating that the IL-2 promoter can serve as a more stringent activation-dependent promoter in T-cells. Based on this data, we generated a self-inactivating retroviral vector with the full-length human IL-2 promoter, namely SINIL-2pr that incorporated the enhanced green fluorescent protein (EGFP fused to herpes simplex virus thymidine kinase as a reporter/suicide "bifunctional" gene. Subsequently, Vesicular Stomatitis Virus-G Protein pseudotyped retroparticles were generated for SINIL-2pr and used to transduce the Jurkat T-cell line and the ZAP-70-deficient P116 cell line. Flow cytometry analysis showed that EGFP expression was markedly enhanced post co-stimulation of the gene-modified cells with 1 μM ionomycin and 10 ng/ml phorbol 12-myristate 13-acetate (PMA. This activation-induced expression was abrogated when the cells were pretreated with 300 nM cyclosporin A. Conclusion These results demonstrate that the SINIL-2pr retrovector leads to activation-inducible transgene expression in Jurkat T-cell lines. We propose that this design can be

  1. Expression and localization of a novel phosducin-like protein from amphioxus Branchiostoma belcheri

    Institute of Scientific and Technical Information of China (English)

    SAREN Gaowa; ZHAO Yonggang

    2009-01-01

    A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP),was identified for the first time from the gut cDNA library of Branchiostoma belched. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histocbemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-NIIAmphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri.

  2. Htr2a gene and 5-HT2A receptor expression in the cerebral cortex studied using genetically modified mice

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    Rodrigo Andrade

    2010-08-01

    Full Text Available Serotonin receptors of the 5-HT2A subtype are robustly expressed in the cerebral cortex where they have been implicated in the pathophysiology and therapeutics of mental disorders and the actions of hallucinogens. Much less is known, however, about the specific cell types expressing 5-HT2A receptors in cortex. In the current study we use immunohistochemical and electrophysiological approaches in genetically modified mice to address the expression of the Htr2a gene and 5-HT2A receptors in cortex. We first use an EGFP expressing BAC transgenic mice and identify three main Htr2A gene expressing neuronal populations in cortex. The largest of these cell populations corresponds to layer V pyramidal cells of the anterior cortex, followed by GABAergic interneurons of the middle layers, and nonpyramidal cells of the subplate/Layer VIb. We then use 5-HT2A receptor knockout mice to identify an antibody capable of localizing 5-HT2A receptors in brain and use it to map these receptors. We find strong laminar expression of 5-HT2A receptors in cortex, especially along a diffuse band overlaying layer Va. This band exhibits a strong anteroposterior gradient that closely matches the localization of Htr2A expressing pyramidal cells of layer V. Finally we use electrophysiological and immunohistochemical approaches to show that most, but not all, GABAergic interneurons of the middle layers are parvalbumin expressing Fast-spiking interneurons and that these cells are depolarized and excited by serotonin, most likely through the activation of 5-HT2A receptors. These results clarify and extend our understanding of the cellular distribution of 5-HT2A receptors in the cerebral cortex.

  3. Post-transcriptional regulation of dopamine D1 receptor expression in caudate-putamen of cocaine-sensitized mice.

    Science.gov (United States)

    Tobón, Krishna E; Catuzzi, Jennifer E; Cote, Samantha R; Sonaike, Adenike; Kuzhikandathil, Eldo V

    2015-07-01

    The dopamine D1 receptor is centrally involved in mediating the effects of cocaine and is essential for cocaine-induced locomotor sensitization. Changes in D1 receptor expression have been reported in various models of cocaine addiction; however, the mechanisms that mediate these changes in D1 receptor expression are not well understood. Using preadolescent drd1a-EGFP mice and a binge cocaine treatment protocol we demonstrate that the D1 receptor is post-transcriptionally regulated in the caudate-putamen of cocaine-sensitized animal. While cocaine-sensitized mice express high levels of steady-state D1 receptor mRNA, the expression of D1 receptor protein is not elevated. We determined that the post-transcriptional regulation of D1 receptor mRNA is rapidly attenuated and D1 receptor protein levels increase within 30 min when the sensitized mice are challenged with cocaine. The rapid increase in D1 receptor protein levels requires de novo protein synthesis and correlates with the cocaine-induced hyperlocomotor activity in the cocaine-sensitized mice. The increase in D1 receptor protein levels in the caudate-putamen inversely correlated with the levels of microRNA 142-3p and 382, both of which regulate D1 receptor protein expression. The levels of these two microRNAs decreased significantly within 5 min of cocaine challenge in sensitized mice. The results provide novel insights into the previously unknown rapid kinetics of D1 receptor protein expression which occurs in a time scale that is comparable to the expression of immediate early genes. Furthermore, the results suggest a potential novel role for inherently labile microRNAs in regulating the rapid expression of D1 receptor protein in cocaine-sensitized animals. PMID:25900179

  4. In vitro study on monitoring the expression of therapy gene via HSV1-sr39tk/FIAU report gene/probe system

    International Nuclear Information System (INIS)

    A recombinant adenovirus Ad5-sr39tk-IRES-VEGF165(Ad5-SIV) was constructed in our lab, using mutant simplex herpes viral thymidine kinase reporter gene (HSV1-sr39tk) as a report gene,and human vascular endothelial growth factor 165 (VEGF165) as a therapeutic gene. A replication-defective adenovirus carrying the enhanced green fluorescent protein gene (Ad5-EGFP) was used as a control.MSCs were infected with Ad5-SIV at different multiplicity of infection(MOI = 0, 10, 25, 50, 75 and 100). The expression of HSV1-sr39tk and VEGF165 was detected by immunofluorescence, and the secretion of VEGF was detected by enzyme-linked immunosorbent assay (ELISA). The cellular uptake of 131I-FIAU was performed to detect the expression of HSV1-sr39tk. The immunofluorescence showed HSV1-sr39tk and VEGF165 protein could express successfully at Ad5-SIV-infected MSCs. The cellular uptake of 131I-FIAU increased with the virus titer(P165 protein secretion increased with the titer(P165 protein secretion correlated highly with the uptake rate of 131I-FIAU(P165 could express successfully after MSCs were infected with Ad5-SIV. The expression of therapy gene correlated significantly with the reporter gene. This provides a theoretical basis for in vitro nuclear reporter gene imaging with this system. (authors)

  5. drd2-cre:ribotag mouse line unravels the possible diversity of dopamine d2 receptor-expressing cells of the dorsal mouse hippocampus.

    Science.gov (United States)

    Puighermanal, Emma; Biever, Anne; Espallergues, Julie; Gangarossa, Giuseppe; De Bundel, Dimitri; Valjent, Emmanuel

    2015-07-01

    Increasing evidences suggest that dopamine facilitates the encoding of novel memories by the hippocampus. However, the role of dopamine D2 receptors (D2R) in such regulations remains elusive due to the lack of the precise identification of hippocampal D2R-expressing cells. To address this issue, mice expressing the ribosomal protein Rpl22 tagged with the hemagglutinin (HA) epitope were crossed with Drd2-Cre mice allowing the selective expression of HA in D2R-containing cells (Drd2-Cre:RiboTag mice). This new transgenic model revealed a more widespread pattern of D2R-expressing cells identified by HA immunoreactivity than the one initially reported in Drd2-EGFP mice, in which the hilar mossy cells were the main neuronal population detectable. In Drd2-Cre:RiboTag mice, scattered HA/GAD67-positive neurons were detected throughout the CA1/CA3 subfields, being preferentially localized in stratum oriens and stratum lacunosum-moleculare. At the cellular level, HA-labeled cells located in CA1/CA3 subfields co-localized with calcium-binding proteins (parvalbumin, calbindin, and calretinin), neuropeptides (neuropeptide Y, somatostatin), and other markers (neuronal nitric oxide synthase, mGluR1α, reelin, coupTFII, and potassium channel-interacting protein 1). These results suggest that in addition to the glutamatergic hilar mossy cells, D2R-expressing cells constitute a subpopulation of GABAergic hippocampal interneurons. PMID:25545461

  6. Heixuedian (heix), a potential melanotic tumor suppressor gene, exhibits specific spatial and temporal expression pattern during Drosophila hematopoiesis.

    Science.gov (United States)

    Xia, Yan; Midoun, Samira Zohra; Xu, Zhiliang; Hong, Ling

    2015-02-15

    The Drosophila heixuedian (heix) is the ortholog of human UBIAD1 gene (a.k.a TERE1). The protein product of UBIAD1/heix has multiple enzymatic activities, including the vitamin K2 and the non-mitochondrial CoQ10 biosynthesis. However, the expression pattern of UBIAD1/Heix during metazoan development has not been systematically studied. In this paper, we found that loss of function of heix resulted in pathological changes of larval hematopoietic system, including lymph gland hypertrophy, hemocyte overproliferation and aberrant differentiation, and melanin mass formation. Overexpression of heix cDNA under the tubulin Gal4 driver rescued the above hematopoietic defects. Interestingly, Heix was specifically expressed in plasmatocyte/macrophage lineage in srp driven EGFP positive cells on the head mesoderm during embryogenesis, while it was highly expressed in crystal cells in the primary lobes of the third instar larval lymph gland. Using qRT-PCR analysis, loss of function of heix caused aberrant activation of multiple hemocyte proliferation-related as well as immune-related pathways, including JAK/STAT pathway, Ras/MAPK pathway, IMD pathway and Toll pathway. These data suggested that heix is a potential melanotic tumor suppressor gene and plays a pivotal role in both hemocytes proliferation and differentiation in Drosophila. PMID:25530181

  7. mTEL-cFms激酶结构域融合蛋白真核表达载体的构建及其对信号转导和转录激活因子核转位的影响%Establishment of mTEL-cFmskd eukaryotic expression vector and its effect on STAT1/3 nuclear translocation

    Institute of Scientific and Technical Information of China (English)

    杨胜乾; 龙隆; 李微; 王莉莉

    2011-01-01

    OBJECTIVE To construct a eukaryotic expression vector of mTEL-cFmskd and study its effect on nuclear translocation of the signal transducer and activator of transcription 1/3 ( STAT1/3 ). METHODS By recombinant DNA technology, the DNA sequence of polypeptide for myristoylation of human c-Src, helix-loop-helix domain of human TEL, kinase domain of macro-phage colony-stimulating factor receptor and c-Myc tag were inserted into pCORON/neo plasmid to generate pCORON/neo-HcSrc-Tel-cfmskd-Myc eukaryotic expression vector. The mTEL-cFmskd expression vector pCORON/neo-HcSrc-Tel-cfmskd-Myc and the c-Fms expression vector pCORON/ neo-cfms were transfected into U2OS ( expressing GFP-STAT1 ) and BHK ( expressing EGFP-STAT3) cells. After 24 h, the cells were fixed, stained and then imaged on the IN Cell Analyzer 1000. The image was analyzed using the Nuclear Trafficking Analysis Module. RESULTS Restriction enzyme digestion and plasmid sequencing confirmed the successful construction of pCORON/neo-HcSrc-Tel-cfmskd-Myc plasmid. mTEL-cFmskd was expressed in cells, and caused nuclear translocation of GFP-STAT1 and EGFP-STAT3 24 h after transfection. C-Fms inhibitors GW2580 and Sutent could block the nuclear translocation of EGFP-STAT3 by mTEL-cFmskd. CONCLUSION mTEL-cFmskd expression vector pCORON/neo-HcSrc-Tel-cfmskd-Myc is successfully constructed and functional mTEL-cFmskd is expressed in GFP-STAT1_U2OS and EGFP-STAT3_BHK cells.%目的 构建激酶盘真核表达载体,观察豆蔻酰化的TEL转录调节因子HLH结构域与c-Fms激酶结构域融合蛋白( mTEL-cFmskd)的表达对信号转导和转录激活因子1(STAT1)和STAT3核转位的影响.方法 利用DNA重组技术,将人的c-Src豆蔻酰化多肽、TEL转录调节因子HLH结构域、c-Fms激酶结构域以及c-Myc标签的DNA序列克隆在pCORON/neo载体上,构建pCORON/neo-HcSrc-Tel-cfmskd-Myc真核表达载体.将载体转染至稳定表达GFP-STAT1的人骨肉瘤细胞(U2OS)和稳定表达EGFP-STAT3

  8. Adenoviral vector mediated-expression of caspase-3 siRNA on apoptosis induced by lipopolysaccharide

    Institute of Scientific and Technical Information of China (English)

    Wu Feixiang; Yu Weifeng; Yuan Yang; Miao Xuerong; Xu Xuewu; Huang Shengdong; Sun Yuming

    2009-01-01

    Objective: To construct the recombinant adenovirus expressing small RNA of rats caspase-3 and observe the down-regulation effect of caspase-3 in neurons induced by lipopolysaccharide(LPS) in vitro. Methods: pShuttleH1-siCas3 containing Oligo DNA of the targeting sequences and pEGFPC1-Cas3 containing caspase-3 and EGFP sequences were constructed respectively, pShuttleH1-siCas3 and pEGFPC1-Cas3 were co-transfected to the 293 cells by liposomes to determine interfering efficacy by flow eytometry, pShuttleH1-siCas3 was linearized and transformed into E. coli BJ5183 cells containing backbone plasmid pAdEasy-1. The recombinant plasmid was transfected into 293 cells to package the adenovirus Ad-siCas3. The titers of adenovirus were determined by the specific 50% tissue culture infection dosage method. After virus infected the cultured hippocampus neurons, LPS-induced apoptosis and caspase-3 mRNA expression were observed. Results: It was identified that the sequence of target gene was correctly inserted into the genome of virus. The expression of green fluorescence protein was reduced by pShuttleH1-siCas3 in 293 cells. The titer of recombinant adenovirus was 1.06×1010 pfu/ml. After virus infection, caspase-3 mRNA was greatly reduced and neurons apoptosis was suppressed. Conclusion: The recombinant adenovirus expressing rats caspase-3 siRNA were successfully constructed, which may probably be further used in pain therapy by its anti-apoptosis effect.

  9. Immune clearance of attenuated rabies virus results in neuronal survival with altered gene expression.

    Directory of Open Access Journals (Sweden)

    Emily A Gomme

    Full Text Available Rabies virus (RABV is a highly neurotropic pathogen that typically leads to mortality of infected animals and humans. The precise etiology of rabies neuropathogenesis is unknown, though it is hypothesized to be due either to neuronal death or dysfunction. Analysis of human brains post-mortem reveals surprisingly little tissue damage and neuropathology considering the dramatic clinical symptomology, supporting the neuronal dysfunction model. However, whether or not neurons survive infection and clearance and, provided they do, whether they are functionally restored to their pre-infection phenotype has not been determined in vivo for RABV, or any neurotropic virus. This is due, in part, to the absence of a permanent "mark" on once-infected cells that allow their identification long after viral clearance. Our approach to study the survival and integrity of RABV-infected neurons was to infect Cre reporter mice with recombinant RABV expressing Cre-recombinase (RABV-Cre to switch neurons constitutively expressing tdTomato (red to expression of a Cre-inducible EGFP (green, permanently marking neurons that had been infected in vivo. We used fluorescence microscopy and quantitative real-time PCR to measure the survival of neurons after viral clearance; we found that the vast majority of RABV-infected neurons survive both infection and immunological clearance. We were able to isolate these previously infected neurons by flow cytometry and assay their gene expression profiles compared to uninfected cells. We observed transcriptional changes in these "cured" neurons, predictive of decreased neurite growth and dysregulated microtubule dynamics. This suggests that viral clearance, though allowing for survival of neurons, may not restore them to their pre-infection functionality. Our data provide a proof-of-principle foundation to re-evaluate the etiology of human central nervous system diseases of unknown etiology: viruses may trigger permanent neuronal

  10. Free Expression or Re-expression?

    Science.gov (United States)

    Gordon, W. Terrence

    1976-01-01

    Presents the notions that: (1) training in free expression skills in a foreign language can be systematized and integrated with the acquisition of fundamental language skills; and (2) that the principle of "re-expression" can be used by the teacher to yield "free expression" in the form of spontaneous oral summary. (AM)

  11. Regulation of gene expression

    International Nuclear Information System (INIS)

    In order to define in molecular terms the mechanisms controlling expression of specific genes in mammalian cells, how gene expression is activated, how tissue-specific expression is effected, how expression is modulated by hormones and other specific effectors, and how genetic control mechanisms are altered in the dysfunction of gene expression in cells transformed to malignancy were studied. Much of this work has focused on expression of the rat liver enzyme tyrosine aminotransferase

  12. Bacteriophage T7 RNA polymerase-based expression in Pichia pastoris.

    Science.gov (United States)

    Hobl, Birgit; Hock, Björn; Schneck, Sandra; Fischer, Reinhard; Mack, Matthias

    2013-11-01

    A novel Pichia pastoris expression vector (pEZT7) for the production of recombinant proteins employing prokaryotic bacteriophage T7 RNA polymerase (T7 RNAP) (EC 2.7.7.6) and the corresponding promoter pT7 was constructed. The gene for T7 RNAP was stably introduced into the P. pastoris chromosome 2 under control of the (endogenous) constitutive P. pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter (pGAP). The gene product T7 RNAP was engineered to contain a nuclear localization signal, which directed recombinant T7 RNAP to the P. pastoris nucleus. To promote translation of uncapped T7 RNAP derived transcripts, the internal ribosomal entry site from hepatitis C virus (HCV-IRES) was inserted directly upstream of the multiple cloning site of pEZT7. A P. pastoris autonomous replicating sequence (PARS1) was integrated into pEZT7 enabling propagation and recovery of plasmids from P. pastoris. Rapid amplification of 5' complementary DNA ends (5' RACE) experiments employing the test plasmid pEZT7-EGFP revealed that transcripts indeed initiated at pT7. HCV-IRES mediated translation of the latter mRNAs, however, was not observed. Surprisingly, HCV-IRES and the reverse complement of PARS1 (PARS1rc) were both found to display significant promoter activity as shown by 5' RACE. PMID:24056257

  13. Rana grylio virus TK and DUT gene locus could be simultaneously used for foreign gene expression.

    Science.gov (United States)

    Huang, Xing; Fang, Jin; Chen, Zhongyuan; Zhang, Qiya

    2016-03-01

    Ranaviruses (family Iridoviridae, genus Ranavirus) have been recognized as emerging infectious pathogens and caused a great loss to the global biodiversity. Thymidine kinase (TK) and deoxyuridine triphosphatase (dUTPase, DUT, encoded by ORF 67R) are ubiquitous, existing in iridoviruses and other organisms. Previous studies showed that TK and DUT could be individually knocked out without impeding viral replication. In this study, we tried to insert two fluorescence genes into the above loci. We started with Δ67R-RGV, a recently generated recombinant Rana grylio virus (RGV) with the whole DUT replaced by enhanced green fluorescence protein (EGFP) gene. Then, a red fluorescence protein (RFP) gene initiated by RGV immediate-early (IE) ICP18 gene promoter was inserted into TK locus through homologous recombination. A novel recombinant virus, ΔDUT, TK-RGV, was generated by nine successive rounds of plaque isolation using RFP selection. All of the plaques produced by this recombinant virus could emit both green and red fluorescence. Furthermore, one-step and multiple-step growth curves of ΔDUT, TK-RGV were similar to those of wt-RGV and Δ67R-RGV. In conclusion, a novel dual-fluorescence labeled recombinant iridovirus in which DUT and TK gene locus were simultaneously used for foreign gene expression was constructed. PMID:26806670

  14. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR

    Directory of Open Access Journals (Sweden)

    Haohan Wang

    2016-03-01

    Full Text Available Abstract As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway.

  15. The Pichia pastoris transmembrane protein GT1 is a glycerol transporter and relieves the repression of glycerol on AOX1 expression.

    Science.gov (United States)

    Zhan, Chunjun; Wang, Songwei; Sun, Yang; Dai, Xiaofeng; Liu, Xiuxia; Harvey, Linda; McNeil, Brian; Yang, Yankun; Bai, Zhonghu

    2016-06-01

    Promoter of alcohol oxidase I (PAOX1) is the most efficient promoter involved in the regulation of recombinant protein expression in Pichia pastoris (P. pastoris). PAOX1 is tightly repressed by the presence of glycerol in the culture medium; thus, glycerol must be exhausted before methanol can be taken up by P. pastoris and the expression of the heterologous protein can be induced. In this study, a candidate glycerol transporter (GT1, GeneID: 8197545) was identified, and its role was confirmed by further studies (e.g. bioinformatics analysis, heterologous complementation in Schizosaccharomyces pombe (S. pombe)). When GT1 is co-expressed with enhanced green fluorescent protein (EGFP), it localizes to the membrane and S. pombe carrying gt1 but not the wild-type strain can grow on medium containing glycerol as the sole carbon source. The present study is the first to report that AOX1 in the X-33Δgt1 mutant can achieve constitutive expression in medium containing glycerol; thus, knocking down gt1 can eliminate the glycerol repression of PAOX1 in P. pastoris These results suggest that the glycerol transporter may participate in the process of PAOX1 inhibition in glycerol medium. PMID:27189360

  16. Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos

    Directory of Open Access Journals (Sweden)

    Lee Okhyun

    2012-06-01

    Full Text Available Abstract Background Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38 in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff, contains three copies of oestrogen response elements (3ERE that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein. Results The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1–2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2, the synthetic oestrogen 17α- ethinyloestradiol (EE2, and the relatively weak environmental oestrogen nonylphenol (NP, and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures. For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish. We

  17. CpG-island fragments from the HNRPA2B1/CBX3 genomic locus reduce silencing and enhance transgene expression from the hCMV promoter/enhancer in mammalian cells

    Directory of Open Access Journals (Sweden)

    Irvine Alistair

    2005-06-01

    Full Text Available Abstract Background The hCMV promoter is very commonly used for high level expression of transgenes in mammalian cells, but its utility is hindered by transcriptional silencing. Large genomic fragments incorporating the CpG island region of the HNRPA2B1 locus are resistant to transcriptional silencing. Results In this report we describe studies on the use of a novel series of vectors combining the HNRPA2B1 CpG island with the hCMV promoter for expression of transgenes in CHO-K1 cells. We show that the CpG island gives at least twenty-fold increases in the levels of EGFP and EPO observed in pools of transfectants, and that transgene expression levels remain high in such pools for more than 100 generations. These novel vectors also allow facile isolation of clonal CHO-K1 cell lines showing stable, high-level transgene expression. Conclusion Vectors incorporating the hnRPA2B1 CpG island give major benefits in transgene expression from the hCMV promoter, including substantial improvements in the level and stability of expression. The utility of these vectors for the improved production of recombinant proteins in CHO cells has been demonstrated.

  18. Recombinant Pseudorabies Virus (PRV Expressing Firefly Luciferase Effectively Screened for CRISPR/Cas9 Single Guide RNAs and Antiviral Compounds

    Directory of Open Access Journals (Sweden)

    Yan-Dong Tang

    2016-03-01

    Full Text Available A Pseudorabies virus (PRV variant has emerged in China since 2011 that is not protected by commercial vaccines, and has not been well studied. The PRV genome is large and difficult to manipulate, but it is feasible to use clustered, regularly interspaced short palindromic repeats (CRISPR/Cas9 technology. However, identification of single guide RNA (sgRNA through screening is critical to the CRISPR/Cas9 system, and is traditionally time and labor intensive, and not suitable for rapid and high throughput screening of effective PRV sgRNAs. In this study, we developed a recombinant PRV strain expressing firefly luciferase and enhanced green fluorescent protein (EGFP as a reporter virus for PRV-specific sgRNA screens and rapid evaluation of antiviral compounds. Luciferase activity was apparent as soon as 4 h after infection and was stably expressed through 10 passages. In a proof of the principle screen, we were able to identify several PRV specific sgRNAs and confirmed that they inhibited PRV replication using traditional methods. Using the reporter virus, we also identified PRV variants lacking US3, US2, and US9 gene function, and showed anti-PRV activity for chloroquine. Our results suggest that the reporter PRV strain will be a useful tool for basic virology studies, and for developing PRV control and prevention measures.

  19. Recombinant Pseudorabies Virus (PRV) Expressing Firefly Luciferase Effectively Screened for CRISPR/Cas9 Single Guide RNAs and Antiviral Compounds

    Science.gov (United States)

    Tang, Yan-Dong; Liu, Ji-Ting; Fang, Qiong-Qiong; Wang, Tong-Yun; Sun, Ming-Xia; An, Tong-Qing; Tian, Zhi-Jun; Cai, Xue-Hui

    2016-01-01

    A Pseudorabies virus (PRV) variant has emerged in China since 2011 that is not protected by commercial vaccines, and has not been well studied. The PRV genome is large and difficult to manipulate, but it is feasible to use clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. However, identification of single guide RNA (sgRNA) through screening is critical to the CRISPR/Cas9 system, and is traditionally time and labor intensive, and not suitable for rapid and high throughput screening of effective PRV sgRNAs. In this study, we developed a recombinant PRV strain expressing firefly luciferase and enhanced green fluorescent protein (EGFP) as a reporter virus for PRV-specific sgRNA screens and rapid evaluation of antiviral compounds. Luciferase activity was apparent as soon as 4 h after infection and was stably expressed through 10 passages. In a proof of the principle screen, we were able to identify several PRV specific sgRNAs and confirmed that they inhibited PRV replication using traditional methods. Using the reporter virus, we also identified PRV variants lacking US3, US2, and US9 gene function, and showed anti-PRV activity for chloroquine. Our results suggest that the reporter PRV strain will be a useful tool for basic virology studies, and for developing PRV control and prevention measures. PMID:27043610

  20. PRL-3 suppresses c-Fos and integrin α2 expression in ovarian cancer cells

    International Nuclear Information System (INIS)

    activation of c-fos, a transcriptional activator of integrin α2, was observed in these PRL-3 knock-down cells. Moreover, forced expression of EGFP-PRL-3 resulted in the suppression of both integrin α2 and c-fos expression in A2780 cells. Significantly, using a xenograft tumor model, we observed a greatly reduced tumorigenicity of A2780 PRL-3 knock-down cells in vivo. These results suggest that PRL-3 plays a critical role in ovarian cancer tumorigenicity and maintaining the malignant phenotype. PRL-3 may inhibit c-fos transcriptional regulation of integrin α2 signaling. Our results strongly support a role for PRL-3 as a promising therapeutic target and potential early biomarker in ovarian cancer progression

  1. Express web application development

    CERN Document Server

    Yaapa, Hage

    2013-01-01

    Express Web Application Development is a practical introduction to learning about Express. Each chapter introduces you to a different area of Express, using screenshots and examples to get you up and running as quickly as possible.If you are looking to use Express to build your next web application, ""Express Web Application Development"" will help you get started and take you right through to Express' advanced features. You will need to have an intermediate knowledge of JavaScript to get the most out of this book.

  2. Facial Expression Recognition

    OpenAIRE

    Neeta Sarode; Prof. Shalini Bhatia

    2010-01-01

    Facial expression analysis is rapidly becoming an area of intense interest in computer science and human-computer interaction design communities. The most expressive way humans display emotions is through facial expressions. In this paper a method is implemented using 2D appearance-based local approach for the extraction of intransient facial features and recognition of four facial expressions. The algorithm implements Radial Symmetry Transform and further uses edge projection analysis for fe...

  3. After Effects expressions

    CERN Document Server

    Geduld, Marcus

    2013-01-01

    Put the power of Expressions to work in your animations with controls and efficiencies impossible to achieve with traditional keyframing techniques. No programming skills are required. Foundation concepts and skills orient the new designer and serve as a handy reference to the experienced one. Basics of creating expressions, variables, commands, and expression helpers precede the leap into javascript and math essentials for more advanced expressions that include randomness, physical simularions and 3D. Full color illustrations display the scripts and the resulti

  4. Stem cells with FGF4-bFGF fused gene enhances the expression of bFGF and improves myocardial repair in rats

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xiang-Qi; Chen, Liang-Long, E-mail: xhzlyx@126.com; Fan, Lin; Fang, Jun; Chen, Zhao-Yang; Li, Wei-Wei

    2014-04-25

    Highlights: • BFGF exists only in the cytoplasm of live cells. • BFGF cannot be secreted into the extracellular space to promote cell growth. • We combine the secretion-promoting signal peptide of FGF4. • We successfully modified BMSCs with the fused genes of FGF4-bFGF. • We promoted the therapeutic effects of transplanted BMSCs in myocardial infarction. - Abstract: The aim of this study was to investigate whether the modification of bone marrow-derived mesenchymal stem cells (BMSCs) with the fused FGF4 (fibroblast growth factor 4)-bFGF (basic fibroblast growth factor) gene could improve the expression and secretion of BFGF, and increase the efficacies in repairing infarcted myocardium. We used In-Fusion technique to construct recombinant lentiviral vectors containing the individual gene of bFGF, enhanced green fluorescent protein (EGFP), or genes of FGF4-bFGF and EGFP, and then transfected these lentiviruses into rat BMSCs. We conducted an in vitro experiment to compare the secretion of bFGF in BMSCs infected by these lentiviruses and also examined their therapeutic effects in the treatment of myocardial infraction in a rodent study. Sixty rats were tested in the following five conditions: Group-SHAM received only sham operation as controls; Group-AMI received only injection of placebo PBS buffer; Group-BMSC, Group-bFGF and Group-FGF4-bFGF received implantation of BMSCs with empty lentivirus, bFGF lentivirus, and FGF4-bFGF lentivirus, respectively. Our results found out that the transplanted FGF4-bFGF BMSCs had the highest survival rate, and also the highest myocardial expression of bFGF and microvascular density as evidenced by Western blotting and immunohistochemistry, respectively. As compared to other groups, the Group-FGF4-BFGF rats had the lowest myocardial fibrotic fraction, and the highest left ventricular ejection fraction. These results suggest that the modification of BMSCs with the FGF4-bFGF fused gene can not only increase the expression of

  5. Tumor cell adhesion to endothelial cells is increased by endotoxin via an upregulation of beta-1 integrin expression.

    LENUS (Irish Health Repository)

    Andrews, E J

    2012-02-03

    BACKGROUND: Recent studies have demonstrated that metastatic disease develops from tumor cells that adhere to endothelial cells and proliferate intravascularly. The beta-1 integrin family and its ligand laminin have been shown to be important in tumor-to-endothelial cell adhesion. Lipopolysaccharide (LPS) has been implicated in the increased metastatic tumor growth that is seen postoperatively. We postulated that LPS increases tumor cell expression of beta-1 integrins and that this leads to increased adhesion. METHODS: The human metastatic colon cancer cell line LS174T was labeled with an enhanced green fluorescent protein (eGFP) using retroviral transfection. Cell cultures were treated with LPS for 1, 2, and 4 h (n = 6 each) and were subsequently cocultured for 30 or 120 min with confluent human umbilical vein endothelial cells (HUVECs), to allow adherence. Adherent tumor cells were counted using fluorescence microscopy. These experiments were carried out in the presence or absence of a functional blocking beta-1 integrin monoclonal antibody (4B4). Expression of beta-1 integrin and laminin on tumor and HUVECs was assessed using flow cytometric analysis. Tumor cell NF-kappaB activation after incubation with LPS was measured. RESULTS: Tumor cell and HUVEC beta-1 integrin expression and HUVEC expression of laminin were significantly (P < 0.05) enhanced after incubation with LPS. Tumor cell adhesion to HUVECs was significantly increased. Addition of the beta-1 integrin blocking antibody reduced tumor cell adhesion to control levels. LPS increased tumor cell NF-kappaB activation. CONCLUSIONS: Exposure to LPS increases tumor cell adhesion to the endothelium through a beta-1 integrin-mediated pathway that is NF-kappaB dependent. This may provide a target for immunotherapy directed at reducing postoperative metastatic tumor growth.

  6. Extrachromosomal inducible expression

    NARCIS (Netherlands)

    Veltman, Douwe M; Van Haastert, Peter J M; Eichinger, L.; Rivero, F.

    2013-01-01

    Inducible expression systems are very convenient for proteins that induce strong side effects such as retardation of growth or development and are essential for the expression of toxic proteins. In this chapter we describe the doxycycline-inducible expression system, optimized for the controlled exp

  7. Effect of GalR2 gene expression on the depression in mice models%GalR2基因的表达对小鼠抑郁症影响的初步探讨

    Institute of Scientific and Technical Information of China (English)

    张丹; 杨春; 何永林; 徐蕾; 靳志栋; 张鹏; 冯鑫

    2011-01-01

    目的 建立C57BL/6J小鼠抑郁症模型,探讨GalR2基因表达对小鼠抑郁症的影响.方法 脂质体转染重组真核表达质粒pEGFP-GalR2至人宫颈癌细胞系Hela细胞,Western blotting检测GalR2基因在细胞内的表达.参照CUMS和CORT建模方法构建小鼠抑郁症模型.侧脑室注射脂质体包裹的pEGFP-GalR2质粒,观察GalR2基因表达对小鼠抑郁症的影响.结果 通过Western blotting鉴定了GalR2基因获得表达.抑郁症模型小鼠体重增加量、摄食量、液体消耗实验中糖水消耗和糖水偏爱百分比均明显下降,纯水消耗显著提高;强迫游泳实验中挣扎时间和游泳时间缩短,不动时间延长.侧脑室注射pEGFP-GalR2后小鼠行为学改变未得到有统计学意义的结果.结论 成功鉴定了GalR2基因在细胞内的表达.成功构建C57BL/6J小鼠抑郁症模型.CORT模型造模效果要优于CUMS模型.GalR2蛋白对抑郁症的影响有待后续实验进一步探讨.%To establish the C57BL/6J mice model of depression and evaluate the influence of GalR2 gene expression on depression. Method The eukaryotic expression recombinant plasmid Pegfp-GofR2 was transiently transfected into Hela cells, and the expression of CaLR2 gene was detected by Western blotting. The mice model of depression was constructed by CUMS and CORT. The plasmid Pegfp-Go2R2 was injected intracerebroventricularly, and the influence of GalK2 gene expression on depression was evaluated. The GalR2 gene expression products in Hela cells were detected by Western blotting. Result Body weight, food intake, sugar consumption and the percentage of preference for sugar of the depressed mice were significantly decreased and pure water consumption was increased compared with control group. In forced swim test, the depressed mice spent less time on swimming and struggling, and more time on immobilit-ing. Behavioral changes after ICV (intracerebrovenlricular injection) had no statistically significant results

  8. Gene Clone, Subcellular Localization of Expression Products of H-FABP and the Preparation of Transgenic Mice in Xuhuai Goat%徐淮山羊H-FABP基因克隆、表达产物亚细胞定位的研究及转基因小鼠的制备

    Institute of Scientific and Technical Information of China (English)

    阴彦辉; 韦光辉; 李伟; 朱才业; 张亚妮; 杜立新; 曹文广; 李碧春

    2012-01-01

    The purpose of this study was to clone heart-type fatty acid binding protein (H-FABP) gene cDNA of Xuhuai goat, and to explore its bioinformatics function and the possibility of preparation of transgenic animals among heterogeneous species. The subcelluar location H-FABP was detected by EGFP fusion protein and its expression was observed in vitro. Reverse transcription PCR (RT-PCR) technology was used to clone the H-FABP gene cDNA of Xuhuai goat, its biological information characteristics was analyzed by online software, then the expression vector pEGFP-H-FABP was constructed. The transfection of goat fibroblasts (GEF) was performed by Liposomes (LTX), and fluorescence was observed under inverted microscope after 48 h. The RT-PCR was conducted to detect mRNA expression of H-FABP in GEF. The pEGFP-H-FABP was injected into mouse testicular and its expression was detected at the level of DNA and protein. The complete CDS size of H-FABP was 402 bp, encoding 133 amino acids with GenBank accession number (AY466498.1). The H-FABP cDNA coding sequence was compared with the corresponding regions of human, chicken, brown rat, cow, wild boar, donkey and zebra fish, the similarity was 89% , 76%, 85% , 84% , 93% , 91% , 70% , respectively, amino acid sequence homol-ogy was 90%, 79%, 88%, 97%, 95%, 94%, 72%, respectively. The signal peptide was not found in H-FABP protein. The RT-PCR results showed the H-FABP mRNA expressed successfully in vitro. pEGFP-H-FABP was successfully constructed, and H-FABP mRNA was expressed. The H-FABP protein was localized in the cytoplasm which was in line with the result of online prediction. The gene can aslo be expressed in mice transiently and persistencely after intravenous and testicular injection. The H-FABP gene cDNA of Xuhuai goat was cloned successfully, and it was conservative during the evolutionary process, there was no signal peptide in protein. The H-FABP protein was located in the cytoplasm, and also could be expressed in mice

  9. Retroviral induction of GSK-3β expression blocks the stimulatory action of physical exercise on the maturation of newborn neurons.

    Science.gov (United States)

    Llorens-Martín, María; Teixeira, Catia M; Jurado-Arjona, Jerónimo; Rakwal, Randeep; Shibato, Junko; Soya, Hideaki; Ávila, Jesús

    2016-09-01

    Adult hippocampal neurogenesis (AHN) is a key process for certain types of hippocampal-dependent learning. Alzheimer's disease (AD) is accompanied by memory deficits related to alterations in AHN. Given that the increased activity of GSK-3β has been related to alterations in the population of hippocampal granule neurons in AD patients, we designed a novel methodology by which to induce selective GSK-3β overexpression exclusively in newborn granule neurons. To this end, we injected an rtTA-IRES-EGFP-expressing retrovirus into the hippocampus of tTO-GSK-3β mice. Using this novel retroviral strategy, we found that GSK-3β caused a cell-autonomous impairment of the morphological and synaptic maturation of newborn neurons. In addition, we examined whether GSK-3β overexpression in newborn neurons limits the effects of physical activity. While physical exercise increased the number of dendritic spines, the percentage of mushroom spines, and the head diameter of the same in tet-OFF cells, these effects were not triggered in tet-ON cells. This observation suggests that GSK-3β blocks the stimulatory actions of exercise. Given that the activity of GSK-3β is increased in the brains of individuals with AD, these data may be relevant for non-pharmacological therapies for AD. PMID:27010990

  10. Cloning and construction of sense and antisense eukaryotic expression vector of human Pin1%正反义人Pin1基因克隆及真核表达载体的构建

    Institute of Scientific and Technical Information of China (English)

    熊文化; 陈安民; 郭风劲

    2006-01-01

    Objective: To clone and construct eukaryotic expressing vectors of sense and antisense human Pin1 (hPin1)genes.Methods: Total RNA was extracted from MG-63 cells,then the hPin1 cDNA was amplified by RT-PCR.The same time the sense and antisense hPin1 genes were formed by binding BamH Ⅰ and Hind Ⅲ in cis and trans-directions.At the end they were cloned into the eukaryotic expressing vector pIRES2-EGFP in cis and trans directions using DNA recombinant technology.The recombinant vectors were further identified by digestion of BamH Ⅰ and Hind Ⅲ.Results: The results of sequencing showed that the orientation of the ligations and the reading frame were correct.After digested by BamH Ⅰ and Hind Ⅲ,two fragments exhibiting 5.3 kb and 0.99 kb were formed in sense and antisense eukaryotic expressing vectors.Electrophoretic results were completely coincident with theoretical calculation.Conclusion: Human Pin1 sense and antisense genes were successfully cloned and eukaryotic expressing vectors were successfully constructed.

  11. Expression, purification, crystallization and preliminary X-ray crystallographic studies of hepatitis B virus core fusion protein corresponding to octahedral particles

    International Nuclear Information System (INIS)

    Novel hepatitis B virus-like particles of recombinant dimeric core–GFP fusion protein were expressed, purified and crystallized. The crystals diffracted to 2.15 Å resolution and belonged to space group F432, with unit-cell parameters a = b = c = 219.7 Å. Recombinant hepatitis B virus core proteins dimerize to form building blocks that are capable of self-assembly into a capsid. A core capsid protein dimer (CPD) linked to a green fluorescent protein variant, EGFP, at the C-terminus has been designed. The recombinant fusion CPD was expressed in Escherichia coli, assembled into virus-like particles (VLPs), purified and crystallized. The single crystal diffracted to 2.15 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 219.7 Å. The fusion proteins assembled into icosahedral VLPs in aqueous solution, but were rearranged into octahedral symmetry through the crystal-packing process under the crystallization conditions

  12. Expression of Mouse SCP2 Gene Adenoviral Vector Carrying Albumin Promoter in Hepa1-6 Cells%固醇携带蛋白2腺病毒载体的构建与鉴定

    Institute of Scientific and Technical Information of China (English)

    贾岩峰; 崔云峰; 崔乃强; 彭雁飞; 宁召臣; 张琚

    2012-01-01

    Objectives To construct the replication defective adenoviral vector of SCP2 gene carrying murine albumin promoter, and study the relations between SCP2 gene and the formation of cholesterol calculus. Methods The cDNA of SCP2 gene was cloned by using RT-PCR technique. The albumin promoter was linked to SCP2 gene's upstream, and the EGFP gene lied in its downstream. The plasmid pDC312-ALB-SCP2-IRES2 -EGFP was constructed by the gene recombination technique. The Admax Adenoviral Vector System was used to generate the replication defective adenoviral vectors, which were purified by CsCl method. The processes of TCID50 were applied to detect the titers of the adenoviral vectors. The RNA and protein were respectively extracted from the infected Hepal-6 cells by the adenoviral vector. The real-time quantitative PCR was employed to detect the mRNA expression levels, and the Western blotting analysis was used to measure the SCP2 protein levels. Result We constructed successfully the replication defective adenoviral vector of SCP2 gene carrying murine albumin promoter. When the mRNA levels of SCP2 gene were overexpressed, CYP7al mRNA levels were down-regulated (t=3.97,p<0.05); and the mRNA levels of HMGCR were up-regulated (t=3.23,p<0.05). Conclusions The SCP2 gene overexpression may affect cholesterol and bile acid metabolism, which could promote the formation of cholesterol calculus.%目的:构建携带白蛋白启动子SCP2 基因腺病毒载体,研究其与胆固醇结石形成的关系.方法:(1)利用RT-PCR技术克隆小鼠SCP2基因,在其上游接入白蛋白(ALB)启动子,下游连接绿色荧光报告基因(EGFP),构建穿梭质粒pDC312-ALB-SCP2-IRES2-EGFP;(2)采用Ad Max TM Adenoviru5 Vector系统包装病毒,CsCl法纯化病毒、TCID50法测定滴度;(3)重组腺病毒感染小鼠hepa-1-6细胞,实时定量PCR检测mRNA的表达;Western印迹检测SCP2蛋白表达情况;结果:成功构建携带白蛋白启动子SCP2基因腺病毒载体;当SCP2

  13. Pleiotrophin Expression during Odontogenesis

    OpenAIRE

    Erlandsen, Heidi; Ames, Jennifer E.; Tamkenath, Amena; Mamaeva, Olga; Stidham, Katherine; Wilson, Mary E; Perez-Pinera, Pablo; Deuel, Thomas F.; Macdougall, Mary

    2012-01-01

    Pleiotrophin (PTN) is an extracellular matrix–associated growth factor and chemokine expressed in mesodermal and ectodermal cells. It plays an important role in osteoblast recruitment and differentiation. There is limited information currently available about PTN expression during odontoblast differentiation and tooth formation, and thus the authors aimed to establish the spatiotemporal expression pattern of PTN during mouse odontogenesis. Immortalized mouse dental pulp (MD10-D3, MD10-A11) an...

  14. Lactococcus lactis expressing either Staphylococcus aureus fibronectin-binding protein A or Listeria monocytogenes internalin A can efficiently internalize and deliver DNA in human epithelial cells.

    Science.gov (United States)

    Innocentin, Silvia; Guimarães, Valeria; Miyoshi, Anderson; Azevedo, Vasco; Langella, Philippe; Chatel, Jean-Marc; Lefèvre, François

    2009-07-01

    Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA(+)), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA(+) and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA(+)) or its fibronectin binding domains C and D (L. lactis CD(+)). L. lactis FnBPA(+) and L. lactis InlA(+) showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD(+) was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA(+), L. lactis CD(+), and L. lactis InlA(+) were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA(+) or L. lactis InlA(+). Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles. PMID

  15. Regular Expression Pocket Reference

    CERN Document Server

    Stubblebine, Tony

    2007-01-01

    This handy little book offers programmers a complete overview of the syntax and semantics of regular expressions that are at the heart of every text-processing application. Ideal as a quick reference, Regular Expression Pocket Reference covers the regular expression APIs for Perl 5.8, Ruby (including some upcoming 1.9 features), Java, PHP, .NET and C#, Python, vi, JavaScript, and the PCRE regular expression libraries. This concise and easy-to-use reference puts a very powerful tool for manipulating text and data right at your fingertips. Composed of a mixture of symbols and text, regular exp

  16. Regular expressions cookbook

    CERN Document Server

    Goyvaerts, Jan

    2009-01-01

    This cookbook provides more than 100 recipes to help you crunch data and manipulate text with regular expressions. Every programmer can find uses for regular expressions, but their power doesn't come worry-free. Even seasoned users often suffer from poor performance, false positives, false negatives, or perplexing bugs. Regular Expressions Cookbook offers step-by-step instructions for some of the most common tasks involving this tool, with recipes for C#, Java, JavaScript, Perl, PHP, Python, Ruby, and VB.NET. With this book, you will: Understand the basics of regular expressions through a

  17. Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo

    International Nuclear Information System (INIS)

    Delivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV) OncoVEXGM-CSF, we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV. To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA) or artificial microRNA (miRNA) against the reporter genes green fluorescent protein (eGFP) and β-galactosidase (lacZ). These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting. Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective. This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen in human clinical trials

  18. The oligodendroglial precursor cell line Oli-neu represents a cell culture system to examine functional expression of the mouse gap junction gene connexin29 (Cx29

    Directory of Open Access Journals (Sweden)

    SonjaHombach

    2013-06-01

    Full Text Available The potential gap junction forming mouse connexin29 (Cx29 protein is concomitantly expressed with connexin32 (Cx32 in peripheral myelin forming Schwann cells and together with both Cx32 and connexin47 (Cx47 in oligodendrocytes of the CNS. To study the genomic structure and functional expression of Cx29, either primary cells or cell culture systems might be selected, from which the latter are easier to cultivate. Both structure and expression of Cx29 is still not fully understood. In the mouse sciatic nerve, brain and the oligodendroglial precursor cell line Oli-neu the Cx29 gene is processed in two transcript isoforms both harbouring a unique reading frame. In contrast to Cx32 and Cx47, only Cx29 protein is abundantly expressed in undifferentiated as well as differentiated Oli-neu cells but the absence of Etbr dye transfer after microinjection concealed the function of Cx29 mediated gap junction communication between those cells. Although HeLa cells stably transfected with Cx29 or Cx29-eGFP neither demonstrated any permeability for Lucifer yellow nor for neurobiotin, blocking of Etbr uptake from the media by gap junction blockers does suppose a role of Cx29 in hemi-channel function. Thus, we conclude that, due to its high abundance of Cx29 expression and its reproducible culture conditions, the oligodendroglial precursor cell line Oli-neu might constitute an appropriate cell culture system to study molecular mechanisms or putative extracellular stimuli to functionally open Cx29 channels or hemi-channels.

  19. Darwin and Emotion Expression

    Science.gov (United States)

    Hess, Ursula; Thibault, Pascal

    2009-01-01

    In his book "The Expression of the Emotions in Man and Animals," Charles Darwin (1872/1965) defended the argument that emotion expressions are evolved and adaptive (at least at some point in the past) and serve an important communicative function. The ideas he developed in his book had an important impact on the field and spawned rich domains of…

  20. Freedom of Expression.

    Science.gov (United States)

    Update on Law-Related Education, 1988

    1988-01-01

    Presents an activity which uses hypothetical situations to explore the proper boundaries of freedom of expression and the role of the U.S. Supreme Court in interpreting its limits. Appropriate for grades 4-12, the lesson includes such topics as the "clear and present danger" clause, student expression, obscenity, and defamation. (GEA)

  1. Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene-Expressing Embryos.

    Science.gov (United States)

    Luchetti, C G; Bevacqua, R J; Lorenzo, M S; Tello, M F; Willis, M; Buemo, C P; Lombardo, D M; Salamone, D F

    2016-08-01

    The use of vesicles co-incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co-incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx-egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p plasmids to allow development to blastocyst stage was 30 ng/μl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p GFP blastocysts. The use of vesicles co-incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids. PMID:27260090

  2. Interactions between beta subunits of the KCNMB family and Slo3: beta4 selectively modulates Slo3 expression and function.

    Directory of Open Access Journals (Sweden)

    Cheng-Tao Yang

    Full Text Available BACKGROUND: The pH and voltage-regulated Slo3 K(+ channel, a homologue of the Ca(2+- and voltage-regulated Slo1 K(+ channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary beta subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct beta subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels. METHODOLOGY/PRINCIPAL FINDINGS: To examine the ability of the KCNMB family of auxiliary beta subunits to regulate Slo3 function, we co-expressed Slo3 and each beta subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The beta4 subunit produced an 8-10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither beta1, beta2, nor beta3 mimicked the ability of beta4 to increase surface expression, although biochemical tests suggested that all four beta subunits are competent to coassemble with Slo3. Fluorescence microscopy from beta4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that beta4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that beta4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm. CONCLUSIONS/SIGNIFICANCE: These results argue that, for native mouse Slo3 channels, the beta4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 alpha subunit.

  3. Expert Oracle application express

    CERN Document Server

    Scott, John Edward

    2011-01-01

    Expert Oracle Application Express brings you groundbreaking insights into developing with Oracle's enterprise-level, rapid-development tool from some of the best practitioners in the field today. Oracle Application Express (APEX) is an entirely web-based development framework that is built into every edition of Oracle Database. The framework rests upon Oracle's powerful PL/SQL language, enabling power users and developers to rapidly develop applications that easily scale to hundreds, even thousands of concurrent users. The 13 authors of Expert Oracle Application Express build their careers aro

  4. Disorder of written expression

    Science.gov (United States)

    ... coordination disorder (includes poor handwriting) Expressive language disorder Mathematics disorder Reading disorder ... JW III, Schor NF, Behrman RE, eds. Nelson Textbook of Pediatrics . 19th ed. Philadelphia, PA: Elsevier Saunders; ...

  5. Neuroglobin over expressing mice

    DEFF Research Database (Denmark)

    Raida, Zindy; Hundahl, Christian Ansgar; Nyengaard, Jens R;

    2013-01-01

    thoroughly validated antibodies and oligos, we give a detailed brain anatomical characterization of transgenic mice over expressing Neuroglobin. Moreover, using permanent middle artery occlusion the effect of elevated levels of Neuroglobin on ischemic damage was studied. Lastly, the impact of mouse strain...... genetic background on ischemic damage was investigated. PRINCIPAL FINDINGS: A four to five fold increase in Neuroglobin mRNA and protein expression was seen in the brain of transgenic mice. A β-actin promoter was used to drive Neuroglobin over expression, but immunohistochemistry and in situ hybridization...... infarct volume 24 hours after ischemia. Immunohistochemistry showed no selective sparing of Neuroglobin expressing cells in the ischemic core or penumbra. A significant difference in infarct volume was found between mice of the same strain, but from different colonies. SIGNIFICANCE: In contrast to some...

  6. Express.js blueprints

    CERN Document Server

    Augarten, Ben; Lin, Eric; Shaikh, Aidha; Soriani, Fabiano Pereira; Tisserand, Geoffrey; Zhang, Chiqing; Zhang, Kan

    2015-01-01

    This book is for beginners to Node.js and also for those who are technically advanced. By the end of this book, every competent developer will have achieved expertise in building web applications with Express.js.

  7. Gene Expression Omnibus (GEO)

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene Expression Omnibus is a public functional genomics data repository supporting MIAME-compliant submissions of array- and sequence-based data. Tools are provided...

  8. 嗜肺军团菌mip基因重组质粒GFP-mip的构建及表达%The Construction and Expression of Recombinant Plasmid GFP -mip of Legionella Pneumophila Macrophage Infectivity Potentiator Gene

    Institute of Scientific and Technical Information of China (English)

    惠英华; 曹秀琴; 杨志伟

    2011-01-01

    Objective To construct recombinant plasmid GFP - mip of Legionella pneumophila macrophage infectivity potentiator gene and observe its expression in the NIH3T3 cells. Methods The macrophage infectivity potentiator gene was amplified from DNA of Legionella pneumophila by polymerase chain reation ( PCR),then cloned into pEGFP - C1 vector. The recombinant plasmid was named as GFP - mip and was analyzed with restriction endonuclease XhoI and BarnHl digestion, PCR and DNA sequencing techniques. The NIH3T3 cell was transfected by recombinant plasmid GFP - mip with lipofection strategy. The stable expression products of macrophage infectivity petentiator gene were observed by the fluorescent microscope. Results 702bp mip gene was amplified . Under the fluorescent microscope, green fluorescent was observed in the cell cytoplasm and on the cell membrane. Conclusion The recombinant plasmid GFP - mip was constructed successfully and expressed in the NIH3T3 cells.%目的构建嗜肺军团菌mip基因的真核重组质粒GFP-mip,并观察其在NIH3T3细胞中的表达.方法 以嗜肺军团菌DNA为模版,通过PCR扩增获得mip基因,将其定向克隆到绿色荧光质粒pEGFP-C1中,构建真核重组质粒GFP-mip.经限制性核酸内切酶XhoI和BamHI酶切鉴定、PCR和核酸序列分析后,通过脂质体法转染到NIH3T3细胞中,利用荧光显微镜观察重组质粒的稳定表达.结果 扩增出了702bpmip基因,在细胞质和细胞膜观察到较强绿色荧光.结论 成功构建了真核重组质粒GFP-mip,并在NIH3T3细胞中得到了表达.

  9. A multi-parameter, high-content, high-throughput screening platform to identify natural compounds that modulate insulin and Pdx1 expression.

    Directory of Open Access Journals (Sweden)

    Jessica A Hill

    Full Text Available Diabetes is a devastating disease that is ultimately caused by the malfunction or loss of insulin-producing pancreatic beta-cells. Drugs capable of inducing the development of new beta-cells or improving the function or survival of existing beta-cells could conceivably cure this disease. We report a novel high-throughput screening platform that exploits multi-parameter high-content analysis to determine the effect of compounds on beta-cell survival, as well as the promoter activity of two key beta-cell genes, insulin and pdx1. Dispersed human pancreatic islets and MIN6 beta-cells were infected with a dual reporter lentivirus containing both eGFP driven by the insulin promoter and mRFP driven by the pdx1 promoter. B-score statistical transformation was used to correct systemic row and column biases. Using this approach and 5 replicate screens, we identified 7 extracts that reproducibly changed insulin and/or pdx1 promoter activity from a library of 1319 marine invertebrate extracts. The ability of compounds purified from these extracts to significantly modulate insulin mRNA levels was confirmed with real-time PCR. Insulin secretion was analyzed by RIA. Follow-up studies focused on two lead compounds, one that stimulates insulin gene expression and one that inhibits insulin gene expression. Thus, we demonstrate that multi-parameter, high-content screening can identify novel regulators of beta-cell gene expression, such as bivittoside D. This work represents an important step towards the development of drugs to increase insulin expression in diabetes and during in vitro differentiation of beta-cell replacements.

  10. Impact of copy number of distinct SV40PolyA segments on expression of a GFP reporter gene

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The presence of Alu repeats downregulates the expression of the green fluorescent protein(GFP) gene.We found that SV40PolyA(PolyA,240 bp),in either orientation,eliminated the inhibition of GFP gene expression induced by Alu repeats when it was placed between the GFP gene and the Alu repeats.In this study,4 different segments(each 60 bp) were amplified from antisense PolyA(PolyAas) by PCR,and inserted upstream of Alu14 in pAlu14 plasmid(14 Alu repeats inserted downstream of the GFP gene in vector pEGFP-C1 in a head-tail tandem manner).Segments 1F1R(the first 60 bp segment at the 5’ end of PolyAas) and 4F4R(the fourth 60 bp segment from the 5’ end of PolyAas) did not activate GFP gene expression,whereas 2F2R and 3F3R(the middle two segments) did(as detected by Northern blot analysis and fluorescent microscopy).Different copy numbers of 2F2R and 3F3R segments,in a head and tail tandem manner,were inserted downstream of the GFP gene in pAlu14.p2F2R*4-Alu28,p3F3R*4-Alu18 and p3F3R*4-Alu28 were used as length controls to verify that the decrease in the expression of GFP was not due to the increased length of the inserted segment in the expression vectors.We found that 2 and 4 copies of 2F2R or 3F3R activated the GFP gene more strongly than one copy of them.However,more than 8 copies of 2F2R or 3F3R reduced the activation of the GFP gene.We concluded that SV40PolyAas contained at least two gene-activating elements(2F2R and 3F3R) and 2-4 copies of 2F2R or 3F3R were optimal for the expression of the GFP gene.

  11. Immunogenicity of recombinant plasmid expressing the PRRSV ORF6 and IL-18 genes%PRRSV ORF6及IL-18基因重组真核质粒试验免疫研究

    Institute of Scientific and Technical Information of China (English)

    牛伟; 王中华; 王晓莉; 宋德武; 母连志; 丁壮

    2011-01-01

    The immunogenicity of recombinant plasmids were further evaluated in the piglets by intramuscular injection,which were PRRSV natural host animal.The results indicated the pEGFP-ORF6 and pEGFP-IL18-ORF6 immune groups produced specific antibodies at 14 d post vaccination,and the production of antibodies increased with time,but the antibody levels were not significant(P0.05)between two groups.Neutralizing antibody analysis showed the pEGFP-N1 induced low titer neutralizing antibodies at 42 d post vaccination.However the pEGFP-IL18-ORF6 could enhance cell-meditated immune response and elicit effective Th1 type immune response by stimulating PRRSV-special T lymphocyte proliferation and promoting the secretion levels of IFN-γ and IL-2.It indicated the IL-18 effectively play the part of molecular adjuvant which could enhance the cell-mediated immune response and Th1 type immune response.%将本实验室构建的重组质粒接种PRRSV的自然宿主断奶仔猪,分析其诱发细胞免疫和体液免疫应答的能力。结果表明:重组质粒pEGFP-ORF6和pEGFP-IL18-ORF6免疫组在首免后14d开始产生特异性抗体,且其产生抗体水平随时间呈上升趋势,但pEGFP-ORF6和pEGFP-IL18-ORF6两组之间产生抗体差异不显著(P〉0.05)。中和抗体检测结果显示只有pEGFP-ORF6在首免后42d有低水平的中和抗体产生。细胞免疫检测结果表明,pEGFP-IL18-ORF6诱导T淋巴细胞增殖和促进IFN-γ和IL-2的分泌的能力显著高于pEGFP-ORF6和pEGFP-IL18(P〈0.05),表明IL-18有效地发挥了分子其佐剂的效用,能增强细胞免疫应答并介导较好的Th1类免疫反应。

  12. Itm2a expression in the developing mouse first lower molar, and the subcellular localization of Itm2a in mouse dental epithelial cells.

    Directory of Open Access Journals (Sweden)

    Makiko Kihara

    Full Text Available Itm2a is a type II transmembrane protein with a BRICHOS domain. We investigated the temporospatial mRNA and protein expression patterns of Itm2a in the developing lower first molar, and examined the subcellular localization of Itm2a in murine dental epithelial (mDE6 cells. From the initiation to the bud stage, the in situ and protein signals of Itm2a were not detected in either the dental epithelial or mesenchymal cells surrounding the tooth bud. However, at the bell stage, these signals of Itm2a were primarily observed in the inner enamel epithelium of the enamel organ. After the initiation of the matrix formation, strong signals were detected in ameloblasts and odontoblasts. Itm2a showed a punctate pattern in the cytoplasm of the mDE6 cells. The perinuclear-localized Itm2a displayed a frequent overlap with the Golgi apparatus marker, GM130. A tiny amount of Itm2a was colocalized with lysosomes and endoplasmic reticulum. Minimal or no overlap between the Itm2a-EGFP signals with the other organelle markers for endoplasmic reticulum, lysosome and mitochondria used in this study noted in the cytoplasm. These findings suggest that Itm2a may play a role in cell differentiation during odontogenesis, rather than during the initiation of tooth germ formation, and may be related to the targeting of proteins associated with enamel and dentin matrices in the secretory pathway.

  13. In Silico Expression Analysis.

    Science.gov (United States)

    Bolívar, Julio; Hehl, Reinhard; Bülow, Lorenz

    2016-01-01

    Information on the specificity of cis-sequences enables the design of functional synthetic plant promoters that are responsive to specific stresses. Potential cis-sequences may be experimentally tested, however, correlation of genomic sequence with gene expression data enables an in silico expression analysis approach to bioinformatically assess the stress specificity of candidate cis-sequences prior to experimental verification. The present chapter demonstrates an example for the in silico validation of a potential cis-regulatory sequence responsive to cold stress. The described online tool can be applied for the bioinformatic assessment of cis-sequences responsive to most abiotic and biotic stresses of plants. Furthermore, a method is presented based on a reverted in silico expression analysis approach that predicts highly specific potentially functional cis-regulatory elements for a given stress. PMID:27557772

  14. Freedom of Expression

    Directory of Open Access Journals (Sweden)

    Guilherme Canela

    2007-06-01

    Full Text Available The freedoms of expression and of the press are basic pillars of the western democracies. The contemporary theoretical framework which gives support to these rights was generated in the wake of the liberal revolutions which took place in Western Europe and in North America starting from the second half of the 1600s. Our purpose in this text is to present the current scene regarding this topic, focusing whenever pertinent on the Brazilian case, and seeking to question the unconditional defense of the freedoms of expression and of the press made by the thinkers who founded these principles vis-á-vis contemporary issues of the communicational universe. Going beyond theoretical-conceptual refl ections, we present and analyze the results of a content analysis showing how 53 Brazilian newspapers and 4 magazines with nationwide circulation report (or not topics relating to freedom of expression and of the press.

  15. Regular Expression Containment

    DEFF Research Database (Denmark)

    Henglein, Fritz; Nielsen, Lasse

    2011-01-01

    We present a new sound and complete axiomatization of regular expression containment. It consists of the conventional axiomatiza- tion of concatenation, alternation, empty set and (the singleton set containing) the empty string as an idempotent semiring, the fixed- point rule E* = 1 + E × E* for...... Kleene-star, and a general coin- duction rule as the only additional rule. Our axiomatization gives rise to a natural computational inter- pretation of regular expressions as simple types that represent parse trees, and of containment proofs as coercions. This gives the axiom- atization a Curry......-Howard-style constructive interpretation: Con- tainment proofs do not only certify a language-theoretic contain- ment, but, under our computational interpretation, constructively transform a membership proof of a string in one regular expres- sion into a membership proof of the same string in another regular expression. We...

  16. The expressions of emotions

    DEFF Research Database (Denmark)

    Vishnivetz, Berta

    Abstract On the broadness of the vast field called “Expressions of Emotions” this study focuses on the whole bodily emotional expression. The main question posed is: Whether there are movement patterns specific to each emotion?. I carried out a thorough review of the theories of emotion and of...... expressions of emotions and movement notation that provided the sources for a careful research plan for the empirical process of this study. On this basis I chose to record onto video the four previously choreographed movements that I considered to correspond each of the following emotions: joy, fear, sadness......, anger. The selection of these four emotions demanded previously to clear up the problems the above named survey ensued. When researchers want to describe a certain movement in the field of psychology and non-verbal communication, it may result in disagreements and misunderstandings which sometimes lead...

  17. Boolean Expression Diagrams

    DEFF Research Database (Denmark)

    Andersen, Henrik Reif; Hulgaard, Henrik

    This paper presents a new data structure called Boolean Expression Diagrams (BEDs) for representing and manipulating Boolean functions. BEDs are a generalization of Binary Decision Diagrams (BDDs) which can represent any Boolean circuit in linear space and still maintain many of the desirable...... properties of BDDs. Two algorithms are described for transforming a BED into a reduced ordered BDD. One closely mimics the BDD apply-operator while the other can exploit the structural information of the Boolean expression. The efficacy of the BED representation is demonstrated by verifying that the...

  18. Transmembrane and ubiquitin-like domain-containing protein 1 (Tmub1/HOPS facilitates surface expression of GluR2-containing AMPA receptors.

    Directory of Open Access Journals (Sweden)

    Hyunjeong Yang

    Full Text Available Some ubiquitin-like (UBL domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported.Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1 protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPS-RNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP were coimmunoprecipitated by the anti-Tmub1/HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane.

  19. Modelling logical expressions exercises

    OpenAIRE

    Ruiz Femenia, José Rubén; Aracil Sáez, Ignacio; Caballero Suárez, José Antonio

    2011-01-01

    Exercises of application of the systematic procedure to derive linear inequalities for logic expressions (Ejercicios de aplicación del método sistemático de obtención de restricciones lineales para expresiones lógicas).

  20. Boolean Expression Diagrams

    DEFF Research Database (Denmark)

    Andersen, Henrik Reif; Hulgaard, Henrik

    2002-01-01

    This paper presents a new data structure called boolean expression diagrams (BEDs) for representing and manipulating Boolean functions. BEDs are a generalization of binary decision diagrams (BDDs) which can represent any Boolean circuit in linear space. Two algorithms are described for transforming...

  1. Facial Expression Analysis

    NARCIS (Netherlands)

    Pantic, Maja; Li, S.; Jain, A.

    2009-01-01

    Facial expression recognition is a process performed by humans or computers, which consists of: 1. Locating faces in the scene (e.g., in an image; this step is also referred to as face detection), 2. Extracting facial features from the detected face region (e.g., detecting the shape of facial compon

  2. Expression of Concern

    Science.gov (United States)

    Delvaux, Damien

    2016-08-01

    This is a note of a temporary expression of concern related to the publication titled, "Sapphirine and fluid inclusions in Tel Thanoun mantle xenoliths, Syria" by Ahmad Bilal, which appeared in Journal of African Earth Sciences, 116 (2016) 105-113.

  3. Collaborating on Referring Expressions

    CERN Document Server

    Heeman, P A; Heeman, Peter A.; Hirst, Graeme

    1995-01-01

    This paper presents a computational model of how conversational participants collaborate in order to make a referring action successful. The model is based on the view of language as goal-directed behavior. We propose that the content of a referring expression can be accounted for by the planning paradigm. Not only does this approach allow the processes of building referring expressions and identifying their referents to be captured by plan construction and plan inference, it also allows us to account for how participants clarify a referring expression by using meta-actions that reason about and manipulate the plan derivation that corresponds to the referring expression. To account for how clarification goals arise and how inferred clarification plans affect the agent, we propose that the agents are in a certain state of mind, and that this state includes an intention to achieve the goal of referring and a plan that the agents are currently considering. It is this mental state that sanctions the adoption of g...

  4. Brandom's Expressive Conception of Logic

    OpenAIRE

    Streed, Adam Joseph

    2015-01-01

    This dissertation is an exploration of the expressive conception of logic, as found in the work of Robert Brandom. I situate the expressive conception against a historical background of thought about logic, investigate the key notion of material inference on which the expressive conception rests, and argue that the expressive conception counts as a form of psychologism about logic which avoids many of the difficulties faced by older forms of psychologism. Finally, I argue that the expressive ...

  5. Rapid Purification of Enhanced Green Fluorescent Protein from Escherichia coli%快速纯化在大肠杆菌中表达的增强型绿色荧光蛋白

    Institute of Scientific and Technical Information of China (English)

    周笑鹏; 史清洪; 邢新会; 孙彦

    2006-01-01

    As an excellent reporter molecule, enhanced green fluorescent protein (eGFP) was widely used for gene expression and regulation and was generally expressed in Escherichia coli strain. A rapid procedure consisting of ammonium sulfate precipitation, size exclusion chromatography, and anion exchange chromatography was developed for the purification of eGFP. Based on the proposed procedure, recombinant eGFP with an electrophoretic purity was achieved in combination with an overall yield of 66% and a purification factor of 17.9. The fluorescent spectrometry of purified eGFP and lysate from E. coli strain expressing eGFP exhibited the same wavelength of excitation and emission maxima, indicating that the purification procedure did not influence the construct and fluorescent characteristics of desired protein. The procedure mentioned was easy to scale up for the purification of large quantities of eGFP.

  6. Regulated expression of a transgene introduced on an oriP/EBNA-1 PAC shuttle vector into human cells

    Directory of Open Access Journals (Sweden)

    Thorsen Jim

    2009-10-01

    Full Text Available Abstract Background Sequencing of the human genome has led to most genes being available in BAC or PAC vectors. However, limited functional information has been assigned to most of these genes. Techniques for the manipulation and transfer of complete functional units on large DNA fragments into human cells are crucial for the analysis of complete genes in their natural genomic context. One limitation of the functional studies using these vectors is the low transfection frequency. Results We have constructed a shuttle vector, pPAC7, which contains both the EBNA-1 gene and oriP from the Epstein-Barr virus allowing stable maintenance of PAC clones in the nucleus of human cells. The pPAC7 vector also contains the EGFP reporter gene, which allows direct monitoring of the presence of PAC constructs in transfected cells, and the Bsr-cassette that allows highly efficient and rapid selection in mammalian cells by use of blasticidin. Positive selection for recombinant PAC clones is obtained in pPAC7 because the cloning sites are located within the SacBII gene. We show regulated expression of the CDH3 gene carried as a 132 kb genomic insert cloned into pPAC7, demonstrating that the pPAC7 vector can be used for functional studies of genes in their natural genomic context. Furthermore, the results from the transfection of a range of pPAC7 based constructs into two human cell lines suggest that the transfection efficiencies are not only dependent on construct size. Conclusion The shuttle vector pPAC7 can be used to transfer large genomic constructs into human cells. The genes transferred could potentially contain all long-range regulatory elements, including their endogenous regulatory promoters. Introduction of complete genes in PACs into human cells would potentially allow complementation assays to identify or verify the function of genes affecting cellular phenotypes.

  7. 荧光标记肿瘤转移体内模型的建立%Establishment of green-fluorescent protein expressing tumor metastasis models

    Institute of Scientific and Technical Information of China (English)

    冯海凉; 刘玉琴; 顾蓓; 卞晓翠; 杨振丽; 杨丽娟

    2009-01-01

    Objective To establish a green-fluorescent protein (GFP) labeled tumor metastasis model and to evaluate its biological characteristics.Methods Human gastric carcinoma cell MGC-803 and murlne cervical carcinoma cell U14 were transfected with the plasmid pEGFP-N1 and the efficiency of transfection was assessed 24 h Iater.Limited dilution was employed to screen and establish menoclonal cell strains,MGC-803-GFP and U14-GFP.The two fluorescent tumor cell stains were transplanted into BALB/c-nu mice and C57BL/6J mice respectively.The latency period of tumor mass appearance and the growth curve in vivo were documented.The tumor growth and metastasis were evaluated in vivo by the Viviperception Fluorescence Imagining System(VFIS).Expressions of CD44 and E-cadherin in tumor tissue were monitored by immunohistochemistry.Results The efficiency of pEGFP-N1 transfeetion of MGC-803 cells and U14 cells were 30%and 60%,respectively.Monoclonal GFP(+)cell strains-MGC-803-GFP and U14-GFP were established.The latency periods of tumor formation of MGC-803-GFP and U14.GFP were 3-5 days and 2-4 days,respectively.Their tumorigenicity rates were 100%in both.The tumor growth of MGC-803-GFP was well defined by the VFIS. Only one mouse was shown to harbor lymphatic metastasis by VFIS.60 days after transplantation.The metastasis process of U14-GFP wag depicted through VFIS on 27.37 and 52 days post-transplantation.The incidence of pulmonary metastasis and lymphatic metastasis of U14-GFP was 67% and 100% respectively when the tumor vo]ume was ≥5 cm3. CD44 was positive and E-cadherin was negative in both tumors by immunohistochemistry.Conclusions Successfully established two monoclonal tumor cell strains stably expressing GFP:MGC-803-GFP and U14-GFP.Transplantation of these cells into mice can establish tumor metastasis models which could be used for future visualized tumor research in vivo.%目的 建立一种带荧光标记的肿瘤转移模型并探讨其应用价值.方法 人胃癌MGC-803

  8. 哺乳动物细胞CDK2系列表达载体的构建与表达%Construction and expression of the CDK2 mammalian cell expression vectors

    Institute of Scientific and Technical Information of China (English)

    吴健谊; 蒋太峰; 谢剑君; 张锴; 杜则澎; 许丽艳

    2011-01-01

    目的:构建一系列细胞周期蛋白依赖激酶2(cyclin-dependent kinase 2,CDK2)在哺乳动物细胞的表达载体,为研究CDK2的功能和修饰提供实验材料.方法:从食管癌细胞中提取总RNA,逆转录PCR扩增CDK2编码区,然后将PCR产物克隆到T载体;扩增后的CDK2片段分别亚克隆入pcDNA3、pcDNA4、pNTAP和pEGFP等4种哺乳动物表达载体;最后,将获得的表达载体PEGFP/CDK2转染小鼠成纤维细胞NIH3T3进行初步的CDK2表达分析.结果:RT-PCR扩增获得约900 bp的目的片段,经T载体克隆和DNA序列分析,显示重组片段是人CDK2基因序列;CDK2片段分别亚克隆入上述4种载体后获得相应表达载体;运用构建的PEGFP/CDK2表达载体,在NIH3T3细胞中表达出CDK2蛋白.结论:成功构建了CDK2的哺乳动物细胞系列表达载体,并在NIH3T3细胞中成功表达目的蛋白.%OBJECTIVE: To construct a series of cyclin-dependent kinase 2 (CDK2) mammalian cell expression vectors and to assess CDK2 expression in NIH3T3 cells. METHODS: RNA was isolated from esophageal cancer cells and the full coding sequence of CDK2 gene was obtained by RT-PCR. The PCR product was then cloned into T vector and subsequently subcloned into four eukaryotic expression vectors (pcDNA3, pcDNA4, pNTAP and pEGFP). The expressing plasmids were transfected into NIH3T3 cells and the expression of CDK2 was detected by western blot. RESULTS: The PCR product was about 900 bp and the sequence analysis showed that it was the full coding sequence of CDK2 gene. The product was subcloned into the eukaryotic expression vectors and four CDK2 expression vectors were constructed. Western blot showed that CDK2 could be expressed in the expression vector-transfected cells.CONCLUSION: Four CDK2 eukaryotic expression vectors were successfully constructed and CDK2 was effectively expressed in NIH3T3 cells.

  9. The Expression of GFP Gene in Transformed and Tumor Cells%绿色荧光蛋白GFP在转化细胞和肿瘤细胞中的表达

    Institute of Scientific and Technical Information of China (English)

    于丽莉; 陈诗书

    2000-01-01

    目的 在肿瘤基因治疗研究中建立一个用绿色荧光蛋白(green fluorescent protein,GFP)为标记基因的合适条件。方法 用带有GFP突变体的质粒转染到表达细胞,观察GFP瞬时表达的情况:1.直接测定GFP在COS-7细胞中的表达并观察表达的稳定性;2.比较不同启动子驱动的pcDNAa-EGFP和pSVKa-S65T两种质粒在不同肿瘤细胞中的转染效率;3.用LacZ基因与GFP基因共转染48h后,通过FACS分选测定两种基因在同一细胞中的表达情况。结果 在不同条件下的活细胞中有GFP基因表达,且表达具有稳定性,表达持续约2周左右;CMV启动子和SV40启动子调控的GFP对不同肿瘤细胞株的转染效率有差异。当两种基因共转染后用FACS分选,选出GFP细胞带有LacZ基因细胞在1:4时可达85%以上。结论 通过GFP表达可连续而直接地观察活细胞中的基因表达,利用GFP可快速地挑选带有靶基因的细胞。%Objective To search the appropriate experimental conditions for using green fluores-cent protein (GFP) as a reporter gene in tumor gene therapy. Methods The plasmids carrying mu-tated GFP gene were transfected into the eukaryotic cells to observe transient gene expression. Thesestudies were conducted to 1. directly determine GFP expression and express stability in the COS- 7cells, 2. compare the transfection efficiency of two plasmids pcDNA3 - EGFP, pSVKa - S65T with dif-ferent promoters in different tumor cell lines, 3. determine two genes expression in a single cell usingLacZ cotransfected with GFP by FACS. Results Fluorescence could be detected in intact viable cellsunder different sets of conditions. The expression of GFP might last two weeks or more and the ex-pressed fluorescence was stable. The transfection rate of pcDNA3 - EGFP expressed was different inthree tumor cell lines examined. But pSVK3 - GFP expressed similarly in four tumor cell lines exam-ined. FACS showed the

  10. HBx、MHBst155真核表达载体构建及在HepG2细胞中的表达%Construction of eukaryotic recombinants HBx, MHBst155 and establishment of hepG2 cell line with stable expression of HBx, MHBst155 fusion protein

    Institute of Scientific and Technical Information of China (English)

    康艳红; 杨林; 麦丽; 张绍全; 胡朝霞; 谢奇峰; 高志良

    2012-01-01

    Objective To establish HepG2 cell lines with stable expression of X protein (HBx) and the carboxyl-terminal truncated molecule surface protein (MHBst) of hepatitis Bvirus(HBV)in order to further explore the roles of HBx and MHBst in hepatocellular carcinogenesis. Methods HBV X gene and eneoding-MHBst55 gene fragments were amplified from the subtype adr of plasmid pHBV DNA by PCR, and the amplified fragments were inserted respectively into Bgt II , Kpn I and Bgt II , BamH I restriction endonuclease sites of the green fluorescent protein (GFP) expression vector pEGFP-Cl to construct the recombinant plasmids pGFP-HBx and pGFP-MHBst55. Then, the pEGFP-C1 and recombinant plasmids were transfected into human hepatoma HepG2 cells by liposome-mediated method. Resistant cell clones were selected with G418, and the expression of GFP in the resistant clones were examined directly with fluorescence microscope. These GFP-expressing resistant clones were expanded. Expression of HBx and MHBst55 proteins in the GFP-expressing resistant cells were detected by RT-PCR and Western blot. Results Recombinant plasmid pGFP-HBx and pGFP-MHBst155 were successfully constructed as judged from the restriction endonuclease analysis and DNA sequencing. After transfecting with pEGFP-C1 and recombinant plasmids, resistant HepG2 cell clones expressing GFP were obtained by selecting with G418 for about 20 days. The resistant HepG2 cells were obtained, and the expression of GFP by these cells was stable for over 40 generations. RT-PCR and Western blot analysis showed that HBx and MHBst155 was only expression in HepG2/GFP-HBx and HepG2/GFP-MHBsst135 cells, respectively. Conclusion The HBx and MHBst155 recombinant expression plasmids pGFP-HBx and pGFP-MHBst155 were successfully constructed. The HepG2 cell lines were found to stably express GFP, GFP-HBx, or GFP-MHBst155 fusion protein. The plasmids may be used for further study on the molecular mechanisms by which HBx and MHBst involve in hepatocellular

  11. GFAP启动子介导放射性131Ⅰ靶向性治疗胶质瘤的实验研究%Glial fibrillary acidic protein promoters directed sodium iodide symporter expression in malignant gioma radioiodine therapy

    Institute of Scientific and Technical Information of China (English)

    李玮; 谭建; 王澎

    2013-01-01

    possibility that the glial fibrillary acidic protein (GFAP) promoters modulate the human sodium iodide symporter (hNIS) expression in glioma and lead transecting hNIS gene into glioma cells for radioactive iodide treatment.Methods PGL3-Basic,PGL3-Control and PGL3-GFAP plasmids were transfected into U251,U87 and MRC-5 cells,respectively,with the help of liposome Lipofectamine 2000; 24 h after that,the reactivity of these cells was detected and the efficiency of GFAP promoter was tested under chemiluminescence apparatus.Recombinant adenovirus vector Ad-GFAP-hNIS in which GFA P promoter could modulate the hNIS gene expression was constructed,and then,the vector was transfected into the U251,U87 and MRC-5 cells; Western blotting was employed to detect the protein expressions of GFAP and hNIS.Ad-CMV-EGFP group (blank control) and Ad-CMV-hNIS group (negative control) and Ad-GFAP-hNIS group were employed; the 125I uptake and effiux abilities and the cell amount after gentian violet staining in the three groups were measured by γ counter; the clonogenecity rate of them was calculated.BALB/c female nude mice (n=20) was divided into four groups:group of injecting Ad-GFAP-hNIS without 131I,group of injecting Ad-GFAP-hNIS with 131I,group of injecting Ad-CMV-EGFP without 131I and group of injecting Ad-CMV-EGFP with 131I (n=5);U87 cells were transfected into the nude mice,and then,the tumor growth was observed and the life cycle of the mice was noted.Nude mice bearing the U87 tumors were injected Ad-GFAP-hNIS and Ad-CMV-EGFP,followed by 1 mCi 99mTcO4 via intraperitoneal injection; single photon emission computed tomography (SPECT) was performed.Results As compared with that of cells being transfected with PGL3-blank plasmid,the relative reactivity of U251 and U87 cells being transfected with PGL3-GFAP plasmid was decreased with significant difference (P<0.05).Western blotting revealed GFAP and hNIS proteins in U87 and U251 cells.125I uptake of U87 and U251 cells after Ad

  12. Cloning of murine BRI3 gene and study on its function for inducing cell death

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To understand the molecular mechanism of TNFα effects, the cDNA of murine BRI3 gene was cloned from the total RNA of murine brain endothelial cells (bEnd.3)treated with hTNFα by using the suppression subtractive hybridization (SSH) and the RT-PCR method. The fusion expression vector harbouring BRI3 gene and enhanced green fluorescence protein (EGFP) thus obtained were designated as pEGFP/I3. Then pEGFP/I3 was transiently transfected into L929 cells and the fusion protein EGFP/I3 was localized in cytoplasm. It is found that the expression of EGFP/I3 could induce cell death in L929 cells detected by TUNEL method and flow cytometry. And the overexpression of Bci-2 in L929 cells can block cell death induced by EGFP/I3, indicating that murine BRI3 gene might related to the TNFα mediated cytotoxicity.

  13. Facial Expressions Recognition Using Eigenspaces

    OpenAIRE

    Senthil Ragavan Valayapalayam Kittusamy; Venkatesh Chakrapani

    2012-01-01

    A challenging research topic is to make the Computer Systems to recognize facial expressions from the face image. A method of facial expression recognition, based on Eigenspaces is presented in this study. Here, the authors recognize the userâs facial expressions from the input images, using a method that was customized from eigenface recognition. Evaluation was done for this method in terms of identification correctness using two different Facial Expressions databases, Cohn-Kanade facial exp...

  14. Association Between the Single Nucleotide Polymorphism and the Level of Aquaporin-4 Protein Expression in Han and Minority Chinese with Inflammatory Demyelinating Diseases of the Central Nervous System.

    Science.gov (United States)

    Chu, Lan; Dai, Qingqing; Xu, Zhu; He, Dian; Wang, Hao; Wang, Qingsong; Zhang, Yifan; Zhu, Yingwu; Li, Yuan; Cai, Gang; Slavica, Krantic; Allan, Kermode

    2016-07-01

    The purpose of this study was to determine whether or not aquaporin-4 (AQP4) gene mutations are related to the pathogenesis of inflammatory demyelinating diseases in the central nervous system. Polymorphisms of AQP4 exons 1-5 were determined by sequencing DNA from 67 patients with central nervous system inflammatory demyelinating diseases, including neuromyelitis optica (NMO), multiple sclerosis, recurrent or simultaneous bilateral optic neuritis, and longitudinally extensive transverse myelitis. A plasmid with the identified new missense mutation was constructed, and human embryonic kidney cells (HEK293A) were transfected with either the pEGFP-N1-AQP4-M23 vector (bearing the identified mutated cDNA sequence) or with the plasmid bearing the wild-type AQP4 gene sequence. AQP4 protein expression was analyzed in both experimental groups using Western Blot analysis following protein extraction from transfected cells. A synonymous mutation (rs1839318) was detected on exon 3, and an additional synonymous mutation was detected on the exon 2-2 (rs72557968). Most importantly, a new missense mutation was detected on exon 2-1. According to Western blot analysis, the mutated cDNA sequence yielded increased AQP4 protein expression in comparison with the wild-type cDNA sequence (P < 0.05). AQP4 gene mutations are uncommon, occurring in only 3 out of 67 patients. Although it is possible that the mutations contributed to an increased risk of inflammatory central nervous system disease in these individuals, it is unlikely that mutations are a significant contributor to most patients with NMO spectrum disorders in China. PMID:25895050

  15. Constitutive expression of pathogen-inducible OsWRKY31 enhances disease resistance and affects root growth and auxin response in transgenic rice plants

    Institute of Scientific and Technical Information of China (English)

    Juan Zhang; Youliang Peng; Zejian Guo

    2008-01-01

    WRKY transcription factors have many regulatory roles in response to biotic and abiotic stresses. In this study, we isolated a rice WRKY gene (OsWRKY31) that is induced by the rice blast fungus Magnaporthe grisea and auxin. This gene encodes a polypeptide of 211 amino-acid residues and belongs to a subgroup of the rice WRKY gene family that probably originated after the divergence of monocot and dicot plants. OsWRKY31 was found to be localized to the nucleus of onion epidermis cells to transiently express OsWRKY31-eGFP fusion protein. Analysis of 0sWRKY31 and its mutants fused with a Cal4 DNA-binding domain indicated that OsWRKY31 has transactivation activity in yeast. Overexpression of the OsWRKY31 gene was found to enhance resistance against infection with M. grisea, and the transgenic lines exhibited reduced lateral root formation and elongation compared with wild-type and RNAi plants. The lines with overexpression showed constitutive expression of many defense-related genes, such as PBZ1 and OsSci2, as well as early auxin-response genes, such as OsIAA4 and OsCrll genes. Furthermore, the plants with overexpression were less sensitive to exogenously supplied IBA, NAA and 2,4-D at high concentrations, suggesting that overexpression of the OsWRKY31 gene might alter the auxin response or transport. These results also suggest that OsWRKY31 might be a common component in the signal transduction pathways of the auxin response and the defense response in rice.

  16. Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat hindlimb of ischemia

    Institute of Scientific and Technical Information of China (English)

    孙晋津

    2012-01-01

    Objective To investigate the effect of plasmid pEGFP-hepatocyte growth factor (HGF)-Cl on rat acute ischemia of hindlimb. Methods The eukaryotic expressed plasmid pEGFP-HGF-Cl carrving human HGF cDNA was constructed. The transfection efficiency and the expression level of HGF were evaluated

  17. Construction of bivalent plant expression vector with cold-induced gene of Chorispora bungeana in sugarcane%高山离子芥冷诱导基因转化甘蔗二元植物表达载体构建

    Institute of Scientific and Technical Information of China (English)

    卢双楠; 刘晓静; 李鸣; 梁俊; 李容柏; 李粲; 滕峥; 刘开雨; 刘芳; 邱永福; 梁朝旭; 方位宽; 何姗珊

    2012-01-01

    [目的]利用载体重组技术分别将高山离子芥冷诱导基因(Cbcor15a)和报告基因eGF插入载体pCambia1300-bar,并引入玉米泛素基因启动子UBi-1代替载体本身启动子CaMV 35s,重组为适合甘蔗转基因的二元植物表达载体pCambia 1300-cbcor 15a-bar.[方法]参照pCambia1300-bar载体多克隆位点和基因Cbcor15a、eGFP和启动子UBi-1的核苷酸序列设计引物,通过载体重组技术将基因和启动子分别插入相应的位点.利用基因枪分别将pCambia1300-bar载体和重组载体导入洋葱表皮细胞,用荧光显微镜和激光共聚焦显微镜观察.[结果]与导入pCambia 1300-bar载体的洋葱表皮细胞相比较,导入重组载体pCambia1300-cbcor15a-bar的洋葱表皮细胞内有强烈的绿色荧光信号.[结论]重组甘蔗转基因二元植物表达载体启动子Ubi-1能够调控下游冷诱导基因Cbcor15a和报告基因eGFP的正常高效表达,为外源基因Cbcor15a转化甘蔗提供保障.%[Objective]A new bivalent plant expression vector, named pCambial300-cbcorl5a-bar, was recombined by inserting two genes, I.e. Cbcor15a and eGFP, into the vector pCambial300-bar and replacing the promoter CaMV 35s with Ubi-1. [Method]Based on the multiple cloning sites of the expression vector pCambial300-bar, the primers were designed according to the nucleotide sequence of gene Cbcorl5a and eGFP and the promoter Ubi-1, and then the fragments of the genes and promoter were inserted into the vector pCambial300-bar. The plasmids of the vector pCambial300-cb-cor15a-bar were transduced into onion epidermal cells via particle bombardment. The results were observed through fluorescence microscopy and confocal laser scanning microscope, respectively. [Result]Compared to onion epidermal cell with pCambia 1300-bar, onion epidermal cell transduced with the recombinant vector plastnid had a very bright green florescence. [Conclusion]Downstream cold-induced gene Cbcorl5a and reporter gene eGFP regulaled by up

  18. Natural Art, False Expressions

    Directory of Open Access Journals (Sweden)

    Oscar Hernando Nossa García

    2011-05-01

    Full Text Available In the documentary My Kid Could Paint That, directed by Bar-Lev, which deals with Marla Olmstead, the child prodigy of painting, several interviews with persons in the art world are conducted, among them an artist who uses a magnifying glass and the thinnest brushes to do his work. This man, although happy for the success of the child’s abstract paintings, saw in the whole spectacle a mockery of art, and stood firmly by her work. The girl’s father, also an artist, was accused of plagiarism. Cameras entered the child’s studio in order to prove that Marla was the real artist. Why should such relevance be given to authorship? What is the cause of the dispute between the expressive and the rational?

  19. Barley beta-glucan promotes MnSOD expression and enhances angiogenesis under oxidative microenvironment

    Science.gov (United States)

    Agostini, Silvia; Chiavacci, Elena; Matteucci, Marco; Torelli, Michele; Pitto, Letizia; Lionetti, Vincenzo

    2015-01-01

    Manganese superoxide dismutase (MnSOD), a foremost antioxidant enzyme, plays a key role in angiogenesis. Barley-derived (1.3) β-d-glucan (β-d-glucan) is a natural water-soluble polysaccharide with antioxidant properties. To explore the effects of β-d-glucan on MnSOD-related angiogenesis under oxidative stress, we tested epigenetic mechanisms underlying modulation of MnSOD level in human umbilical vein endothelial cells (HUVECs) and angiogenesis in vitro and in vivo. Long-term treatment of HUVECs with 3% w/v β-d-glucan significantly increased the level of MnSOD by 200% ± 2% compared to control and by 50% ± 4% compared to untreated H2O2-stressed cells. β-d-glucan-treated HUVECs displayed greater angiogenic ability. In vivo, 24 hrs-treatment with 3% w/v β-d-glucan rescued vasculogenesis in Tg (kdrl: EGFP) s843Tg zebrafish embryos exposed to oxidative microenvironment. HUVECs overexpressing MnSOD demonstrated an increased activity of endothelial nitric oxide synthase (eNOS), reduced load of superoxide anion (O2−) and an increased survival under oxidative stress. In addition, β-d-glucan prevented the rise of hypoxia inducible factor (HIF)1-α under oxidative stress. The level of histone H4 acetylation was significantly increased by β-d-glucan. Increasing histone acetylation by sodium butyrate, an inhibitor of class I histone deacetylases (HDACs I), did not activate MnSOD-related angiogenesis and did not impair β-d-glucan effects. In conclusion, 3% w/v β-d-glucan activates endothelial expression of MnSOD independent of histone acetylation level, thereby leading to adequate removal of O2−, cell survival and angiogenic response to oxidative stress. The identification of dietary β-d-glucan as activator of MnSOD-related angiogenesis might lead to the development of nutritional approaches for the prevention of ischemic remodelling and heart failure. PMID:25388628

  20. Remembering Faces with Emotional Expressions

    Directory of Open Access Journals (Sweden)

    Chang Hong eLiu

    2014-12-01

    Full Text Available It is known that happy faces create more robust identity recognition memory than faces with some other expressions. However, this advantage was not verified against all basic expressions. Moreover, no research has assessed whether similar differences also exist among other expressions. To tackle these questions, we compared the effects of six basic emotional expressions on recognition memory using a standard old/new recognition task. The experiment also examined whether exposure to different emotional expressions at training creates variable effects on transfer of the trained faces to a new/neutral expression. Our results suggest that happy faces produced better identity recognition relative to disgusted faces, regardless of whether they were tested in the same image or a new image displaying a neutral expression. None of the other emotional expressions created measurable advantage for recognition memory. Overall, our data lend further support for the happy face advantage for long-term recognition memory. However, our detailed analyses also show that the advantage of happy expression on identity recognition may not be equally discernible from all other emotional expressions.

  1. Facial Expressivity at 4 Months: A Context by Expression Analysis.

    Science.gov (United States)

    Bennett, David S; Bendersky, Margaret; Lewis, Michael

    2002-01-01

    The specificity predicted by differential emotions theory (DET) for early facial expressions in response to 5 different eliciting situations was studied in a sample of 4-month-old infants (n = 150). Infants were videotaped during tickle, sour taste, jack-in-the-box, arm restraint, and masked-stranger situations and their expressions were coded second by second. Infants showed a variety of facial expressions in each situation; however, more infants exhibited positive (joy and surprise) than negative expressions (anger, disgust, fear, and sadness) across all situations except sour taste. Consistent with DET-predicted specificity, joy expressions were the most common in response to tickling, and were less common in response to other situations. Surprise expressions were the most common in response to the jack-in-the-box, as predicted, but also were the most common in response to the arm restraint and masked-stranger situations, indicating a lack of specificity. No evidence of predicted specificity was found for anger, disgust, fear, and sadness expressions. Evidence of individual differences in expressivity within situations, as well as stability in the pattern across situations, underscores the need to examine both child and contextual factors in studying emotional development. The results provide little support for the DET postulate of situational specificity and suggest that a synthesis of differential emotions and dynamic systems theories of emotional expression should be considered. PMID:16878184

  2. Inhibition of RAW264.7 Macrophage Inflammatory Cytokines Release by Small Haparin RNAi Targeting TLR4

    Institute of Scientific and Technical Information of China (English)

    WANG Hui; ZHANG Jinxiang; WU Heshui; JIANG Chunfang; ZHENG Qichang; LI Zhuoya

    2006-01-01

    In order to construct an expression vector carrying small hairpin (sh) RNA (shRNA) for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS)stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism, the reporter gene plasmid pEGFP-C1 (4.7 kb) and psiRNA-hHlneo (2979 bp) were used. The H1 promotor and double Bbs Ⅰ restrict endoenzyme site were cloned from plasmid psiRNA-hHlneo and reconstructed them into plasmid pEGFP-C1 in the Mlu Ⅰ restrict endoenzymic site, forming plasmid pEGFP-H1/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H 1/siRNA forming plasmid pEGFP-H 1/TLR4-siRNA. After transfection of pEGFP-H1/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha (TNF-α) release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was (50.37±8.23) %and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA the level of TNF-α released by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-α release by RAW264.7 cells evoked by LPS.

  3. Altered Astrocytic Swelling in the Cortex of α-Syntrophin-Negative GFAP/EGFP Mice

    Czech Academy of Sciences Publication Activity Database

    Anděrová, Miroslava; Benešová, Jana; Mikešová, Michaela; Džamba, Dávid; Honsa, Pavel; Kriška, Ján; Butenko, Olena; Novosadová, Vendula; Valihrach, Lukáš; Kubista, Mikael; Dmytrenko, L.; Cicanič, M.; Vargová, L.

    2014-01-01

    Roč. 9, č. 11 (2014), e113444. E-ISSN 1932-6203 R&D Projects: GA ČR GA13-02154S; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) ED1.1.00/02.0109 Grant ostatní: GA MŠk(CZ) CZ.1.07/2.3.00/30.0045 Institutional support: RVO:68378041 ; RVO:86652036 Keywords : astrocyte volume regulation * aquaporines * extracellular space Subject RIV: FH - Neurology Impact factor: 3.234, year: 2014

  4. Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo

    Directory of Open Access Journals (Sweden)

    Anesti Anna-Maria

    2010-09-01

    Full Text Available Abstract Background Delivery of small interfering RNA (siRNA to tumours remains a major obstacle for the development of RNA interference (RNAi-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV OncoVEXGM-CSF, we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV. Methods To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA or artificial microRNA (miRNA against the reporter genes green fluorescent protein (eGFP and β-galactosidase (lacZ. These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting. Results Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective. Conclusions This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen

  5. Techniques in Facial Expression Recognition

    OpenAIRE

    Avinash Prakash Pandhare; Umesh Balkrishna Chavan

    2016-01-01

    Facial expression recognition is gaining widespread importance as the applications related to Human – Computer interactions are increasing. This paper mentions various techniques and approaches that have been used in the field of facial expression recognition. Facial expression recognition takes place in various stages and these stages have been implemented by various approaches. Viola and Jones for face detection, Gabor filters for feature extraction, SVM classifiers for classifi...

  6. Natural Editing of Algebraic Expressions

    OpenAIRE

    Nicaud, Jean-François

    2007-01-01

    We call “natural editing of algebraic expressions” the editing of algebraic expressions in their natural representation, the one that is used on paper and blackboard. This is an issue we have investigated in the Aplusix project, a project which develops a system aiming at helping students to learn algebra. The paper summarises first the Aplusix project. Second it presents a notion of algebraic expressions, of representations of algebraic expressions. The last section develops ideas about natu...

  7. XPD对SMMC-7721肝癌细胞中DNp73和GADD45β的调控及意义%Overexpression of XPD decreases DNp73 expression and increases GADD45β expression in SMMC-7721 hepatoma cells

    Institute of Scientific and Technical Information of China (English)

    王芬芬; 张吉翔

    2012-01-01

    AIM: To evaluate the impact of transfection with the XPD gene on the expression of DNp73 and GADD45p and cell proliferation and apoptosis in human hepatoma cell line SMMC-7721.METHODS: After SMMC-7721 cells were trans-fected with SMMC-7721-pEGFP-N2-XPD, the mRNA and protein expression of DNp73 and GADD45β was detected by RT-PCR and Western blot, respectively; cell proliferation was assessed by MTT assay; and the changes in cell apoptosis were evaluated by flow cytometry.RESULTS: Compared to control cells, the expres-sion of DNp73 mRNA decreased significantly and that of XPD and GADD45β mRNAs was enhanced obviously in cells transfected with XPD (all P < 0.01). Similar results were obtained for the expression of XPD, DNp73 and GADD45p proteins. The proliferation of SMMC-7721 cells was markedly inhibited and the apoptosis of SMMC-7721 cells was increased after transfection with XPD (both P< 0.01).CONCLUSION: The wild-type XPD plays an important inhibitory role in the carcinogenesis of HCC. Overexpression of XPD decreases the expression of DNp73 and increases the expression of GADD45p, which suggests that both DNp73 and GADD45p may play a key role in the inhibitory effect of XPD on the carcinogenesis of HCC.%目的:观察人剪切修复基因人类着色性干皮病D组基因(xeroderma pigmentosum group D,XPD)转染至人肝癌细胞株SMMC-7721细胞后XPD、DNp73和GADD45p基因的表达变化以及对肝癌细胞生长的影响.方法:实验分4组:重组质粒SMMC-7721-pEGFP-N2-XPD(XPD组)、空载质粒SMMC-7721-pEGFP-N2组(N2组),脂质体组和SMMC-7721细胞空白对照组.应用Lipofectamine2000脂质体瞬时转染,逆转录聚合酶链反应(RT-PCR)和蛋白印迹(Westernblot)法检测转XPD基因后,人肝癌细胞株SMMC-7721细胞中DNp73以及GADD45β的mRNA和蛋白质的表达量变化,并用四甲基偶氮唑盐(MTT)法检测细胞增殖的活力,流式细胞仪检测细胞凋亡的变化.结果:荧光显微镜下,XPD组和N2组细胞中观察到

  8. Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA

    OpenAIRE

    Watkins, Craig; Hopkins, John; Harkiss, Gordon

    2005-01-01

    Dendritic cells (DC) play an integral role in plasmid DNA vaccination. However, the interaction between plasmid DNA and DC in vivo is incompletely understood. In this report, we utilise the sheep pseudoafferent cannulation model to examine the interaction between plasmid DNA encoding enhanced green fluorescent protein (pEGFP) and afferent lymph DC (ALDC) following gene gun administration. The results show that peaks of fluorescent ALDC tended to appear around days 1-4 and 9-13, then erratical...

  9. Photodynamic therapy cures green fluorescent protein expressing RIF1 tumors in mice

    Science.gov (United States)

    Castano, Ana P.; Liu, Qingde; Hamblin, Michael R.

    2004-07-01

    Cancer is a leading cause of death among modern people, largely due to metastatic disease. The ideal cancer treatment should destroy both the primary tumor and distant metastases with minimal toxicity to normal tissue. This is best accomplished by educating the body's immune system to recognize the tumor as foreign so that after the primary tumor is destroyed, distant metastases will also be eradicated. Photodynamic therapy (PDT) involves the IV administration of photosensitizers followed by illumination of the tumor with red light producing reactive oxygen species that eventually cause vascular shutdown and tumor cell apoptosis. Anti-tumor immunity is stimulated after PDT due to the acute inflammatory response, generation of tumor-specific antigens, and induction of heat-shock proteins. Combination regimens are likely to emerge in the future to even further enhance immunity. Green fluorescent protein is used as an optical reporter to non-invasively image the progression of mouse tumors, and in addition, may act as a foreign (jellyfish) antigen. We asked whether the response of tumor bearing mice to PDT differed when a non-immunogenic tumor cell line was transfected with GFP? We injected RIF-1 or RIF1-EGFP cells in the leg of C3H/HeN mice and both the cells and tumors grew equally well. We used two PDT protocols (benzoporphyrin derivative (BPD) with 15-minute interval or Photofrin with 24-hour interval). The results showed significant differences between the responses of RIF1 or RIF1-EGFP tumors after BPD or Photofrin PDT and complete cures and mouse survival when RIF-1 EGFP tumors were treated with BPD. This increased tumor response may be due to antibody-mediated cytotoxicity and the presence of an artificial tumor antigen (GFP) that can produce a CD8 T-cell response against the whole tumor. The presence of antibodies against EGFP in mouse serum correlates with the hypothesis.

  10. Emotional Expression in Reality TV

    DEFF Research Database (Denmark)

    Rasmussen, Tove Arendt

    , that information ‘given off’ in lying behavior will must often be found in non-verbal mikro expressions and mikro gestures. I will end by discussing the strategic emotional expressions as production of floating identities in a broader framework on the basis of among others Gergen, Lipovetsky and Baumann...... are being treated as ordinary people. My article will discuss different presentations of selves and especially the emotional verbal and nonverbal expressions in reality TV communication. Aspects of the intimate self and its emotional expressions seem to be strategically managed in reality TV and even...

  11. Generational Differences of Emotional Expression

    Institute of Scientific and Technical Information of China (English)

    李学勇

    2014-01-01

    As a kind of subjective psychological activity, emotion can only be known and perceived by a certain expressive form. Varies as the different main bodies, difference of emotional expression can be reflected not only among individuals but between generations. The old conceals their emotions inside, the young express their emotions boldly, and the middle-aged are rational and deep in their expressions. Facing and understanding such differences is the premise and foundation of the con-struction of a harmonious relationship between different generations.

  12. Oracle Application Express 4 Recipes

    CERN Document Server

    Zehoo, Edmund

    2011-01-01

    Oracle Application Express 4 Recipes provides an example-based approach to learning Application Express - the ground-breaking, rapid application development platform included with every Oracle Database license. The recipes format is ideal for the quick-study who just wants a good example or two to kick start their thinking and get pointed in the right direction. The recipes cover the gamut of Application Express development. Author and Application Express expert Edmund Zehoo shows how to create data entry screens, visualize data in the form of reports and charts, implement validation and back-

  13. Expression modeling for expression-invariant face recognition

    NARCIS (Netherlands)

    Haar, F.B. Ter; Veltkamp, R.C.

    2010-01-01

    Morphable face models have proven to be an effective tool for 3D face modeling and face recognition, but the extension to 3D face scans with expressions is still a challenge. The two main difficulties are (1) how to build a new morphable face model that deals with expressions, and (2) how to fit thi

  14. Tumor-specific gene expression patterns with gene expression profiles

    Institute of Scientific and Technical Information of China (English)

    RUAN; Xiaogang; LI; Yingxin; LI; Jiangeng; GONG; Daoxiong

    2006-01-01

    Gene expression profiles of 14 common tumors and their counterpart normal tissues were analyzed with machine learning methods to address the problem of selection of tumor-specific genes and analysis of their differential expressions in tumor tissues. First, a variation of the Relief algorithm, "RFE_Relief algorithm" was proposed to learn the relations between genes and tissue types. Then, a support vector machine was employed to find the gene subset with the best classification performance for distinguishing cancerous tissues and their counterparts. After tissue-specific genes were removed, cross validation experiments were employed to demonstrate the common deregulated expressions of the selected gene in tumor tissues. The results indicate the existence of a specific expression fingerprint of these genes that is shared in different tumor tissues, and the hallmarks of the expression patterns of these genes in cancerous tissues are summarized at the end of this paper.

  15. Generation and evaluation of a chimeric classical swine fever virus expressing a visible marker gene.

    Science.gov (United States)

    Li, Yongfeng; Wang, Xiao; Sun, Yuan; Li, Lian-Feng; Zhang, Lingkai; Li, Su; Luo, Yuzi; Qiu, Hua-Ji

    2016-03-01

    Classical swine fever virus (CSFV) is a noncytopathogenic virus, and the incorporation of an enhanced green fluorescent protein (EGFP) tag into the viral genome provides a means of direct monitoring of viral infection without immunostaining. It is well established that the 3' untranslated region (3'-UTR) of the CSFV plays an important role in viral RNA replication. Although CSFV carrying a reporter gene and chimeric CSFV have been generated and evaluated, a chimeric CSFV with a visible marker has not yet been reported. Here, we generated and evaluated a chimeric virus containing the EGFP tag and the 3'-UTR from vaccine strain HCLV (C-strain) in the genetic background of the highly virulent CSFV Shimen strain. The chimeric marker CSFV was fluorescent and had an approximately 100-fold lower viral titer, lower replication level of viral genome, and weaker fluorescence intensity than the recombinant CSFV with only the EGFP tag or the parental virus. Furthermore, the marker chimera was avirulent and displayed no viremia in inoculated pigs, which were completely protected from lethal CSFV challenge as early as 15 days post-inoculation. The chimeric marker virus was visible in vitro and attenuated in vitro and in vivo, which suggests that CSFV can be engineered to produce attenuated variants with a visible marker to facilitate in vitro studies of CSFV infection and replication and to develop of novel vaccines against CSF. PMID:26614259

  16. Human papillomavirus gene expression

    International Nuclear Information System (INIS)

    To determine the role of tissue differentiation on expression of each of the papillomavirus mRNA species identified by electron microscopy, the authors prepared exon-specific RNA probes that could distinguish the alternatively spliced mRNA species. Radioactively labeled single-stranded RNA probes were generated from a dual promoter vector system and individually hybridized to adjacent serial sections of formalin-fixed, paraffin-embedded biopsies of condylomata. Autoradiography showed that each of the message species had a characteristic tissue distribution and relative abundance. The authors have characterized a portion of the regulatory network of the HPVs by showing that the E2 ORF encodes a trans-acting enhancer-stimulating protein, as it does in BPV-1 (Spalholz et al. 1985). The HPV-11 enhancer was mapped to a 150-bp tract near the 3' end of the URR. Portions of this region are duplicated in some aggressive strains of HPV-6 (Boshart and zur Hausen 1986; Rando et al. 1986). To test the possible biological relevance of these duplications, they cloned tandem arrays of the enhancer and demonstrated, using a chloramphenicol acetyltransferase (CAT) assay, that they led to dramatically increased transcription proportional to copy number. Using the CAT assays, the authors found that the E2 proteins of several papillomavirus types can cross-stimulate the enhancers of most other types. This suggests that prior infection of a tissue with one papillomavirus type may provide a helper effect for superinfection and might account fo the HPV-6/HPV-16 coinfections in condylomata that they have observed

  17. Organisation and expression of a cluster of female-specific genes in the Australian sheep blowfly, Lucilia cuprina

    International Nuclear Information System (INIS)

    . cuprina can be made with a piggyBac vector containing the PUbnlsEGFP marker gene and the hsp70-piggyBachelper. The results from these expression studies will be presented at the meeting. We will also outline a sheep blowfly genome project that will be initiated late 2004 with the initial goals of: - Sequencing 30,000 embryonic and larval EST clones; - Construction and fingerprinting of a BAC genomic library; - Improving procedures for germ-line transformation of L. cuprina. (author)

  18. A preclinical model for noninvasive imaging of hypoxia-induced gene expression; comparison with an exogenous marker of tumor hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Wen Bixiu; Burgman, Paul; Zanzonico, Pat; O' Donoghue, Joseph; Li, Gloria C.; Ling, C. Clifton [Memorial Sloan-Kettering Cancer Center, Department of Medical Physics, New York (United States); Cai Shangde; Finn, Ron [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York (United States); Serganova, Inna [Memorial Sloan-Kettering Cancer Center, Department of Neurology, New York (United States); Blasberg, Ronald; Gelovani, Juri [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York (United States); Memorial Sloan-Kettering Cancer Center, Department of Neurology, New York (United States)

    2004-11-01

    Hypoxia is associated with tumor aggressiveness and is an important cause of resistance to radiation therapy and chemotherapy. Assays of tumor hypoxia could provide selection tools for hypoxia-modifying treatments. The purpose of this study was to develop and characterize a rodent tumor model with a reporter gene construct that would be transactivated by the hypoxia-inducible molecular switch, i.e., the upregulation of HIF-1. The reporter gene construct is the herpes simplex virus 1-thymidine kinase (HSV1-tk) fused with the enhanced green fluorescent protein (eGFP) under the regulation of an artificial hypoxia-responsive enhancer/promoter. In this model, tumor hypoxia would up-regulate HIF-1, and through the hypoxia-responsive promoter transactivate the HSV1-tkeGFPfusion gene. The expression of this reporter gene can be assessed with the {sup 124}I-labeled reporter substrate 2'-fluoro-2'-deoxy-1-{beta}-d-arabinofuranosyl-5-iodouracil ({sup 124}I-FIAU), which is phosphorylated by the HSV1-tk enzyme and trapped in the hypoxic cells. Animal positron emission tomography (microPET) and phosphor plate imaging (PPI) were used in this study to visualize the trapped {sup 124}I-FIAU, providing a distribution of the hypoxia-induced molecular events. The distribution of {sup 124}I-FIAU was also compared with that of an exogenous hypoxic cell marker, {sup 18}F-fluoromisonidazole (FMISO). Our results showed that {sup 124}I-FIAU microPET imaging of the hypoxia-induced reporter gene expression is feasible, and that the intratumoral distributions of {sup 124}I-FIAU and {sup 18}F-FMISO are similar. In tumor sections, detailed radioactivity distributions were obtained with PPI which also showed similarity between {sup 124}I-FIAU and {sup 18}F-FMISO. This reporter system is sufficiently sensitive to detect hypoxia-induced transcriptional activation by noninvasive imaging and might provide a valuable tool in studying tumor hypoxia and in validating existing and future

  19. A preclinical model for noninvasive imaging of hypoxia-induced gene expression; comparison with an exogenous marker of tumor hypoxia

    International Nuclear Information System (INIS)

    Hypoxia is associated with tumor aggressiveness and is an important cause of resistance to radiation therapy and chemotherapy. Assays of tumor hypoxia could provide selection tools for hypoxia-modifying treatments. The purpose of this study was to develop and characterize a rodent tumor model with a reporter gene construct that would be transactivated by the hypoxia-inducible molecular switch, i.e., the upregulation of HIF-1. The reporter gene construct is the herpes simplex virus 1-thymidine kinase (HSV1-tk) fused with the enhanced green fluorescent protein (eGFP) under the regulation of an artificial hypoxia-responsive enhancer/promoter. In this model, tumor hypoxia would up-regulate HIF-1, and through the hypoxia-responsive promoter transactivate the HSV1-tkeGFPfusion gene. The expression of this reporter gene can be assessed with the 124I-labeled reporter substrate 2'-fluoro-2'-deoxy-1-β-d-arabinofuranosyl-5-iodouracil (124I-FIAU), which is phosphorylated by the HSV1-tk enzyme and trapped in the hypoxic cells. Animal positron emission tomography (microPET) and phosphor plate imaging (PPI) were used in this study to visualize the trapped 124I-FIAU, providing a distribution of the hypoxia-induced molecular events. The distribution of 124I-FIAU was also compared with that of an exogenous hypoxic cell marker, 18F-fluoromisonidazole (FMISO). Our results showed that 124I-FIAU microPET imaging of the hypoxia-induced reporter gene expression is feasible, and that the intratumoral distributions of 124I-FIAU and 18F-FMISO are similar. In tumor sections, detailed radioactivity distributions were obtained with PPI which also showed similarity between 124I-FIAU and 18F-FMISO. This reporter system is sufficiently sensitive to detect hypoxia-induced transcriptional activation by noninvasive imaging and might provide a valuable tool in studying tumor hypoxia and in validating existing and future exogenous markers for tumor hypoxia. (orig.)

  20. Precise Analysis of String Expressions

    DEFF Research Database (Denmark)

    Christensen, Aske Simon; Møller, Anders; Schwartzbach, Michael Ignatieff

    2003-01-01

    , including statically checking the syntax of dynamically generated expressions, such as SQL queries. Our analysis constructs flow graphs from class files and generates a context-free grammar with a nonterminal for each string expression. The language of this grammar is then widened into a regular language...

  1. Spontaneous Emotional Facial Expression Detection

    Directory of Open Access Journals (Sweden)

    Zhihong Zeng

    2006-08-01

    Full Text Available Change in a speaker’s emotion is a fundamental component in human communication. Automatic recognition of spontaneous emotion would significantly impact human-computer interaction and emotion-related studies in education, psychology and psychiatry. In this paper, we explore methods for detecting emotional facial expressions occurring in a realistic human conversation setting—the Adult Attachment Interview (AAI. Because non-emotional facial expressions have no distinct description and are expensive to model, we treat emotional facial expression detection as a one- class classification problem, which is to describe target objects (i.e., emotional facial expressions and distinguish them from outliers (i.e., non-emotional ones. Our preliminary experiments on AAI data suggest that one-class classification methods can reach a good balance between cost (labeling and computing and recognition performance by avoiding non-emotional expression labeling and modeling.

  2. A lentiviral sponge for miR-101 regulates RanBP9 expression and amyloid precursor protein metabolism in hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Francesca Ruberti

    2014-02-01

    Full Text Available Neurodegeneration associated with amyloid β (Aβ peptide accumulation, synaptic loss, and memory impairment are pathophysiological features of Alzheimer's disease (AD. Numerous microRNAs regulate amyloid precursor protein (APP expression and metabolism. We previously reported that miR-101 is a negative regulator of APP expression in cultured hippocampal neurons. In this study, a search for predicted APP metabolism-associated miR-101 targets led to the identification of a conserved miR-101 binding site within the 3’ untranslated region (UTR of the mRNA encoding Ran-binding protein 9 (RanBP9. RanBP9 increases APP processing by β-amyloid converting enzyme 1 (BACE1, secretion of soluble APPβ (sAPPβ, and generation of Aβ. MiR-101 significantly reduced reporter gene expression when co-transfected with a RanBP9 3'-UTR reporter construct, while site-directed mutagenesis of the predicted miR-101 target site eliminated the reporter response. To investigate the effect of stable inhibition of miR-101 both in vitro and in vivo, a microRNA sponge was developed to bind miR-101 and derepress its targets. Four tandem bulged miR-101 responsive elements (REs, located downstream of the enhanced green fluorescence protein (EGFP open reading frame and driven by the synapsin promoter, were placed in a lentiviral vector to create the pLSyn-miR-101 sponge. Delivery of the sponge to primary hippocampal neurons significantly increased both APP and RanBP9 expression, as well as sAPPβ levels in the conditioned medium. Importantly, silencing of endogenous RanBP9 reduced sAPPβ levels in miR-101 sponge-containing hippocampal cultures, indicating that miR-101 inhibition may increase amyloidogenic processing of APP by RanBP9. Lastly, the impact of miR-101 on its targets was demonstrated in vivo by intrahippocampal injection of the pLSyn-miR-101 sponge into C57BL6 mice. This study thus provides the basis for studying the consequences of long-term miR-101 inhibition on

  3. Cortical control of facial expression.

    Science.gov (United States)

    Müri, René M

    2016-06-01

    The present Review deals with the motor control of facial expressions in humans. Facial expressions are a central part of human communication. Emotional face expressions have a crucial role in human nonverbal behavior, allowing a rapid transfer of information between individuals. Facial expressions can be either voluntarily or emotionally controlled. Recent studies in nonhuman primates and humans have revealed that the motor control of facial expressions has a distributed neural representation. At least five cortical regions on the medial and lateral aspects of each hemisphere are involved: the primary motor cortex, the ventral lateral premotor cortex, the supplementary motor area on the medial wall, and the rostral and caudal cingulate cortex. The results of studies in humans and nonhuman primates suggest that the innervation of the face is bilaterally controlled for the upper part and mainly contralaterally controlled for the lower part. Furthermore, the primary motor cortex, the ventral lateral premotor cortex, and the supplementary motor area are essential for the voluntary control of facial expressions. In contrast, the cingulate cortical areas are important for emotional expression, because they receive input from different structures of the limbic system. J. Comp. Neurol. 524:1578-1585, 2016. © 2015 Wiley Periodicals, Inc. PMID:26418049

  4. TempoExpress: An Expressivity-Preserving Musical Tempo Transformation

    OpenAIRE

    Grachten, Maarten; Arcos, Josep Ll.; Lopez De Mantaras, Ramon

    2006-01-01

    The research described in this paper focuses on global tempo transformations of monophonic audio recordings of saxophone jazz performances. More concretely, we have investigated the problem of how a performance played at a particular tempo can be automatically rendered at another tempo while preserving its expressivity. To do so we have developed a case-based reasoning system called TempoExpress. The results we have obtained have been extensively compared against a sta...

  5. The expressive stance: intentionality, expression, and machine art

    OpenAIRE

    Linson, Adam

    2013-01-01

    This paper proposes a new interpretive stance for interpreting artistic works and performances that is relevant to artificial intelligence research but also has broader implications. Termed the expressive stance, this stance makes intelligible a critical distinction between present-day machine art and human art, but allows for the possibility that future machine art could find a place alongside our own. The expressive stance is elaborated as a response to Daniel Dennett's notion of the intent...

  6. Shuffling Yeast Gene Expression Data

    CERN Document Server

    Bilke, S

    2000-01-01

    A new method to sort gene expression patterns into functional groups is presented. The method is based on a sorting algorithm using a non-local similarity score, which takes all other patterns in the dataset into account. The method is therefore very robust with respect to noise. Using the expression data for yeast, we extract information about functional groups. Without prior knowledge of parameters the cell cycle regulated genes in yeast can be identified. Furthermore a second, independent cell clock is identified. The capability of the algorithm to extract information about signal flow in the regulatory network underlying the expression patterns is demonstrated.

  7. Expression in E. coli systems

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Kristiansen, Karsten; Nøhr, Jane

    2003-01-01

    Owing to cost advantage, speed of production, and often high product yield (up to 50% of total cell protein), expression in Escherichia coli is generally the first choice when attempting to express a recombinant protein. Expression systems exist to produce recombinant protein intracellularly...... intracellularly in soluble form. In E. coli, proteins containing disulfide bonds are best produced by secretion because the disulfide forming foldases reside in the periplasm. Likewise, a correct N-terminus is more likely to be obtained upon secretion. Moreover, potentially toxic proteins are more likely to be...

  8. 75 FR 51823 - Government-Owned Inventions; Availability for Licensing

    Science.gov (United States)

    2010-08-23

    ... model features a TGF- 1 transgene for which expression is blocked by a floxed enhanced green fluorescent protein (EGFP) gene downstream of the promoter. Excision of the EGFP gene by Cre recombinase allows... Human Recombinant MATER Protein Description of Technology: The inventors have identified MATER, a...

  9. A Tattoo Is Expression, Too.

    Science.gov (United States)

    Dowling-Sendor, Benjamin

    1997-01-01

    In "Stephenson v. Davenport Community School District," the U.S. Eighth Circuit Court of Appeals ruled that schools cannot adopt unduly vague policies to regulate student expression, in this case, a cross-shaped tattoo. (LMI)

  10. Leptospira Protein Expression During Infection

    Science.gov (United States)

    We are characterizing protein expression in vivo during experimental leptospirosis using immunofluorescence microscopy. Coding regions for several proteins were identified through analysis of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovar Hardjo genomes. In addition, codi...

  11. Race, Reparations, and Free Expression.

    Science.gov (United States)

    Brownstein, Andrew

    2001-01-01

    Describes how a controversial newspaper ad opposing slavery reparations and the subsequent trashing of the student daily have set off a debate at Brown University about the competing values of sensitivity and free expression. (EV)

  12. Gene expression in colorectal cancer

    DEFF Research Database (Denmark)

    Birkenkamp-Demtroder, Karin; Christensen, Lise Lotte; Olesen, Sanne Harder;

    2002-01-01

    Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each...... high frequency of loss of heterozygosity. The genes and ESTs presented in this study encode new potential tumor markers as well as potential novel therapeutic targets for prevention or therapy of CRC....

  13. Shuffling Yeast Gene Expression Data

    OpenAIRE

    Bilke, Sven

    2000-01-01

    A new method to sort gene expression patterns into functional groups is presented. The method is based on a sorting algorithm using a non-local similarity score, which takes all other patterns in the dataset into account. The method is therefore very robust with respect to noise. Using the expression data for yeast, we extract information about functional groups. Without prior knowledge of parameters the cell cycle regulated genes in yeast can be identified. Furthermore a second, independent ...

  14. Expressive alignment language and implementation

    OpenAIRE

    Euzenat, Jérôme; Scharffe, François; Zimmermann, Antoine

    2007-01-01

    euzenat2007e This deliverable provides the description of an alignment language which is both expressive and independent from ontology languages. It defines the language through its abstract syntax and semantics depending on ontology language semantics. It then describes two concrete syntax: an exchange syntax in RDF/XML and a surface syntax for human consumption. Finally, it presents the current implementation of this expressive language within the Alignment API taking advantage of the OM...

  15. Measuring facial expression of emotion

    OpenAIRE

    Wolf, Karsten

    2015-01-01

    Research into emotions has increased in recent decades, especially on the subject of recognition of emotions. However, studies of the facial expressions of emotion were compromised by technical problems with visible video analysis and electromyography in experimental settings. These have only recently been overcome. There have been new developments in the field of automated computerized facial recognition; allowing real-time identification of facial expression in social environments. This rev...

  16. Spontaneous Emotional Facial Expression Detection

    OpenAIRE

    Zhihong Zeng; Yun Fu; Roisman, Glenn I.; Zhen Wen; Yuxiao Hu; Thomas S. Huang

    2006-01-01

    Change in a speaker’s emotion is a fundamental component in human communication. Automatic recognition of spontaneous emotion would significantly impact human-computer interaction and emotion-related studies in education, psychology and psychiatry. In this paper, we explore methods for detecting emotional facial expressions occurring in a realistic human conversation setting—the Adult Attachment Interview (AAI). Because non-emotional facial expressions have no distinct description and are exp...

  17. Vascular gene expression: a hypothesis

    OpenAIRE

    Martínez-Navarro, Angélica C.; Galván-Gordillo, Santiago V.; Xoconostle-Cázares, Beatriz; Ruiz-Medrano, Roberto

    2013-01-01

    The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular ti...

  18. Zipf's Law in Gene Expression

    OpenAIRE

    Furusawa, Chikara; Kaneko, Kunihiko

    2002-01-01

    Using data from gene expression databases on various organisms and tissues, including yeast, nematodes, human normal and cancer tissues, and embryonic stem cells, we found that the abundances of expressed genes exhibit a power-law distribution with an exponent close to -1, i.e., they obey Zipf's law. Furthermore, by simulations of a simple model with an intra-cellular reaction network, we found that Zipf's law of chemical abundance is a universal feature of cells where such a network optimize...

  19. On Fuzzy Expressions in English

    Institute of Scientific and Technical Information of China (English)

    张桂君

    2005-01-01

    In this paper Ⅰ attempt to explain my general understanding of fuzzy expressions in language, and I take English as an example.Fuzziness is one of the basic characteristics of language.It differs from generality, ambiguity and vagueness.There are many examples of fuzzy expressions in English.I supply examples of color words, some adjectives and linguistic hedges.Finally I attempt to explain fuzziness from pragmatic point of view.

  20. Epimorphin expression in interstitial pneumonia

    Directory of Open Access Journals (Sweden)

    Suga Moritaka

    2005-01-01

    Full Text Available Abstract Epimorphin modulates epithelial morphogenesis in embryonic mouse organs. We previously suggested that epimorphin contributes to repair of bleomycin-induced pulmonary fibrosis in mice via epithelium-mesenchyme interactions. To clarify the role of epimorphin in human lungs, we evaluated epimorphin expression and localization in normal lungs, lungs with nonspecific interstitial pneumonia (NSIP, and lungs with usual interstitial pneumonia (UIP; we also studied the effect of recombinant epimorphin on cultured human alveolar epithelial cells in vitro. Northern and Western blotting analyses revealed that epimorphin expression in NSIP samples were significantly higher than those in control lungs and lungs with UIP. Immunohistochemistry showed strong epimorphin expression in mesenchymal cells of early fibrotic lesions and localization of epimorphin protein on mesenchymal cells and extracellular matrix of early fibrotic lesions in the nonspecific interstitial pneumonia group. Double-labeled fluorescent images revealed expression of matrix metalloproteinase 2 in re-epithelialized cells overlying epimorphin-positive early fibrotic lesions. Immunohistochemistry and metalloproteinase activity assay demonstrated augmented expression of metalloproteinase induced by recombinant epimorphin in human alveolar epithelial cells. These findings suggest that epimorphin contributes to repair of pulmonary fibrosis in nonspecific interstitial pneumonia, perhaps partly by inducing expression of matrix metalloproteinase 2, which is an important proteolytic factor in lung remodeling.

  1. Referring Expressions: A Unified Approach

    Institute of Scientific and Technical Information of China (English)

    K.M. Jaszczolt

    2001-01-01

    @@ 0. Introduction Expressions used by speakers to refer are commonly divided into two categories: that of directly referring expressions and that of expressions whose referring function is secured by the context of utterance. Directly referring expressions are normally said to include proper names, some pronouns including demonstrative, and demonstrative phrases. The other category comprises mainly definite and indefinite descriptions, the first being their most acclaimed representative. Definite descriptions are widely acknowledged to have referential uses. They are not referring expressions, so to speak, by default,but rather as a result of a contextually determined interpretation. As a category, they are frequently said to belong with quantifiers (Neale 1990; Recanati 1993). However, the arguments for classifying them with referring expressions are ample (Bach 1987a; Larson & Segal 1995; Brown 1995; Jaszczolt 1997a,1997b, 1999b). It is argued in Part I of this paper that although definite descriptions exhibit an ambiguity of use between the referential and the attributive reading, they also exhibit the property of having an unmarked, salient interpretation which makes them akin to directly referential terms. This salient reading is the referential interpretation, arrived at with the help of the hearer′s presumption of the presence of strong referential intention that supports the speaker′s utterance.

  2. Electrophysiological, morphological, and topological properties of two histochemically distinct subpopulations of cerebellar unipolar brush cells.

    Science.gov (United States)

    Kim, Jin-Ah; Sekerková, Gabriella; Mugnaini, Enrico; Martina, Marco

    2012-12-01

    Unipolar brush cells (UBCs) are excitatory cerebellar granular layer interneurons whose brush-like dendrites receive one-to-one mossy fiber inputs. Subclasses of UBCs differ primarily by expressing metabotropic glutamate receptor (mGluR) 1α or calretinin. We used GENSAT Tg(Grp-EGFP) BAC transgenic mice, which selectively express enhanced green fluorescent protein (EGFP) in mGluR1α-positive UBCs to compare the functional properties of the two subclasses. Compared to EGFP-negative UBCs, which include the calretinin-positive cells, EGFP-positive UBCs had smaller somata (area 48 vs 63 μm(2)), lower specific membrane resistance (6.4 vs. 13.7 KΩ cm(2)), were less prone to intrinsic firing, and showed more irregular firing (in cell-attached ~49 % were firing vs. ~88 %, and the CV was 0.53 vs. 0.32 for EGFP-negative cells). Some of these differences are attributable to higher density of background K(+) currents in EGFP-positive cells (at -120 mV, the barium-sensitive current was 94 vs. 37 pA in EGFP-negative cells); Ih, on the contrary, was more abundantly expressed in EGFP-negative cells (at -140 mV, it was -122 vs. -54 pA in EGFP-positive neurons); furthermore, while group II mGluR modulation of the background potassium current in EGFP-negative UBCs was maintained after intracellular dialysis, mGluR modulation in EGFP-positive UBCs was lost in whole-cell recordings. Finally, cell-attached firing was reversibly abolished by the GABA(B) activation in EGFP-positive, but not in EGFP-negative UBCs. Immunohistochemistry showed that EGFP-negative UBCs express GIRK2 at high density, while mGluR1α UBCs are GIRK2 negative, suggesting that GIRK2 mediates the mGluR-sensitive current in EGFP-negative UBCs. These data suggest that the two subclasses perform different functions in the cerebellar microcircuits. PMID:22528965

  3. Extracting expression modules from perturbational gene expression compendia

    Directory of Open Access Journals (Sweden)

    Van Dijck Patrick

    2008-04-01

    Full Text Available Abstract Background Compendia of gene expression profiles under chemical and genetic perturbations constitute an invaluable resource from a systems biology perspective. However, the perturbational nature of such data imposes specific challenges on the computational methods used to analyze them. In particular, traditional clustering algorithms have difficulties in handling one of the prominent features of perturbational compendia, namely partial coexpression relationships between genes. Biclustering methods on the other hand are specifically designed to capture such partial coexpression patterns, but they show a variety of other drawbacks. For instance, some biclustering methods are less suited to identify overlapping biclusters, while others generate highly redundant biclusters. Also, none of the existing biclustering tools takes advantage of the staple of perturbational expression data analysis: the identification of differentially expressed genes. Results We introduce a novel method, called ENIGMA, that addresses some of these issues. ENIGMA leverages differential expression analysis results to extract expression modules from perturbational gene expression data. The core parameters of the ENIGMA clustering procedure are automatically optimized to reduce the redundancy between modules. In contrast to the biclusters produced by most other methods, ENIGMA modules may show internal substructure, i.e. subsets of genes with distinct but significantly related expression patterns. The grouping of these (often functionally related patterns in one module greatly aids in the biological interpretation of the data. We show that ENIGMA outperforms other methods on artificial datasets, using a quality criterion that, unlike other criteria, can be used for algorithms that generate overlapping clusters and that can be modified to take redundancy between clusters into account. Finally, we apply ENIGMA to the Rosetta compendium of expression profiles for

  4. Transcriptional regulation of mouse alpha A-crystallin gene in a 148kb Cryaa BAC and its derivates

    Directory of Open Access Journals (Sweden)

    Yang Ying

    2008-09-01

    Full Text Available Abstract Background αA-crystallin is highly expressed in the embryonic, neonatal and adult mouse lens. Previously, we identified two novel distal control regions, DCR1 and DCR3. DCR1 was required for transgenic expression of enhanced green fluorescent protein, EGFP, in lens epithelium, whereas DCR3 was active during "late" stages of lens primary fiber cell differentiation. However, the onset of transgenic EGFP expression was delayed by 12–24 hours, compared to the expression of the endogenous Cryaa gene. Results Here, we used bacterial artificial chromosome (BAC and standard transgenic approaches to examine temporal and spatial regulation of the mouse Cryaa gene. Two BAC transgenes, with EGFP insertions into the third coding exon of Cryaa gene, were created: the intact αA-crystallin 148 kb BAC (αA-BAC and αA-BAC(ΔDCR3, which lacks approximately 1.0 kb of genomic DNA including DCR3. Expression of EGFP in the majority of both BAC transgenics nearly recapitulated the endogenous expression pattern of the Cryaa gene in lens, but not outside of the lens. The number of cells expressing αA-crystallin in the lens pit was higher compared to the number of cells expressing EGFP. Next, we generated additional lines using a 15 kb fragment of αA-crystallin locus derived from αA-BAC(ΔDCR3, 15 kb Cryaa/EGFP. A 15 kb region of Cryaa/EGFP supported the expression pattern of EGFP also in the lens pit. However, co-localization studies of αA-crystallin and EGFP indicated that the number of cells that showed transgenic expression was higher compared to cells expressing αA-crystallin in the lens pit. Conclusion We conclude that a 148 kb αA-BAC likely contains all of the regulatory regions required for αA-crystallin expression in the lens, but not in retina, spleen and thymus. In addition, while the 15 kb Cryaa/EGFP region also supported the expression of EGFP in the lens pit, expression in regions such as the hindbrain, indicate that additional genomic

  5. Studying Emotional Expression in Music Performance.

    Science.gov (United States)

    Gabrielsson, Alf

    1999-01-01

    Explores the importance of emotional expression in music performance. Performers played music to express different emotions and then listening tests were conducted in order to determine whether the intended expressions were perceived. Presents and discusses the results. (CMK)

  6. NF-kB activation as a biomarker of light injury using a transgenic mouse model

    Science.gov (United States)

    Pocock, Ginger M.; Boretsky, Adam; Wang, Heuy-Ching; Golden, Dallas; Gupta, Praveena; Vargas, Gracie; Oliver, Jeffrey W.; Motamedi, Massoud

    2012-03-01

    The spatial and temporal activation of NF-kB (p65) was monitored in the retina of a transgenic mouse model (cis-NFkB-EGFP) in vivo after receiving varying grades of laser induced thermal injury in one eye. Baseline images of the retinas from 26 mice were collected prior to injury and up to five months post-exposure using a Heidelberg Spectralis HRA confocal scanning laser ophthalmoscope (cSLO) with a spectral domain optical coherence tomographer (SDOCT). Injured and control eyes were enucleated at discrete time points following laser exposure for cryosectioning to determine localization of NF-kB dependent enhanced green fluorescent protein (EGFP) reporter gene expression within the retina using fluorescence microscopy. In addition, EGFP basal expression in brain and retinal tissue from the cis-NFkB-EGFP was characterized using two-photon imaging. Regions of the retina exposed to threshold and supra-threshold laser damage evaluated using fluorescence cSLO showed increased EGFP fluorescence localized to the exposed region for a duration that was dependent upon the degree of injury. Fluorescence microscopy of threshold damage revealed EGFP localized to the outer nuclear region and retinal pigment epithelial layer. Basal expression of EGFP imaged using two-photon microscopy was heterogeneously distributed throughout brain tissue and confined to the inner retina. Results show cis-NF-kB-EGFP reporter mouse can be used for in vivo studies of light induced injury to the retina and possibly brain injury.

  7. Construction of recombinant adenovirus co-expressing FMDV serotype O capsid and GFP proteins%共表达O型FMDV衣壳蛋白和绿色荧光蛋白重组腺病毒的构建

    Institute of Scientific and Technical Information of China (English)

    刘蒙蒙; 杨德成; 周国辉; 梁特; 于力

    2012-01-01

    To generate the recombinant adenovirus expressing GFP and P12A3C gene of serotype O foot and mouth disease virus (FMDV), the foreign fusion gene was constructed by insertion of EGFP gene between PI and 3C genes with a linker sequence encoding selfprocessing peptide 2A derived from the FMDV. The recombinant adenovirus expressing the P12A3C of FMDV and GFP proteins was generated and identified by PCR, fluorescence assay, western blot. The one-step growth curve indicated that the recombinant adenovirus had no difference with the parental adenovirus. To the best of our knowledge, this report was the first description of the recombinant human adenovirus serotype 5 co-expressing P12A3C of FMDV and GFP proteins, which might be used as an attractive way to improve the FMDV vaccine delivered by adenovirus vector.%为构建表达O型口蹄疫病毒(FMDV)的P12A3C基因及GFP基因的重组腺病毒rAdV-P12aEGFP2a3C,本研究以FMDV的2A基因序列为Linker,将报告基因EGFP插入FMDV的P12A与3C之间.重组腺病毒感染HEK-293细胞后可以观察到绿色荧光,表明EGFP蛋白获得表达.应用FMDV的VP2单克隆抗体4B2对重组病毒感染细胞进行western blot检测,反应条带与FMDV衣壳蛋白VP0和VP3的分子量大小相符,表明FMDV的完整衣壳蛋白和3C蛋白酶也均获得表达,而且EGFP的插入并未影响P1蛋白的表达和3C蛋白酶对P1的正确切割.重组腺病毒的生长特性分析表明,EGFP的插入也未影响该重组腺病毒的增殖特性.上述研究结果显示,表达FMDV衣壳蛋白P12A3C的重组腺病毒可以作为载体,以2A蛋白作为Linker表达一个小分子蛋白,为改进以腺病毒为载体的口蹄疫基因工程疫苗提供了新思路.

  8. The motivation to express prejudice.

    Science.gov (United States)

    Forscher, Patrick S; Cox, William T L; Graetz, Nicholas; Devine, Patricia G

    2015-11-01

    Contemporary prejudice research focuses primarily on people who are motivated to respond without prejudice and the ways in which unintentional bias can cause these people to act in a manner inconsistent with this motivation. However, some real-world phenomena (e.g., hate speech, hate crimes) and experimental findings (e.g., Plant & Devine, 2001, 2009) suggest that some prejudice is intentional. These phenomena and findings are difficult to explain solely from the motivations to respond without prejudice. We argue that some people are motivated to express prejudice, and we develop the Motivation to Express Prejudice Scale (MP) to measure this motivation. In 7 studies involving more than 6,000 participants, we demonstrate that, across scale versions targeted at Black people and gay men, the MP has good reliability and convergent, discriminant, and predictive validity. In normative climates that prohibit prejudice, the internal and external motivations to express prejudice are functionally nonindependent, but they become more independent when normative climates permit more prejudice toward a target group. People high in the motivation to express prejudice are relatively likely to resist pressure to support programs promoting intergroup contact and to vote for political candidates who support oppressive policies. The motivation to express prejudice predicted these outcomes even when controlling for attitudes and the motivations to respond without prejudice. This work encourages contemporary prejudice researchers to give greater consideration to the intentional aspects of negative intergroup behavior and to broaden the range of phenomena, target groups, and samples that they study. PMID:26479365

  9. Monoallelic expression of olfactory receptors.

    Science.gov (United States)

    Monahan, Kevin; Lomvardas, Stavros

    2015-01-01

    The sense of smell collects vital information about the environment by detecting a multitude of chemical odorants. Breadth and sensitivity are provided by a huge number of chemosensory receptor proteins, including more than 1,400 olfactory receptors (ORs). Organizing the sensory information generated by these receptors so that it can be processed and evaluated by the central nervous system is a major challenge. This challenge is overcome by monogenic and monoallelic expression of OR genes. The single OR expressed by each olfactory sensory neuron determines the neuron's odor sensitivity and the axonal connections it will make to downstream neurons in the olfactory bulb. The expression of a single OR per neuron is accomplished by coupling a slow chromatin-mediated activation process to a fast negative-feedback signal that prevents activation of additional ORs. Singular OR activation is likely orchestrated by a network of interchromosomal enhancer interactions and large-scale changes in nuclear architecture. PMID:26359778

  10. Facial Asymmetry and Emotional Expression

    CERN Document Server

    Pickin, Andrew

    2011-01-01

    This report is about facial asymmetry, its connection to emotional expression, and methods of measuring facial asymmetry in videos of faces. The research was motivated by two factors: firstly, there was a real opportunity to develop a novel measure of asymmetry that required minimal human involvement and that improved on earlier measures in the literature; and secondly, the study of the relationship between facial asymmetry and emotional expression is both interesting in its own right, and important because it can inform neuropsychological theory and answer open questions concerning emotional processing in the brain. The two aims of the research were: first, to develop an automatic frame-by-frame measure of facial asymmetry in videos of faces that improved on previous measures; and second, to use the measure to analyse the relationship between facial asymmetry and emotional expression, and connect our findings with previous research of the relationship.

  11. Homeobox gene expression in Brachiopoda

    DEFF Research Database (Denmark)

    Altenburger, Andreas; Martinez, Pedro; Wanninger, Andreas

    2011-01-01

    . Not is a homeobox containing gene that regulates the formation of the notochord in chordates, while Cdx (caudal) is a ParaHox gene involved in the formation of posterior tissues of various animal phyla. The T. transversa homolog, TtrNot, is expressed in the ectoderm from the beginning of gastrulation until...... (ectoderm) specification with co-opted functions in notochord formation in chordates and left/right determination in ambulacrarians and vertebrates. The caudal ortholog, TtrCdx, is first expressed in the ectoderm of the gastrulating embryo in the posterior region of the blastopore. Its expression stays...... metazoans, where genes belonging to the Cdx/caudal family are predominantly localized in posterior domains during gastrulation. Later in development this gene will play a fundamental role in the formation of posterior tissues....

  12. VEGF expression and FDG kinetics

    International Nuclear Information System (INIS)

    Aim: The kinetics of dynamic PET FDG studies in musculoskeletal tumors was compared with the quantitative measurements of VEGF expression. Methods: Dynamic PET FDG studies were performed in 35 patients with sarcomas prior to surgery. The PET data were acquired for 60 minutes using a dedicated PET ring system. Following iterative image reconstruction, the data were quantified using a dedicated software program. Besides the calculation of SUV, a two compartment model was used to retrieve the parameters VB (vessel density) and the FDG transport constants K1-k4. Furthermore, a non-compartment model was applied to gain information about the tumor heterogeneity using the fractal dimension (FD). Following surgery, tissue samples were processed by quantitative RT-PCR using a lightcycler system for VEGF, the vascular endothelial growth factor, important for the tumor angiogenesis. Results: Scatter plots demonstrate, that the VEGF expression was generally associated with higher K1 values. The vessel density was independent from the VEGF expression. While the VEGF expression was variable at low SUV, the expression of VEGF was generally increased when the SUV exceeded 4.0. The simple linear correlation analysis of the parameters provided no significant correlations. A multi-factorial regression function was calculated, using SUV, K1-k4, VB and FD as predictor variables and the measured VEGF expression as the target variable. We noted a correlation of r=0.88 for these parameters, demonstrating that the dynamic of FDG accumulation is associated with angiogenetic activity. Conclusion: The results demonstrate, that tumor angiogenesis modulate the kinetics of FDG accumulation

  13. Extracting expression modules from perturbational gene expression compendia

    OpenAIRE

    Van Dijck Patrick; Maere Steven; Kuiper Martin

    2008-01-01

    Abstract Background Compendia of gene expression profiles under chemical and genetic perturbations constitute an invaluable resource from a systems biology perspective. However, the perturbational nature of such data imposes specific challenges on the computational methods used to analyze them. In particular, traditional clustering algorithms have difficulties in handling one of the prominent features of perturbational compendia, namely partial coexpression relationships between genes. Biclus...

  14. Detection of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

    International Nuclear Information System (INIS)

    The C-terminal domain (D4) of perfringolysin O binds selectively to cholesterol in cholesterol-rich microdomains. To address the issue of whether cholesterol-rich microdomains exist in the inner leaflet of the plasma membrane, we expressed D4 as a fusion protein with EGFP in MEF cells. More than half of the EGFP-D4 expressed in stable cell clones was bound to membranes in raft fractions. Depletion of membrane cholesterol with β-cyclodextrin reduced the amount of EGFP-D4 localized in raft fractions, confirming EGFP-D4 binding to cholesterol-rich microdomains. Subfractionation of the raft fractions showed most of the EGFP-D4 bound to the plasma membrane rather than to intracellular membranes. Taken together, these results strongly suggest the existence of cholesterol-rich microdomains in the inner leaflet of the plasma membrane

  15. Correction of gene expression data

    DEFF Research Database (Denmark)

    Darbani Shirvanehdeh, Behrooz; Stewart, C. Neal, Jr.; Noeparvar, Shahin;

    2014-01-01

    This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies. For...... maximal reliability of analysis, therefore, comparisons should be performed at the cellular level. This could be accomplished using an appropriate correction method that can detect and remove the inter-treatment bias for cell-number. Based on inter-treatment variations of reference genes, we introduce an...

  16. Advanced express web application development

    CERN Document Server

    Keig, Andrew

    2013-01-01

    A practical book, guiding the reader through the development of a single page application using a feature-driven approach.If you are an experienced JavaScript developer who wants to build highly scalable, real-world applications using Express, this book is ideal for you. This book is an advanced title and assumes that the reader has some experience with node, Javascript MVC web development frameworks, and has heard of Express before, or is familiar with it. You should also have a basic understanding of Redis and MongoDB. This book is not a tutorial on Node, but aims to explore some of the more

  17. Zipf's Law in Gene Expression

    CERN Document Server

    Furusawa, C; Furusawa, Chikara; Kaneko, Kunihiko

    2002-01-01

    Using data from gene expression databases on various organisms and tissues, including yeast, nematodes, human normal and cancer tissues, and embryonic stem cells, we found that the abundances of expressed genes exhibit a power-law distribution with an exponent close to -1, i.e., they obey Zipf's law. Furthermore, by simulations of a simple model with an intra-cellular reaction network, we found that Zipf's law of chemical abundance is a universal feature of cells where such a network optimizes the efficiency and faithfulness of self-reproduction. These findings provide novel insights into the nature of the organization of reaction dynamics in living cells.

  18. Reconhecimento Expressões Faciais

    OpenAIRE

    Costa, Fernando Gil Abreu Mesquita da

    2010-01-01

    Dissertação de Mestrado em Engenharia Electrotécnica de Computadores A expressão facial é uma forma de comunicação não-verbal que permite a partilha de sentimentos e emoções, numa linguagem subentendida entre seres humanos. A detecção facial e reconhecimento automático de expressões baseia-se no processamento de imagem e reconhecimento de padrões. O desenvolvimento nestas áreas deve-se a avanços multidisciplinares recentes, desde a aquisição e processamento digital de imagem, caracteriz...

  19. Microsoft Expression Web for dummies

    CERN Document Server

    Hefferman, Linda

    2013-01-01

    Expression Web is Microsoft's newest tool for creating and maintaining dynamic Web sites. This FrontPage replacement offers all the simple ""what-you-see-is-what-you-get"" tools for creating a Web site along with some pumped up new features for working with Cascading Style Sheets and other design options. Microsoft Expression Web For Dummies arrives in time for early adopters to get a feel for how to build an attractive Web site. Author Linda Hefferman teams up with longtime FrontPage For Dummies author Asha Dornfest to show the easy way for first-time Web designers, FrontPage ve

  20. Mapping and Manipulating Facial Expression

    Science.gov (United States)

    Theobald, Barry-John; Matthews, Iain; Mangini, Michael; Spies, Jeffrey R.; Brick, Timothy R.; Cohn, Jeffrey F.; Boker, Steven M.

    2009-01-01

    Nonverbal visual cues accompany speech to supplement the meaning of spoken words, signify emotional state, indicate position in discourse, and provide back-channel feedback. This visual information includes head movements, facial expressions and body gestures. In this article we describe techniques for manipulating both verbal and nonverbal facial…

  1. An Expressive Extension of TLC

    DEFF Research Database (Denmark)

    Henriksen, Jesper Gulmann

    2002-01-01

    this game prove that the chosen property is not definable in TLC. We then show that the same property is definable in TLC*. We prove in fact the stronger result that TLC* is expressively stronger than TLC exactly when the dependency relation associated with the underlying trace alphabet is not...

  2. Ascidian gene-expression profiles

    OpenAIRE

    William R Jeffery

    2002-01-01

    With the advent of gene-expression profiling, a large number of genes can now be investigated simultaneously during critical stages of development. This approach will be particularly informative in studies of ascidians, basal chordates whose genomes and embryology are uniquely suited for mapping developmental gene networks.

  3. Neuroticism Delays Detection of Facial Expressions

    Science.gov (United States)

    Sawada, Reiko; Sato, Wataru; Uono, Shota; Kochiyama, Takanori; Kubota, Yasutaka; Yoshimura, Sayaka; Toichi, Motomi

    2016-01-01

    The rapid detection of emotional signals from facial expressions is fundamental for human social interaction. The personality factor of neuroticism modulates the processing of various types of emotional facial expressions; however, its effect on the detection of emotional facial expressions remains unclear. In this study, participants with high- and low-neuroticism scores performed a visual search task to detect normal expressions of anger and happiness, and their anti-expressions within a crowd of neutral expressions. Anti-expressions contained an amount of visual changes equivalent to those found in normal expressions compared to neutral expressions, but they were usually recognized as neutral expressions. Subjective emotional ratings in response to each facial expression stimulus were also obtained. Participants with high-neuroticism showed an overall delay in the detection of target facial expressions compared to participants with low-neuroticism. Additionally, the high-neuroticism group showed higher levels of arousal to facial expressions compared to the low-neuroticism group. These data suggest that neuroticism modulates the detection of emotional facial expressions in healthy participants; high levels of neuroticism delay overall detection of facial expressions and enhance emotional arousal in response to facial expressions. PMID:27073904

  4. DNA expressions - A formal notation for DNA

    NARCIS (Netherlands)

    Vliet, Rudy van

    2015-01-01

    We describe a formal notation for DNA molecules that may contain nicks and gaps. The resulting DNA expressions denote formal DNA molecules. Different DNA expressions may denote the same molecule. Such DNA expressions are called equivalent. We examine which DNA expressions are minimal, which

  5. Construction of a regulable gene therapy vector targeting for hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Shao-Ying Lu; Yan-Fang Sui; Zeng-Shan Li; Cheng-En Pan; Jing Ye; Wen-Yong Wang

    2003-01-01

    AIM: To construct a gene modified hepatocellular carcinoma (HCC) specific EGFP expression vector regulated by abbreviated cis-acting element of AFP gene.METHODS: The minimal essential DNA segments of AFP gene enhancer and promoter were synthesized through PCR from Genome DNA of HepG2 cells. Gene fragments were then cloned into the multiple cloning site of non-promoter EGFP vector pEGFP-t. Recombinant plasmid was transferred into positive or negative AFP cell lines by means of lipofectamine. The expression of EGFP was tested by fluorescence microscope and flow cytometry. The effect of all-trans retinoic acid (ATRA) on the expression of EGFP was tested in different concentrations.RESULTS: By the methods of restriction digestion and sequence analyses we confirmed that the length, position and orientation of inserted genes of cis-acting element of AFP were all correct. The transcription of EGFP was under the control of AFP cis-acting element. The expressing EGFP can only been detected in AFP producing hepatoma cells.The expression rate of EGFP in G418 screened cell line was 34.9±4.1%. 48 h after adding 1×10-7M retinoic acid, EGFP expression rate was 14.7±3.5%. The activity of AFP gene promoter was significantly suppressed by addition of 1×10-7M retinoic acid (P<0.05, P=0.003, t=6.488).CONCLUSION: This recombinant expression vector can be used as a gene therapy vector for HCC. The expression of tumor killing gene will be confined within the site of tumor and the activity of which can be regulated by retinoic acid.

  6. Vascular Gene Expression: A Hypothesis

    Directory of Open Access Journals (Sweden)

    Angélica Concepción eMartínez-Navarro

    2013-07-01

    Full Text Available The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular tissues. This tissue-specific expression in Arabidopsis is predicted by the overrepresentation of GA/CT-rich motifs in gene promoters. In this work we have searched for common motifs in upstream regions of the homologous genes from plants considered to possess a primitive vascular tissue (a lycophyte, as well as from others that lack a true vascular tissue (a bryophyte, and finally from chlorophytes. Both lycophyte and bryophyte display motifs similar to those found in Arabidopsis with a significantly low E-value, while the chlorophytes showed either a different conserved motif or no conserved motif at all. These results suggest that these same genes are expressed coordinately in non- vascular plants; this coordinate expression may have been one of the prerequisites for the development of conducting tissues in plants. We have also analyzed the phylogeny of conserved proteins that may be involved in phloem function and development. The presence of CmPP16, APL, FT and YDA in chlorophytes suggests the recruitment of ancient regulatory networks for the development of the vascular tissue during evolution while OPS is a novel protein specific to vascular plants.

  7. 荧光标记人卵巢癌细胞株的建立%Establishment of a fluorescence-labeled human ovarian cancer cell line

    Institute of Scientific and Technical Information of China (English)

    张小梅; 方廖琼; 王智彪

    2012-01-01

    Objective To establish a human ovarian cancer cell line stably expressing enhanced green fluorescent protein (EGFP), so as to carry out visualized research on whole ovarian cancer. Methods We transfected human ovarian cancer cell line HO8910PM by gene transfection, and obtained cells stably expressing EGFP by sub-cloning amplification and selection with G418-resistance. The expression rate of EGFP was analyzed by flow cytometry (FC). The growth curve, adhesion, migration and invasion experiments were employed to study the biological behaviors of the cells transfected with EGFP. Results Flow cytometry results showed that EGFP positive rate of screened EGFP-HO8910PM cells was higher than 99%. The cell growth, adhesion, invasion and migration abilities of cells were not significantly changed after transfection. Conclusion We have successfully established a cell line EGFP-HO8910PM stably expressing EGFP and at mean time maintaining the characteristics of the parent cell line, which lays a foundation for whole-body visualization research of human ovarian cancer in vivo.%目的 建立稳定高表达增强型绿色荧光蛋白(EGFP)的人卵巢癌细胞株.方法 采用基因转染的方法,将EGFP基因导入人卵巢癌细胞HO8910PM中,通过G418筛选、亚克隆扩增获得稳定表达绿色荧光蛋白的EGFP-HO8910PM细胞株,并用流式细胞术检测所获细胞株的EGFP表达率,通过细胞生长曲线、黏附实验、侵袭迁移实验比较EGFP-HO8910PM细胞和HO8910PM细胞的生物学行为.结果 筛选出的EGFP-HO8910PM细胞经流式细胞仪检测EGFP阳性表达率达99%以上,EGFP-HO8910PM细胞和HO8910PM细胞生长、黏附及侵袭迁移能力无统计学差异.结论 成功建立了稳定表达EGFP且保持母株细胞特性的人卵巢癌细胞株EGFP-HO8910PM,为人卵巢癌整体活体应用中可视化研究打下基础.

  8. Microarray Data Analysis of Gene Expression Evolution

    OpenAIRE

    Honghuang Lin

    2009-01-01

    Microarrays are becoming a widely used tool to study gene expression evolution. A recent paper by Wang and Rekaya describes a comprehensive study of gene expression evolution by microarray.1 The work provides a perspective to study gene expression evolution in terms of functional enrichment and promoter conservation. It was found that gene expression patterns are highly conserved in some biological processes, but the correlation between promoter and gene expression is insignificant. This scop...

  9. Dynamic Facial Expression of Emotion Made Easy

    OpenAIRE

    Broekens, Joost; Qu, Chao; Brinkman, Willem-Paul

    2012-01-01

    Facial emotion expression for virtual characters is used in a wide variety of areas. Often, the primary reason to use emotion expression is not to study emotion expression generation per se, but to use emotion expression in an application or research project. What is then needed is an easy to use and flexible, but also validated mechanism to do so. In this report we present such a mechanism. It enables developers to build virtual characters with dynamic affective facial expressions. The mecha...

  10. Renal tumor with alpha b crystallin expression

    OpenAIRE

    Kim, Mee-Seon; Lee, Hyoun Wook; Lee, Eun Hee

    2015-01-01

    In human, proximal convoluted tubules and thin limbs of Henle show expression of αB crystallin. Renal cell carcinoma also showed expression of αB crystallin in previous reports. We aimed to study the association between αB crystallin expression and renal cell carcinoma and urothelial carcinoma. Furthermore, we also investigated αB crystallin expression depending on the subtype of renal cell carcinoma and examined the relationship between αB crystallin expression and survival in patients with ...

  11. Facial Expressions with Some Mixed Expressions Recognition Using Neural Networks

    Directory of Open Access Journals (Sweden)

    Dr.R.Parthasarathi

    2011-01-01

    Full Text Available Facial feature extraction is the essential step of facial expression recognition. The automatic facial impression evaluation applies for wide area use. The important facial feature vectors for expressionanalysis are analyzed. The extracted feature vector loads all known feature vectors and trains the NN using as input training vectors while PCA is used for dimensionality reduction. The method is effective for both dimension reduction and good recognition performance in comparison with other proposed methods as shown in experiment results.

  12. Facial Expressions with Some Mixed Expressions Recognition Using Neural Networks

    OpenAIRE

    Dr.R.Parthasarathi; V.Lokeswar Reddy,; K.Vishnuthej,; G.Vishnu Vandan

    2011-01-01

    Facial feature extraction is the essential step of facial expression recognition. The automatic facial impression evaluation applies for wide area use. The important facial feature vectors for expressionanalysis are analyzed. The extracted feature vector loads all known feature vectors and trains the NN using as input training vectors while PCA is used for dimensionality reduction. The method is effective for both dimension reduction and good recognition performance in comparison with other p...

  13. Recombination-ready Sindbis replicon expression vectors for transgene expression

    Directory of Open Access Journals (Sweden)

    Olson Ken E

    2007-10-01

    Full Text Available Abstract Background Sindbis viruses have been widely used as tools to study gene function in cells. Despite the utility of these systems, the construction and production of alphavirus replicons is time consuming and inefficient due to potential additional restriction sites within the insert region and lack of directionality for insert ligation. In this report, we present a system useful for producing recombinant Sindbis replicons that uses lambda phage recombination technology to rapidly and specifically construct replicon expression plasmids that contain insert regions in the desired orientation. Results Recombination of the gene of interest with the replicon plasmid resulted in nearly 100% recombinants, each of which contained a correctly orientated insert. Replicons were easily produced in cell culture and packaged into pseudo-infectious viral particles. Insect and mammalian cells infected with pseudo-infectious viral particles expressed various transgenes at high levels. Finally, inserts from persistently replicating replicon RNA were easily isolated and recombined back into entry plasmids for sequencing and subsequent analysis. Conclusion Replication-ready replicon expression plasmids make the use of alphavirus replicons fast and easy as compared to traditional replicon production methods. This system represents a significant step forward in the utility and ease of use of alphavirus replicons in the study of gene function.

  14. Nestin Reporter Transgene Labels Multiple Central Nervous System Precursor Cells

    Directory of Open Access Journals (Sweden)

    Avery S. Walker

    2010-01-01

    Full Text Available Embryonic neuroepithelia and adult subventricular zone (SVZ stem and progenitor cells express nestin. We characterized a transgenic line that expresses enhanced green fluorescent protein (eGFP specified to neural tissue by the second intronic enhancer of the nestin promoter that had several novel features. During embryogenesis, the dorsal telencephalon contained many and the ventral telencephalon few eGFP+ cells. eGFP+ cells were found in postnatal and adult neurogenic regions. eGFP+ cells in the SVZ expressed multiple phenotype markers, glial fibrillary acidic protein, Dlx, and neuroblast-specific molecules suggesting the transgene is expressed through the lineage. eGFP+ cell numbers increased in the SVZ after cortical injury, suggesting this line will be useful in probing postinjury neurogenesis. In non-neurogenic regions, eGFP was strongly expressed in oligodendrocyte progenitors, but not in astrocytes, even when they were reactive. This eGFP+ mouse will facilitate studies of proliferative neuroepithelia and adult neurogenesis, as well as of parenchymal oligodendrocytes.

  15. ERG protein expression over time

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Brasso, Klaus; Thomsen, Frederik Birkebæk;

    2015-01-01

    AIMS: We evaluated the consistency in ERG protein expression from diagnostic specimens through rebiopsies to radical prostatectomies in patients with clinically localised prostate cancer to investigate the validity of ERG status in biopsies. METHODS: ERG expression was assessed by...... immunohistochemistry (IHC) in 625 biopsy sets and 86 radical prostatectomy specimens from 265 patients with prostate cancer managed on active surveillance. For IHC, a rabbit monoclonal primary antibody was used (clone: EPR3864). TMPRSS2-ERG fluorescence in situ hybridisation (FISH) analyses were performed in 74...... biopsies using the FISH ZytoLight TriCheck Probe (SPEC ERG/TMPRSS2). FISH results were correlated with IHC findings. RESULTS: The concordance between FISH and IHC was 97.3% and IHC demonstrated a sensitivity and specificity for ERG rearrangement of 100% and 95.5%, respectively. Applying IHC, 38.1% of...

  16. User Expressions Translated Into Requirements

    Directory of Open Access Journals (Sweden)

    Birgitta Bergvall-Kåreborn

    2010-01-01

    Full Text Available Grounding the development of mobile and ubiquitous services on actual needs and behaviors of users, rather than on designers' intuition, is a well-established tradition. However, gathering data about users in different contexts usually results in large amounts of data that have to be analyzed and translated into requirements. This crucial activity and its outcome are often shaped by the preconceptions of the developers or researchers. Despite this subjectivity, the translation process is seldom transparent. The aim of this paper, therefore, is to contribute to the field by presenting a process for translating user expressions into needs and later into requirements using Reiss' taxonomy of human needs. By adopting this process of translation, we were able to identify two hierarchical levels of needs: needs of a service and needs in a service. These two levels provide a transparent bridge between user expressions and system requirements.

  17. Summer Oral Expression English Course

    CERN Document Server

    HR Department

    2011-01-01

    An English Oral Expression course will take place between 15 August and 30 September 2011. Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enrol here. Or contact: Kerstin FUHRMEISTER (70896) Tessa OSBORNE (72957)  

  18. The motivation to express prejudice

    OpenAIRE

    Forscher, Patrick S.; Cox, William T. L.; Graetz, Nicholas; Devine, Patricia G.

    2015-01-01

    Contemporary prejudice research focuses primarily on people who are motivated to respond without prejudice and the ways in which unintentional bias can cause these people to act inconsistent with this motivation. However, some real-world phenomena (e.g., hate speech, hate crimes) and experimental findings (e.g., Plant & Devine, 2001; 2009) suggest that some expressions of prejudice are intentional. These phenomena and findings are difficult to explain solely from the motivat...

  19. Cultural expression, creativity and innovation

    OpenAIRE

    Anheier, Helmut K.; Isar, Yudhishthir Raj

    2010-01-01

    Volume 3 of the Cultures & Globalization series, Creativity and Innovations, explores the interactions between globalization and the forms of cultural expression that are their basic resource. Bringing together over 25 high-profile authors from around the world, this volume addresses such questions as: What impacts does globalization have on cultural creativity and innovation? How is the evolving world 'map' of creativity related to the drivers and patterns of globalization? What are the rela...

  20. Parsing Expression Grammars Made Practical

    OpenAIRE

    Laurent, Nicolas; Mens, Kim

    2015-01-01

    Parsing Expression Grammars (PEGs) define languages by specifying recursive-descent parser that recognises them. The PEG formalism exhibits desirable properties, such as closure under composition, built-in disambiguation, unification of syntactic and lexical concerns, and closely matching programmer intuition. Unfortunately, state of the art PEG parsers struggle with left-recursive grammar rules, which are not supported by the original definition of the formalism and can lead to infinite recu...

  1. Vénus version Express

    Science.gov (United States)

    Nazé, Yaël

    2010-04-01

    En avril 2006, Vénus a "capturé" un objet d'un genre particulier: une sonde robotique européenne, baptisée Venus Express et destinée à scruter cette planète sous tous les angles. Bilan de cette mission 5 ans après le lancement de la sonde, dont 4 d'observations vénusiennes.

  2. Professional expression of technology teacher's

    OpenAIRE

    Jacevič, Jolanta

    2005-01-01

    Professional expression of technology teacher’s is important in nowadays society and educational system, especially when new educational paradigm is becoming stronger. The teacher of technology has to help pupils to understand the value of present time life: to set pupils’ sights on country’s production and use policy, individual usage culture, healthy living, ability to develop creatively the national ethnic cultural traditions and their integration into work and housekeeping, and also to...

  3. The Expression of a Need

    OpenAIRE

    Alfort, Esben

    2013-01-01

    This thesis provides a framework for information retrieval based on a set of models which together illustrate how users of search engines come to express their needs in a particular way. With such insights, we may be able to improve systems’ capabilities of understanding users’ requests and through that eventually the ability to satisfy their needs. Developing the framework necessitates discussion of context, relevance, need development, and the cybernetics of search, all of which...

  4. Transgenic Arabidopsis Gene Expression System

    Science.gov (United States)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  5. Expressive Sound Synthesis for Animation

    OpenAIRE

    Picard-Limpens, Cécile

    2009-01-01

    The main objective of this thesis is to provide tools for an expressive and real-time synthesis of sounds resulting from physical interactions of various objects in a 3D virtual environment. Indeed, these sounds, such as collisions sounds or sounds from continuous interaction between surfaces, are difficult to create in a pre-production process since they are highly dynamic and vary drastically depending on the interaction and objects. To achieve this goal, two approaches are proposed; the fi...

  6. Misrecognition of facial expressions in delinquents

    Directory of Open Access Journals (Sweden)

    Matsuura Naomi

    2009-09-01

    Full Text Available Abstract Background Previous reports have suggested impairment in facial expression recognition in delinquents, but controversy remains with respect to how such recognition is impaired. To address this issue, we investigated facial expression recognition in delinquents in detail. Methods We tested 24 male adolescent/young adult delinquents incarcerated in correctional facilities. We compared their performances with those of 24 age- and gender-matched control participants. Using standard photographs of facial expressions illustrating six basic emotions, participants matched each emotional facial expression with an appropriate verbal label. Results Delinquents were less accurate in the recognition of facial expressions that conveyed disgust than were control participants. The delinquents misrecognized the facial expressions of disgust as anger more frequently than did controls. Conclusion These results suggest that one of the underpinnings of delinquency might be impaired recognition of emotional facial expressions, with a specific bias toward interpreting disgusted expressions as hostile angry expressions.

  7. Gene expression profile of pulpitis.

    Science.gov (United States)

    Galicia, J C; Henson, B R; Parker, J S; Khan, A A

    2016-06-01

    The cost, prevalence and pain associated with endodontic disease necessitate an understanding of the fundamental molecular aspects of its pathogenesis. This study was aimed to identify the genetic contributors to pulpal pain and inflammation. Inflamed pulps were collected from patients diagnosed with irreversible pulpitis (n=20). Normal pulps from teeth extracted for various reasons served as controls (n=20). Pain level was assessed using a visual analog scale (VAS). Genome-wide microarray analysis was performed using Affymetrix GeneTitan Multichannel Instrument. The difference in gene expression levels were determined by the significance analysis of microarray program using a false discovery rate (q-value) of 5%. Genes involved in immune response, cytokine-cytokine receptor interaction and signaling, integrin cell surface interactions, and others were expressed at relatively higher levels in the pulpitis group. Moreover, several genes known to modulate pain and inflammation showed differential expression in asymptomatic and mild pain patients (⩾30 mm on VAS) compared with those with moderate to severe pain. This exploratory study provides a molecular basis for the clinical diagnosis of pulpitis. With an enhanced understanding of pulpal inflammation, future studies on treatment and management of pulpitis and on pain associated with it can have a biological reference to bridge treatment strategies with pulpal biology. PMID:27052691

  8. Gene Expression in Trypanosomatid Parasites

    Directory of Open Access Journals (Sweden)

    Santiago Martínez-Calvillo

    2010-01-01

    Full Text Available The parasites Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are the trypanosomatid protozoa that cause the deadly human diseases leishmaniasis, African sleeping sickness, and Chagas disease, respectively. These organisms possess unique mechanisms for gene expression such as constitutive polycistronic transcription of protein-coding genes and trans-splicing. Little is known about either the DNA sequences or the proteins that are involved in the initiation and termination of transcription in trypanosomatids. In silico analyses of the genome databases of these parasites led to the identification of a small number of proteins involved in gene expression. However, functional studies have revealed that trypanosomatids have more general transcription factors than originally estimated. Many posttranslational histone modifications, histone variants, and chromatin modifying enzymes have been identified in trypanosomatids, and recent genome-wide studies showed that epigenetic regulation might play a very important role in gene expression in this group of parasites. Here, we review and comment on the most recent findings related to transcription initiation and termination in trypanosomatid protozoa.

  9. The Gene Expression Omnibus database

    Science.gov (United States)

    Clough, Emily; Barrett, Tanya

    2016-01-01

    The Gene Expression Omnibus (GEO) database is an international public repository that archives and freely distributes high-throughput gene expression and other functional genomics data sets. Created in 2000 as a worldwide resource for gene expression studies, GEO has evolved with rapidly changing technologies and now accepts high-throughput data for many other data applications, including those that examine genome methylation, chromatin structure, and genome–protein interactions. GEO supports community-derived reporting standards that specify provision of several critical study elements including raw data, processed data, and descriptive metadata. The database not only provides access to data for tens of thousands of studies, but also offers various Web-based tools and strategies that enable users to locate data relevant to their specific interests, as well as to visualize and analyze the data. This chapter includes detailed descriptions of methods to query and download GEO data and use the analysis and visualization tools. The GEO homepage is at http://www.ncbi.nlm.nih.gov/geo/. PMID:27008011

  10. Emotional context influences micro-expression recognition.

    Directory of Open Access Journals (Sweden)

    Ming Zhang

    Full Text Available Micro-expressions are often embedded in a flow of expressions including both neutral and other facial expressions. However, it remains unclear whether the types of facial expressions appearing before and after the micro-expression, i.e., the emotional context, influence micro-expression recognition. To address this question, the present study used a modified METT (Micro-Expression Training Tool paradigm that required participants to recognize the target micro-expressions presented briefly between two identical emotional faces. The results of Experiments 1 and 2 showed that negative context impaired the recognition of micro-expressions regardless of the duration of the target micro-expression. Stimulus-difference between the context and target micro-expression was accounted for in Experiment 3. Results showed that a context effect on micro-expression recognition persists even when the stimulus similarity between the context and target micro-expressions was controlled. Therefore, our results not only provided evidence for the context effect on micro-expression recognition but also suggested that the context effect might result from both the stimulus and valence differences.

  11. Trafficking of Na,K-ATPase fused to enhanced green fluorescent protein is mediated by protein kinase A or C

    DEFF Research Database (Denmark)

    Kristensen, B; Birkelund, Svend; Jørgensen, PL

    2003-01-01

    Fusion of enhanced green fluorescent protein (EGFP) to the C-terminal of rat Na,K-ATPase a1-subunit is introduced as a novel procedure for visualizing trafficking of Na,K-pumps in living COS-1 renal cells in response to PKA or PKC stimulation. Stable, functional expression of the fluorescent...... along the plasma membrane of COS cells. In unstimulated COS cells, Na,K-EGFP was also present in lysosomes and in vesicles en route from the endoplasmic reticulum to the plasma membrane, but it was almost absent from recycling endosomes labelled with fluorescent transferrin. After activation of protein...... chimera (Na,K-EGFP) was achieved in COS-1 cells using combined puromycin and ouabain selection procedures. Na,K-pump activities were unchanged after fusion with EGFP, both in basal and regulated states. In confocal laser scanning and fluorescence microscopes, the Na,K-EGFP chimera was distributed mainly...

  12. Optimal Expression Condition of Recombinant RAP

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jie; ZHANG Hong; BI Hao; LIU Zhiguo; GUO Jianli; QU Shen

    2007-01-01

    In order to construct the expression recombinant of human receptor associated protein (RAP), optimize its expression condition and obtain the recombinant protein after expression with high efficiency, two prokaryotic expression vectors-pT7-PL and pET-28a(+) were used to construct the expression recombinant containing RAP cDNA, and the expression efficiency of two kinds of expression E. coli of BL21 strains was compared. The effect of different induction conditions on the expression of recombinant RAP was observed. After recombinant protein was purified with Ni+-nitrilotriacetic acid (Ni+-NTA) affinity chromatogram, its binding ability with microphage was observed. The results showed that two recombinant plasmids both obtained high expression of RAP. The expression levels of RAP in plasmid pT7-PL-RAP in BL21 (DE3, plysS) strain were significantly higher than in BL21 (DE3) strain. The expression of pT7-PL-RAP in the presence of chloramphenicol was higher than in the absence of chloramphenicol, and most of the inducible expressed RAP was soluble. The RAP which was purified by Ni+-NTA resin could strongly bind with the RAW264.7 cells rich in low density lipoprotein receptor (LDLR) family receptors. It was concluded that the expression condition of recombinant RAP was optimized and functional RAP was obtained, which offered a good foundation for the further production of RAP as research tool.

  13. THE ENHANCED GREEN FLUORESCENT PROTEIN AS A MARKER FOR HUMAN TUMOR CELLS LABELLED BY RETROVIRAL TRANSDUCTION

    Institute of Scientific and Technical Information of China (English)

    傅建新; 王玮; 白霞; 卢大儒; 阮长耿; 陈子兴

    2002-01-01

    Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSN encoding EGFP was constructed and three human tumor cell lines were infected with LGSN amphotropic virus. Tumor cell lines that stably express EGFP were selected with G418. The integration and expression of EGFP gene were analyzed by polymerase chain reaction, and flow cytometry (FCM). Results: After gene transfection and ping-pong transduction, amphotropic producer line Am12/LGSN was generated with a stable green fluorescence signal readily detectable by FCM in up to 97% of examined cells. The viral titer in the supernatants was up to 8.2×105CFU/ml. After transduction and selection, G418-resistant leukemia K562, mammary carcinoma MCF-7, and bladder cancer 5637 cells were developed, in which the integration of both EGFP and neomycin resistance gene was confirmed by DNA amplification. In comparison with uninfected cells, FCM analysis revealed EGFP expression in up to 90% (range 85.5%~90.0%) of tumor cells containing LGSN provirus. Conclusion: The retroviral vector LGSN can effectively mark the human tumor cells with a stably EGFP expression which may be in studying tumor growth, metastasis and angiogenesis.

  14. Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes

    KAUST Repository

    Horiuchi, Youko

    2015-12-23

    Background Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue specific expression differences. However, different types of gene expression alteration should have different effects on an organism, the evolutionary forces that act on them might be different, and different types of genes might show different types of differential expression between species. To confirm this, we studied differentially expressed (DE) genes among closely related groups that have extensive gene expression atlases, and clarified characteristics of different types of DE genes including the identification of regulating loci for differential expression using expression quantitative loci (eQTL) analysis data. Results We detected differentially expressed (DE) genes between rice subspecies in five homologous tissues that were verified using japonica and indica transcriptome atlases in public databases. Using the transcriptome atlases, we classified DE genes into two types, global DE genes and changed-tissues DE genes. Global type DE genes were not expressed in any tissues in the atlas of one subspecies, however changed-tissues type DE genes were expressed in both subspecies with different tissue specificity. For the five tissues in the two japonica-indica combinations, 4.6 ± 0.8 and 5.9 ± 1.5 % of highly expressed genes were global and changed-tissues DE genes, respectively. Changed-tissues DE genes varied in number between tissues, increasing linearly with the abundance of tissue specifically expressed genes in the tissue. Molecular evolution of global DE genes was rapid, unlike that of changed-tissues DE genes. Based on gene ontology, global and changed-tissues DE genes were different, having no common GO terms. Expression differences of most global DE genes were regulated by cis-eQTLs. Expression

  15. 甲胎蛋白对耐药基因MDR1表达及肝癌细胞化疗敏感性的影响%Influence of alpha-fetoprotein on the expression of drug-resistance gene MDR1 and chemotherapeutic sensitivity in hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    吴超; 杨健; 张金玲; 金涛; 何前进; 李常海

    2015-01-01

    Objective To explore the influence of alpha-fetoprotein (AFP) on the expression of drug-resistance gene MDR1 and chemotherapeutic sensitivity in hepatocellular carcinoma (HCC) cells.Methods A HCC cell line SMMC-7721/AFP, which was stably transfected with AFP gene, was established.mRNA and protein expressions of AFP and MDR1 were detected by real-time PCR and Western Blot,respectively.The sensitivity of SMMC-7721/AFP and SMMC-7721/EGFP cells with or without MDR1 silencing by siRNA to doxorubicin was tested by MTT assay.Immunohistochemistry was used to detect the expression of MDR1 genes-coded protein Pgp in 60 cases of HCC tissues, and the relationship between Pgp expression and serum AFP levels was analyzed.Results AFP mRNA and protein could be detected in SMMC-7721/AFP cells, but not in control cells, indicating that the AFP stably transfected cell line was successfully established.MDR1 mRNA and protein levels were higher in SMMC-7721/AFP cells than those in SMMC-7721/EGFP cells.MDR1 mRNA level in SMMC-7721/AFP cells was (52.7 ± 1.5) times as high as that in SMMC-7721/EGFP cells (P < 0.05).The resistance to doxorubicin was increased by (12.8 ± 1.1) times after AFP transfection (P < 0.05).The chemosensitivity to doxorubicin was increased after the expression of MDR1 was knocked down by siRNA.The expression of Pgp in HCC tissues was positively correlated with the serum AFP levels.Conclusion AFP could induce drug-resistance to doxorubicin in HCC cells by increasing the expression of MDR1.%目的 探讨甲胎蛋白(AFP)对耐药基因MDR1表达和肝癌细胞化疗敏感性的影响.方法 建立稳定表达AFP的肝癌细胞系SMMC-7721/AFP,分别通过Real-time PCR和蛋白印迹检测转染前后AFP和MDR1的表达.MTT法测定SMMC-7721/AFP和SMMC-7721/EGFP细胞对阿霉素的化疗敏感性.siRNA沉默SMMC-7721/AFP细胞中MDR1的表达,观察细胞对阿霉素化疗敏感性的变化.采用免疫组织化学染色法检测60例肝癌组织中MDR1编码蛋

  16. Summer Oral Expression English course

    CERN Multimedia

    2012-01-01

    An English Oral Expression course will take place this summer at some time between 25 June and 28 September. The exact dates will be decided according to the preferences of the students.   Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enroll through this link. Please be sure to indicate your planned absences in the comments field so we can schedule the course. If you need more information please send a message to English.training@cern.ch

  17. Summer Oral Expression English course

    CERN Document Server

    2013-01-01

    An English Oral Expression course will take place this summer at some time between August 19 and October 4.   Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enroll through this link. Please be sure to indicate your planned absences in the comments field so we can schedule the course. If you need more information please send a message to English.training@cern.ch.

  18. Summer Oral Expression English Course

    CERN Document Server

    HR Department

    2011-01-01

    An English Oral Expression course will take place between 15 August and 30 September 2011. Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enrol through the following link https://cta.cern.ch/cta2/f?p=110:9:1576796470009589::::X_STATUS,XS_COURSE_NAME,XS_PROGRAMME,XS_SUBCATEGORY,X_COURSE_ID,XS_LANGUAGE,XS_SESSION:D,,1,,4368,B, Or contact: Kerstin FUHRMEISTER (70896) Tessa OSBORNE (72957)  

  19. Summer Oral Expression English course

    CERN Multimedia

    2012-01-01

    An English Oral Expression course will take place this summer from 20 August to 29 September.   Schedule: to be determined (2 sessions of 2 hours per week). Please note that this course is for learners who have a good knowledge of English (CERN level 7 upwards). If you are interested in following this course, please enroll through this link. Please be sure to indicate your planned absences in the comments field so we can schedule the course. If you need more information please send a message to English.training@cern.ch

  20. Adipocyte differentiation and leptin expression

    DEFF Research Database (Denmark)

    Hwang, C S; Loftus, T M; Mandrup, S;

    1997-01-01

    , most notably those of the C/EBP and PPAR families, which combine to regulate each other and to control the expression of adipocyte-specific genes. One such gene, i.e. the obese gene, was recently identified and found to encode a hormone, referred to as leptin, that plays a major role in the regulation...... of energy intake and expenditure. The hormonal and transcriptional control of adipocyte differentiation is discussed, as is the role of leptin and other factors secreted by the adipocyte that participate in the regulation of adipose homeostasis....

  1. PRENOVA SPLETNEGA PORTALA SŽ EXPRESS

    OpenAIRE

    Šetina, Jadran

    2016-01-01

    Sprva so podjetja šele spoznavala možnosti, ki jih lahko prinese internet. S konkurenčnega vidika je bila pomembna že sama prisotnost na svetovnem spletu. S hitrim širjenjem in razvojem interneta ter odprtokodnih tehnologij pa je vse več spletnih portalov, katerih namen je predstaviti podjetje in preko elektronskega poslovanja sodelovati z uporabniki. Tako poslovanje zahteva zanesljive, varne in uporabniku prijazne rešitve. Portal SŽ Express potrebuje celovito prenovo. S trenutno uporabljenim...

  2. General expression of manipulator kinematics

    International Nuclear Information System (INIS)

    In this paper, a general description for kinematic modelling of all types of manipulators is presented by expanding the concept of the homogeneous transformation (Ai-matrix) based on Denavit-Hartenberg notation. Unlike the previous matrix method, the expression of recursive relationships which is suitable for computer-aided design makes it possible to derive automatically the position and orientation of manipulator hand. Two algorithms which take into consideration of the order of operation result in identical relations with respect to the kinematics between the reference system and the final link, which was proved by an application to a six-link manipulator. (author)

  3. Impact of money on emotional expression

    OpenAIRE

    Jiang, Y.; Chen, Z.; Wyer, RS

    2014-01-01

    Activating the concept of money can influence people's own expressions of emotion as well as their reactions to the emotional expressions of others. Thinking about money increases individuals' disposition to perceive themselves in a business-like relationship with others in which transactions are based on objective criteria and the expression of emotion is considered inappropriate. Therefore, these individuals express less emotion in public and expect others to do likewise. Six experiments sh...

  4. Survey on Human Face Expression Recognition

    OpenAIRE

    Atul Chinchamalatpure; Prof. Anurag Jain

    2014-01-01

    Facial expression recognition plays an important and effective role in the interaction in Human. There are seven basic facial expressions namely fear, surprise, happy, sad, disgust, Neutral and anger. Facial Expression Recognition is process performed by computers which consist of detect the face in the image and pre-process the face region, extracting facial expression features from image by analyzing the motion of facial features or change in the appearance of facial features and classifyin...

  5. Computer-enhanced emotion in facial expressions.

    OpenAIRE

    Calder, A J; Young, A W; Rowland, D.; Perrett, D.I

    1997-01-01

    Benson & Perrett's (1991 b) computer-based caricature procedure was used to alter the positions of anatomical landmarks in photographs of emotional facial expressions with respect to their locations in a reference norm face (e.g. a neutral expression). Exaggerating the differences between an expression and its norm produces caricatured images, whereas reducing the differences produces 'anti-caricatures'. Experiment 1 showed that caricatured (+50% different from neutral) expressions were recog...

  6. Reconnaissance de l'expression du visage

    OpenAIRE

    Zhao, Xi

    2010-01-01

    This Ph.D thesis work is dedicated to automatic facial analysis in 3D, including facial landmarking and facial expression recognition. Indeed, facial expression plays an important role both in verbal and non verbal communication, and in expressing emotions. Thus, automatic facial expression recognition has various purposes and applications and particularly is at the heart of "intelligent" human-centered human/computer(robot) interfaces. Meanwhile, automatic landmarking provides aprior knowled...

  7. Kivy and Langer on expressiveness in music

    OpenAIRE

    van der Schoot Albert

    2013-01-01

    From 1980 onwards, Peter Kivy has put forward that music does not so much express emotions but rather is expressive of emotions. The character of the music does not represent the character or mood of the composer, but reflects his knowledge of emotional life. Unfortunately, Kivy fails to give credit to Susanne Langer, who brought these views to the fore as early as 1942, claiming that the vitality of music lies in expressiveness, not in expression.

  8. Kivy and Langer on expressiveness in music

    Directory of Open Access Journals (Sweden)

    van der Schoot Albert

    2013-01-01

    Full Text Available From 1980 onwards, Peter Kivy has put forward that music does not so much express emotions but rather is expressive of emotions. The character of the music does not represent the character or mood of the composer, but reflects his knowledge of emotional life. Unfortunately, Kivy fails to give credit to Susanne Langer, who brought these views to the fore as early as 1942, claiming that the vitality of music lies in expressiveness, not in expression.

  9. Electrophysiological, morphological and topological properties of two histochemically distinct subpopulations of cerebellar unipolar brush cells

    OpenAIRE

    Kim, Jin-Ah; Sekerková, Gabriella; Mugnaini, Enrico; Martina, Marco

    2012-01-01

    Unipolar brush cells (UBCs) are excitatory cerebellar granular layer interneurons whose brush-like dendrites receive one-to-one mossy fiber inputs. Subclasses of UBCs differ primarily by expressing metabotropic glutamate receptor (mGluR) 1α or calretinin. We used GENSAT Tg(Grp-EGFP) BAC-transgenic mice, which selectively express EGFP in mGluR1α-positive UBCs to compare the functional properties of the two subclasses. Compared to EGFP-negative UBCs, which include the calretinin-positive cells,...

  10. Deformation Expression for Elements of Algebra

    OpenAIRE

    Omori, H.; Y. Maeda; Miyazaki, N.; Yoshioka, A.

    2011-01-01

    The purpose of this paper is to give a notion of deformation of expressions for elements of algebra. Deformation quantization (cf.[BF]) deforms the commutative world to a non-commutative world. However, this involves deformation of expression of elements of algebras even from a commutative world to another commutative world. This is indeed a deformation of expressions for elements of algebra.

  11. Mitochondrial RNA granules: Compartmentalizing mitochondrial gene expression.

    Science.gov (United States)

    Jourdain, Alexis A; Boehm, Erik; Maundrell, Kinsey; Martinou, Jean-Claude

    2016-03-14

    In mitochondria, DNA replication, gene expression, and RNA degradation machineries coexist within a common nondelimited space, raising the question of how functional compartmentalization of gene expression is achieved. Here, we discuss the recently characterized "mitochondrial RNA granules," mitochondrial subdomains with an emerging role in the regulation of gene expression. PMID:26953349

  12. 7 CFR 27.99 - Values; expression.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Values; expression. 27.99 Section 27.99 Agriculture... CLASSIFICATION UNDER COTTON FUTURES LEGISLATION Regulations Price Quotations and Differences § 27.99 Values; expression. For the purpose of this subpart values shall be expressed in terms of cents and hundredths of...

  13. Bit-coded regular expression parsing

    DEFF Research Database (Denmark)

    Nielsen, Lasse; Henglein, Fritz

    2011-01-01

    Regular expression parsing is the problem of producing a parse tree of a string for a given regular expression. We show that a compact bit representation of a parse tree can be produced efficiently, in time linear in the product of input string size and regular expression size, by simplifying the...

  14. Technology Corner: A Regular Expression Training App

    Directory of Open Access Journals (Sweden)

    Nick Flor

    2012-12-01

    Full Text Available Regular expressions enable digital forensic analysts to find information in files. The best way for an analyst to become proficient in writing regular expressions is to practice. This paper presents the code for an app that allows an analyst to practice writing regular expressions.

  15. Facial Expressivity in Infants of Depressed Mothers.

    Science.gov (United States)

    Pickens, Jeffrey; Field, Tiffany

    1993-01-01

    Facial expressions were examined in 84 3-month-old infants of mothers classified as depressed, nondepressed, or low scoring on the Beck Depression Inventory. Infants of both depressed and low-scoring mothers showed significantly more sadness and anger expressions and fewer interest expressions than infants of nondepressed mothers. (Author/MDM)

  16. A constructive approach to gene expression dynamics

    International Nuclear Information System (INIS)

    Recently, experiments on mRNA abundance (gene expression) have revealed that gene expression shows a stationary organization described by a scale-free distribution. Here we propose a constructive approach to gene expression dynamics which restores the scale-free exponent and describes the intermediate state dynamics. This approach requires only one assumption: Markov property

  17. Expressiveness modulo Bisimilarity of Regular Expressions with Parallel Composition (Extended Abstract

    Directory of Open Access Journals (Sweden)

    Jos C. M. Baeten

    2010-11-01

    Full Text Available The languages accepted by finite automata are precisely the languages denoted by regular expressions. In contrast, finite automata may exhibit behaviours that cannot be described by regular expressions up to bisimilarity. In this paper, we consider extensions of the theory of regular expressions with various forms of parallel composition and study the effect on expressiveness. First we prove that adding pure interleaving to the theory of regular expressions strictly increases its expressiveness up to bisimilarity. Then, we prove that replacing the operation for pure interleaving by ACP-style parallel composition gives a further increase in expressiveness. Finally, we prove that the theory of regular expressions with ACP-style parallel composition and encapsulation is expressive enough to express all finite automata up to bisimilarity. Our results extend the expressiveness results obtained by Bergstra, Bethke and Ponse for process algebras with (the binary variant of Kleene's star operation.

  18. Expressiveness modulo Bisimilarity of Regular Expressions with Parallel Composition (Extended Abstract)

    CERN Document Server

    Baeten, Jos C M; Muller, Tim; van Tilburg, Paul; 10.4204/EPTCS.41.1

    2010-01-01

    The languages accepted by finite automata are precisely the languages denoted by regular expressions. In contrast, finite automata may exhibit behaviours that cannot be described by regular expressions up to bisimilarity. In this paper, we consider extensions of the theory of regular expressions with various forms of parallel composition and study the effect on expressiveness. First we prove that adding pure interleaving to the theory of regular expressions strictly increases its expressiveness up to bisimilarity. Then, we prove that replacing the operation for pure interleaving by ACP-style parallel composition gives a further increase in expressiveness. Finally, we prove that the theory of regular expressions with ACP-style parallel composition and encapsulation is expressive enough to express all finite automata up to bisimilarity. Our results extend the expressiveness results obtained by Bergstra, Bethke and Ponse for process algebras with (the binary variant of) Kleene's star operation.

  19. Hepcidin expression in psoriasis patients

    Directory of Open Access Journals (Sweden)

    Nursel Dilek

    2014-01-01

    Full Text Available Background: Iron is an essential nutrient for mammals. Accelerated loss of nutrients through hyperproliferation and desquamation from the skin in psoriasis is known. Hepcidin is an important and recently discovered regulator of iron homeostasis. Aims and Objectives: The present study was undertaken to investigate the hepcidin expression in psoriasis patients. Materials and Methods: We examined peripheral blood cell counts, serum Fe, ferritin, interleukin-6 (IL-6 and hepcidin levels using respectively automated hematology analyzer, Iron assay on the AEROSET system, chemiluminescent microparticle immunoassay with automated analyzer, and enzyme-linked immunosorbent assay. Results: The independent comparison of Fe, ferritin, IL-6 and hepcidin levels in psoriasis patients and control group (healthy volunteers revealed lower Fe and higher IL-6, hepcidin levels in psoriasis patients. No significant difference was seen in the ferritin level between the psoriasis and the control group. Conclusions: We think that studies on hepcidin expression in psoriatic plaques will contribute to our understanding the role of iron and hepcidin in the pathogenesis of psoriasis.

  20. Moxibustion upregulates hippocampal progranulin expression

    Directory of Open Access Journals (Sweden)

    Tao Yi

    2016-01-01

    Full Text Available In China, moxibustion is reported to be useful and has few side effects for chronic fatigue syndrome, but its mechanisms are largely unknown. More recently, the focus has been on the wealth of information supporting stress as a factor in chronic fatigue syndrome, and largely concerns dysregulation in the stress-related hypothalamic-pituitary-adrenal axis. In the present study, we aimed to determine the effect of moxibustion on behavioral symptoms in chronic fatigue syndrome rats and examine possible mechanisms. Rats were subjected to a combination of chronic restraint stress and forced swimming to induce chronic fatigue syndrome. The acupoints Guanyuan (CV4 and Zusanli (ST36, bilateral were simultaneously administered moxibustion. Untreated chronic fatigue syndrome rats and normal rats were used as controls. Results from the forced swimming test, open field test, tail suspension test, real-time PCR, enzyme-linked immunosorbent assay, and western blot assay showed that moxibustion treatment decreased mRNA expression of corticotropin-releasing hormone in the hypothalamus, and adrenocorticotropic hormone and corticosterone levels in plasma, and markedly increased progranulin mRNA and protein expression in the hippocampus. These findings suggest that moxibustion may relieve the behavioral symptoms of chronic fatigue syndrome, at least in part, by modulating the hypothalamic-pituitary-adrenal axis and upregulating hippocampal progranulin.

  1. Expressing Model Constraints Visually with VMQL

    DEFF Research Database (Denmark)

    Störrle, Harald

    OCL is the de facto standard language for expressing constraints and queries on UML models. However, OCL expressions are very difficult to create, understand, and maintain, even with the sophisticated tool support now available. In this paper, we propose to use the Visual Model Query Language (VMQL......) for specifying constraints on UML models. We examine VMQL's usability by controlled experiments and its expressiveness by a representative sample. We conclude that VMQL is less expressive than OCL, although expressive enough for most of the constraints in the sample. In terms of usability, however...

  2. Analysis of Gene Expression Patterns Using Biclustering.

    Science.gov (United States)

    Roy, Swarup; Bhattacharyya, Dhruba K; Kalita, Jugal K

    2016-01-01

    Mining microarray data to unearth interesting expression profile patterns for discovery of in silico biological knowledge is an emerging area of research in computational biology. A group of functionally related genes may have similar expression patterns under a set of conditions or at some time points. Biclustering is an important data mining tool that has been successfully used to analyze gene expression data for biologically significant cluster discovery. The purpose of this chapter is to introduce interesting patterns that may be observed in expression data and discuss the role of biclustering techniques in detecting interesting functional gene groups with similar expression patterns. PMID:26350227

  3. The labeling of C57BL/6j derived embryonic stem cells with enhanced green fluorescent protein

    Institute of Scientific and Technical Information of China (English)

    滕路; 张崇本; 尤洁芳; 尚克刚; 顾军

    2003-01-01

    Objective To labele MESPU35, a embryonic stem (ES) cell line derived from C57BL/6j mouse, with enhanced green fluorescent protein (EGFP) for further application.Methods The EGFP gene was controlled by the hybrid CA promoter/enhancer (CMV enhancer/ chicken beta-actin promoter/ beta-actin intron) to construct the vector of the transgene, pCA-EGFP. The vector was transfected into MESPU35 by electroporation.Results We generated EGFP expressing ES cells demonstrating normal properties. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages as well as in differentiated cells. Cultured in suspension, the "green" ES cells aggregated, and formed embryoid bodies maintaining the green fluorescence at varying developmental stages. The "green" embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence. Conclusions The hybrid CA promoter/enhancer used to control the EGFP expressing ES cells, resulted in more intense and ubiquitous activity. The EGFP transfected cells yield bright green fluorescence, which can be visualized in real time and in situ. In addition, the ES cells, MESPU35, are derived from C57BL/6j mice, which are the most widely used in oncology, physiology and genetics. Compared to 129 substrains, C57BL/6j mice avoid a number of potential problems apparent in the other strains.

  4. Increased fibroblast telomerase expression precedes myofibroblast α-smooth muscle actin expression in idiopathic pulmonary fibrosis

    Directory of Open Access Journals (Sweden)

    Daniel Reis Waisberg

    2012-09-01

    Full Text Available OBJECTIVE: This study sought to identify the relationship between fibroblast telomerase expression, myofibroblasts, and telomerase-mediated regulatory signals in idiopathic pulmonary fibrosis. METHODS: Thirty-four surgical lung biopsies, which had been obtained from patients with idiopathic pulmonary fibrosis and histologically classified as usual interstitial pneumonia, were examined. Immunohistochemistry was used to evaluate fibroblast telomerase expression, myofibroblast α-smooth muscle actin expression and the tissue expression of inter leu kin-4, transforming growth factor-β, and basic fibroblast growth factor. The point-counting technique was used to quantify the expression of these markers in unaffected, collapsed, mural fibrosis, and honeycombing areas. The results were correlated to patient survival. RESULTS: Fibroblast telomerase expression and basic fibroblast growth factor tissue expression were higher in collapsed areas, whereas myofibroblast expression and interleukine-4 tissue expression were higher in areas of mural fibrosis. Transforming growth factor-β expression was higher in collapsed, mural fibrosis and honeycombing areas in comparison to unaffected areas. Positive correlations were found between basic fibroblast growth factor tissue expression and fibroblast telomerase expression and between interleukin-4 tissue expression and myofibroblast α-smooth muscle actin expression. Negative correlations were observed between interleukin-4 expression and basic fibroblast growth factor tissue expression in areas of mural fibrosis. Myofibroblast α-smooth muscle actin expression and interleukin-4 tissue expression in areas of mural fibrosis were negatively associated with patient survival. CONCLUSION: Fibroblast telomerase expression is higher in areas of early remodeling in lung tissues demonstrating typical interstitial pneumonia, whereas myofibroblast α-smooth muscle actin expression predominates in areas of late remodeling

  5. Cyclooxygenase-2 expression in the normal human eye and its expression pattern in selected eye tumours

    DEFF Research Database (Denmark)

    Wang, Jinmei; Wu, Yazhen; Heegaard, Steffen;

    2011-01-01

    retina. The COX-2 expression was less in tumours deriving from the ciliary epithelium and also in retinoblastoma. Conclusion: Cyclooxygenase-2 is constitutively expressed in normal human eyes. The expression of COX-2 is much lower in selected eye tumours involving COX-2 expressing cells....

  6. Definition, Detection and Generation of Iyashi Expressions

    Science.gov (United States)

    Kitaoka, Tetsuko; Diago, Luis A.; Hagiwara, Ichiro; Kitazaki, Satoshi; Yamane, Shigeru

    This paper concerns the engineering analysis of “Iyashi”, a peculiar concept to the Japanese, which affect person's heart and may change their expression and behavior. We have integrated the advocator's view of “Iyashi”, analyzed the social background of “Iyashi” and have defined Iyashi and also the Iyashi expression. As the facial expression is the special and important stimulus for both observers and people who show expressions, we want to prove the existence of expressions that change the observer's emotion with Iyashi. We have developed the system to clarify the combination of facial features important for Iyashi through the psychological experiments and the analysis by Holographic Neural Networks (HNN). HNN analysis gave the structure of the Iyashi expression, that is the important combination of the physical facial parameters contributing to the high degree of Iyashi. Based on the structure of Iyashi we are able to generate the Iyashi expression appropriate for each person.

  7. Expression of non-muscle type myosin heavy polypeptide 9 (MYH9 in mammalian cells

    Directory of Open Access Journals (Sweden)

    T Takubo

    2009-06-01

    Full Text Available Myosin is a functional protein associated with cellular movement, cell division, muscle contraction and other functions. Members of the myosin super-family are distinguished from the myosin heavy chains that play crucial roles in cellular processes. Although there are many studies of myosin heavy chains in this family, there are fewer on non-muscle myosin heavy chains than of muscle myosin heavy chains. Myosin is classified as type I (myosin I or type II (myosin II. Myosin I, called unconventional myosin or mini-myosin, has one head, while myosin II, called conventional myosin, has two heads. We transfected myosin heavy polypeptide 9 (MYH9 into HeLa cells as a fusion protein with enhanced green fluorescent protein (EGFP and analyzed the localization and distribution of MYH9 in parallel with those of actin and tubulin. The results indicate that MYH9 colocalizes with actin stress fibers. Since it has recently been shown by genetic analysis that autosomal dominant giant platelet syndromes are MYH9-related disorders, our development of transfected EGFP-MYH9 might be useful for predicting the associations between the function of actin polymerization, the MYH9 motor domain, and these disorders.

  8. Cyclooxygenase-2 expression in the normal human eye and its expression pattern in selected eye tumours

    DEFF Research Database (Denmark)

    Wang, Jinmei; Wu, Yazhen; Heegaard, Steffen;

    2011-01-01

    Purpose: Cyclooxygenase-2 (COX-2) is an enzyme involved in neoplastic processes. The purpose of the present study is to investigate COX-2 expression in the normal human eye and the expression pattern in selected eye tumours involving COX-2 expressing cells. Methods: Immunohistochemical staining...... using antibodies against COX-2 was performed on paraffin sections of normal human eyes and selected eye tumours arising from cells expressing COX-2. Results: Cyclooxygenase-2 expression was found in various structures of the normal eye. Abundant expression was seen in the cornea, iris, ciliary body and...... retina. The COX-2 expression was less in tumours deriving from the ciliary epithelium and also in retinoblastoma. Conclusion: Cyclooxygenase-2 is constitutively expressed in normal human eyes. The expression of COX-2 is much lower in selected eye tumours involving COX-2 expressing cells....

  9. Abstract Expression Grammar Symbolic Regression

    Science.gov (United States)

    Korns, Michael F.

    This chapter examines the use of Abstract Expression Grammars to perform the entire Symbolic Regression process without the use of Genetic Programming per se. The techniques explored produce a symbolic regression engine which has absolutely no bloat, which allows total user control of the search space and output formulas, which is faster, and more accurate than the engines produced in our previous papers using Genetic Programming. The genome is an all vector structure with four chromosomes plus additional epigenetic and constraint vectors, allowing total user control of the search space and the final output formulas. A combination of specialized compiler techniques, genetic algorithms, particle swarm, aged layered populations, plus discrete and continuous differential evolution are used to produce an improved symbolic regression sytem. Nine base test cases, from the literature, are used to test the improvement in speed and accuracy. The improved results indicate that these techniques move us a big step closer toward future industrial strength symbolic regression systems.

  10. Process Expression of Security Automaton

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Security is an essential aspect for mobile systems. Usually, mobile system modeling and its security policies specification are realized in different techniques. So when constructed a mobile system using formal methods it is difficult to verify if the system comply with any given security policies. A method was introduced to express security automata which specifying enforceable security policies as processes in an extended π-calculus. In this extended π-calculus, an exception termination process was introduced, called bad. Any input which violating a security automaton will correspond to a step of transformation of the process that specifying the security automaton to exception termination process. Our method shows that any security automata which specifying enforceable security policies would decide a process in the extended π-calculus.

  11. Generalized Expression for Polarization Density

    Energy Technology Data Exchange (ETDEWEB)

    Lu Wang and T.S. Hahm

    2009-04-23

    A general polarization density which consists of classical and neoclassical parts is system-atically derived via modern gyrokinetics and bounce-kinetics by employing a phase-space Lagrangian Lie-transform perturbation method. The origins of polarization density are further elucidated. Extending the work on neoclassical polarization for long wavelength compared to ion banana width [M. N. Rosenbluth and F. L. Hinton, Phys. Rev. Lett. 80, 724 (1998)], an analytical formula for the generalized neoclassical polarization including both finite-banana-width (FBW) and finite-Larmor-radius (FLR) effects for arbitrary radial wavelength in comparison to banana width and gyroradius is derived. In additional to the contribution from trapped particles, the contribution of passing particles to the neoclassical polarization is also explicitly calculated. Our analytic expression agrees very well with the previous numerical results for a wide range of radial wavelength.

  12. E-mail: Outlook Express

    Directory of Open Access Journals (Sweden)

    Zainul Bakri

    2012-10-01

    Full Text Available Salah satu layanan Internet yang sangat penting adalah electronic mail atau sering hanya disebut sebagai e-mail. Untuk menggunakan e-mail, diperlukan piranti lunak khusus supaya pengguna dapat mengirim dan menerima e-mail. Jenis piranti lunak e-mail diantaranya adalah Outlook Express yang merupakan satu paket yang didistribusikan bersama Internet Explorer versi 4. Piranti lunak ini dijalankan pada PC yang mempunyai sistem operasi Windows 95 atau 98. Jenis piranti lunak e-mail yang lain adalah Eudora, Pegasus dan sebagainya. Bahkan ada yang diintegrasikan dengan Web Browser (alat untuk menelusuri situs Web misalnya IE,dan Netscape.Sebagai layaknya pelayanan pos, maka setiap pengguna e-mail mempunyai alamat tertentu yang tidak mungkin dipunyai oleh pengguna lainnya diseluruh dunia. Untuk keperluan pendistribusian, maka e-mail mempunyai semacam kantor pos yang ditempatkan dalam sebuah komputer server (mail server atau sering disebut sebagai host. 

  13. Role of clathrin in the regulated secretory pathway of pancreatic beta-cells

    OpenAIRE

    Molinete, M.; Dupuis, S.; Brodsky, F M; Halban, Philippe A.

    2001-01-01

    The role of clathrin in the sorting of proinsulin to secretory granules, the formation of immature granules and their subsequent maturation is not known. To this end, primary rat pancreatic beta-cells were infected with a recombinant adenovirus co-expressing the Hub fragment, a dominant-negative peptide of the clathrin heavy chain and enhanced green fluorescent protein (EGFP as a marker of infected cells). A population of cells expressing the highest levels of EGFP (and thus Hub) was obtained...

  14. Active Sulforhodamine 101 Uptake into Hippocampal Astrocytes

    OpenAIRE

    Christian Schnell; Yohannes Hagos; Swen Hülsmann

    2012-01-01

    Sulforhodamine 101 (SR101) is widely used as a marker of astrocytes. In this study we investigated labeling of astrocytes by SR101 in acute slices from the ventrolateral medulla and the hippocampus of transgenic mice expressing EGFP under the control of the astrocyte-specific human GFAP promoter. While SR101 efficiently and specifically labeled EGFP-expressing astrocytes in hippocampus, we found that the same staining procedure failed to label astrocytes efficiently in the ventrol...

  15. L'(E)TABLISSEMENT D'UN MOD(E)LE CELLULAIRE DE L'OLIGONUCL(E)OTIDE MUTANT

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objectif Pour établir un modèle cellulaire de l'oligonucléotide mutant. Méthodes Par rapport la prise du gène de la protéine de fluorescence verte et agrandisse (EGFP), nous induissons une mutation par la technique du point mutant de PCR pour changer du codon de 98ème position du gène egfp GAA au codon d' arrêt TAA , formant le gène egfp-D de mutant, donc on construit deux vecteurs de plasmides : pcDNA3-egfp et pcDNA3-egfp-D. Puis les plasmides sont transférés dans les cellules NIH-3T3 par le calcium de phosphate. Résultats Sous le microscope de fluorescence, on n'a pas vu l'expression de la EGFP dans tous les cellules transférées par l' egfp-D de mutant, et en même temps, les cellules transférées par l'egfp normal peuvent exprimer la EGFP. Ensuite on obtient les cellules stables par le criblage de G418. PCR confirme l'intégration du gène egfp-D dedans le génome ADN des cellules. RT-PCR et Northern blot analysent les gènes egfp et egfp-D peuvent transcrire normalement, il montre les cellules transférées par le gène egfp-D de mutant n'exprimant pas la fluorescence verte est parce que la traduction est terminée en avant. Conclusion Le modèle cellulaire de l'oligonucléotide mutant rapporté par le gène egfp est établi avec succès, ce modèle peut employer en recherche du système réparant thérapeutique via le chimeric ARN /ADN oligonucléotide (RDOs).

  16. Anticausatives are weak scalar expressions, not reflexive expressions

    Directory of Open Access Journals (Sweden)

    Florian Schäfer

    2016-07-01

    Full Text Available We discuss conceptual and empirical arguments from Germanic, Romance and Slavic languages against an analysis treating anticausative verbs as derived from their lexical causative counterparts under reflexivization. Instead, we defend the standard account to the semantics of the causative alternation according to which anticausatives in general, and anticausatives marked with reflexive morphology in particular, denote simple one-place inchoative events that are logically entailed by their lexical causative counterparts. Under such an account, anticausative verbs are weak scalar expressions that stand in a semantico-pragmatic opposition to their strong lexical causative counterparts. Due to this scalar relation, the use of an anticausative can trigger the implicature that the use of its lexical causative counterpart is too strong. As usual with implicatures, they can be ‘metalinguistically’ denied, cancelled, or reinforced and we argue that these mechanisms explain all central empirical facts brought up in the literature in favor of a treatment of anticausatives as semantically reflexive predicates. Our results reinforce the view that the reflexive morphemes used in many (Indo-European languages to mark anticausatives do not necessarily trigger reflexive semantics. However, we also show that a string involving a reflexively marked (anti-causative verb can be forced into a semantically reflexive construal under particular conceptual or grammatical circumstances.

  17. Construction of the eukaryotic expression vector encoding IT15 gene fragment and its expression in SH-SY5Y cells%亨廷顿舞蹈病的IT15基因片段真核表达载体构建及其在SH-SY5Y细胞中的表达定位

    Institute of Scientific and Technical Information of China (English)

    田均; 李盈枝; 殷鑫浈; 张宝荣

    2008-01-01

    Objective To construct the eukaryotic expression vector encoding IT15 gene and detect its expression in SH-SY5Y cells.Methods The exon1 of IT15 gene was amplified from genomic DNA isolated from a healthy adult and a Huntington disease patient lymphoblastoid cell lines by PCR.PCR products were subcloned into eukaryotic expression vector pEGFP-C1.The SH-SY5Y cells were transiently transfected with the recombinant plasmids and observed under fluorescence microscope.Western blot was used to detect the expression of the fusion protein.Results PCR products had approximately 170 and 310 bp specific segments.The recombinant eukaryotie expression plasmids were confirmed by restriction endonuclease analysis and sequencing.The N-terminal fragment of normal huntingtin primarily localized in the cytoplasim of SH-SY5Y cells,while the N-terminal fragment of mutant huntingtin aggregated both perinuclearly and intranuelearly.Conclusions The recombinant eukaryotic expression vectors encoding wild type and mutant IT15 gene fragments are useful for studying pathogenesis of Huntington disease.The Nterminal fragment of mutant huntingtin aggregats both perinuclearly and intranuclearly,which could be important for Huntington disease pathophysiology.%目的 构建亨廷顿舞蹈病的致病基因IT15基因片段的真核表达载体,观察其在SH-SY5Y细胞内的表达分布情况.方法 以PCR方法扩增出健康人和亨廷顿舞蹈病患者的IT15基因1号外显子片段,应用基因重组技术与绿色荧光蛋白(GFP)基因融合构建以GFP为报告基因的重组真核表达质粒GFP-Htt-19Q与GFP-mHtt-70Q,并经酶切分析和测序证实.利用脂质体瞬时转染人神经母细胞瘤细胞SH-SY5Y细胞;Western blot证实亨廷顿蛋白氨基末端片段在SH-SY5Y细胞内表达;荧光显微镜观察正常与变异亨廷顿蛋白氨基末端片段在细胞内的表达定位情况.结果 健康人和亨廷顿舞蹈病患者的IT15基因1号外显子的PCR产物分别为170

  18. Androgen Receptor Is Expressed in Genital Warts

    Institute of Scientific and Technical Information of China (English)

    Jiang Haiyang; Zhang Li; Fan Min; Yang Dexiu

    2003-01-01

    Objective:To study the expression of androgen receptor(AR) in genital warts. Methods:The expressions of AR weredetected in 40 samples of genital warts from 28 males and 12 females and 9 normal foreskins by immunohistochemical stain S-Pmethod. The status of AR expression in wart and normal foreskin were compared. Results:The AR expression was revealed in all 40samples of genital wart and 9 samples of normal foreskin.There weren's any differences in AR expression between the genital wartsand normal foreskins. Conclusions:It' s supposed that androgens may play an important role in regulating the metabolism of GW andthe HPV might be one of viruses which addicts to the tissues expressing AR properly.

  19. Recognizing Facial Expressions Automatically from Video

    Science.gov (United States)

    Shan, Caifeng; Braspenning, Ralph

    Facial expressions, resulting from movements of the facial muscles, are the face changes in response to a person's internal emotional states, intentions, or social communications. There is a considerable history associated with the study on facial expressions. Darwin [22] was the first to describe in details the specific facial expressions associated with emotions in animals and humans, who argued that all mammals show emotions reliably in their faces. Since that, facial expression analysis has been a area of great research interest for behavioral scientists [27]. Psychological studies [48, 3] suggest that facial expressions, as the main mode for nonverbal communication, play a vital role in human face-to-face communication. For illustration, we show some examples of facial expressions in Fig. 1.

  20. Roles of PI3K/Akt and c-Jun signaling pathways in human papillomavirus type 16 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression and in vitro angiogenesis in non-small cell lung cancer cells.

    Directory of Open Access Journals (Sweden)

    Erying Zhang

    Full Text Available Human papillomavirus (HPV-16 infection may be related to non-smoking associated lung cancer. Our previous studies have found that HPV-16 oncoproteins promoted angiogenesis via enhancing hypoxia-inducible factor-1α (HIF-1α, vascular endothelial growth factor (VEGF, and interleukin-8 (IL-8 expression in non-small cell lung cancer (NSCLC cells. In this study, we further investigated the roles of PI3K/Akt and c-Jun signaling pathways in it.Human NSCLC cell lines, A549 and NCI-H460, were stably transfected with pEGFP-16 E6 or E7 plasmids. Western blotting was performed to analyze the expression of HIF-1α, p-Akt, p-P70S6K, p-P85S6K, p-mTOR, p-JNK, and p-c-Jun proteins. VEGF and IL-8 protein secretion and mRNA levels were determined by ELISA and Real-time PCR, respectively. The in vitro angiogenesis was observed by human umbilical vein endothelial cells (HUVECs tube formation assay. Co-immunoprecipitation was performed to analyze the interaction between c-Jun and HIF-1α.HPV-16 E6 and E7 oncoproteins promoted the activation of Akt, P70S6K, P85S6K, mTOR, JNK, and c-Jun. LY294002, a PI3K inhibitor, inhibited HPV-16 oncoprotein-induced activation of Akt, P70S6K, and P85S6K, expression of HIF-1α, VEGF, and IL-8, and in vitro angiogenesis. c-Jun knockdown by specific siRNA abolished HPV-16 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression and in vitro angiogenesis. Additionally, HPV-16 oncoproteins promoted HIF-1α protein stability via blocking proteasome degradation pathway, but c-Jun knockdown abrogated this effect. Furthermore, HPV-16 oncoproteins increased the quantity of c-Jun binding to HIF-1α.PI3K/Akt signaling pathway and c-Jun are involved in HPV-16 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression and in vitro angiogenesis. Moreover, HPV-16 oncoproteins promoted HIF-1α protein stability possibly through enhancing the interaction between c-Jun and HIF-1α, thus making a contribution to angiogenesis in NSCLC cells.

  1. Gene expression in the Parkinson's disease brain

    OpenAIRE

    Lewis, Patrick A.; Cookson, Mark R.

    2012-01-01

    The study of gene expression has undergone a transformation in the past decade as the benefits of the sequencing of the human genome have made themselves felt. Increasingly, genome wide approaches are being applied to the analysis of gene expression in human disease as a route to understanding the underlying pathogenic mechanisms. In this review, we will summarise current state of gene expression studies of the brain in Parkinson's disease, and examine how these techniques can be used to gain...

  2. Comparative gene expression between two yeast species

    Directory of Open Access Journals (Sweden)

    Guan Yuanfang

    2013-01-01

    Full Text Available Abstract Background Comparative genomics brings insight into sequence evolution, but even more may be learned by coupling sequence analyses with experimental tests of gene function and regulation. However, the reliability of such comparisons is often limited by biased sampling of expression conditions and incomplete knowledge of gene functions across species. To address these challenges, we previously systematically generated expression profiles in Saccharomyces bayanus to maximize functional coverage as compared to an existing Saccharomyces cerevisiae data repository. Results In this paper, we take advantage of these two data repositories to compare patterns of ortholog expression in a wide variety of conditions. First, we developed a scalable metric for expression divergence that enabled us to detect a significant correlation between sequence and expression conservation on the global level, which previous smaller-scale expression studies failed to detect. Despite this global conservation trend, between-species gene expression neighborhoods were less well-conserved than within-species comparisons across different environmental perturbations, and approximately 4% of orthologs exhibited a significant change in co-expression partners. Furthermore, our analysis of matched perturbations collected in both species (such as diauxic shift and cell cycle synchrony demonstrated that approximately a quarter of orthologs exhibit condition-specific expression pattern differences. Conclusions Taken together, these analyses provide a global view of gene expression patterns between two species, both in terms of the conditions and timing of a gene's expression as well as co-expression partners. Our results provide testable hypotheses that will direct future experiments to determine how these changes may be specified in the genome.

  3. Willingness to Express Emotions to Caregiving Spouses

    OpenAIRE

    Monin, Joan K.; Martire, Lynn M.; Schulz, Richard; Clark, Margaret S.

    2009-01-01

    This study examined the association between care-recipients’ willingness to express emotions to spousal caregivers and caregiver’s well-being and support behaviors. Using self-report measures in the context of a larger study, 262 care-recipients with osteoarthritis reported on their willingness to express emotions to caregivers, and caregivers reported on their stress and insensitive responding to care-recipients. Results revealed that care-recipients’ willingness to express happiness was ass...

  4. Bayesian biclustering of gene expression data

    OpenAIRE

    Liu Jun S; Gu Jiajun

    2008-01-01

    Abstract Background Biclustering of gene expression data searches for local patterns of gene expression. A bicluster (or a two-way cluster) is defined as a set of genes whose expression profiles are mutually similar within a subset of experimental conditions/samples. Although several biclustering algorithms have been studied, few are based on rigorous statistical models. Results We developed a Bayesian biclustering model (BBC), and implemented a Gibbs sampling procedure for its statistical in...

  5. Quality Measures for Gene Expression Biclusters

    OpenAIRE

    Beatriz Pontes; Ral Girldez; Aguilar-Ruiz, Jess S.

    2015-01-01

    An noticeable number of biclustering approaches have been proposed proposed for the study of gene expression data, especially for discovering functionally related gene sets under different subsets of experimental conditions. In this context, recognizing groups of co-expressed or co-regulated genes, that is, genes which follow a similar expression pattern, is one of the main objectives. Due to the problem complexity, heuristic searches are usually used instead of exhaustive algorithms. Further...

  6. Enhancing Regular Expressions For Polish Text Processing

    OpenAIRE

    Krzysztof Dorosz; Anna Szczerbińska

    2009-01-01

    The paper presents proposition of regular expressions engine based on the modified Thompson’salgorithm dedicated to the Polish language processing. The Polish inflectional dictionaryhas been used for enhancing regular expressions engine and syntax. Instead of usingcharacters as a basic element of regular expressions patterns (as it takes place in BRE orERE standards) presented tool gives possibility of using words from a natural language orlabels describing words grammar properties in regex s...

  7. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy; Bachkirova, Elena; Rey, Michael

    2013-10-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  8. Foxp3 expression in human cancer cells

    OpenAIRE

    Gourgoulianis Konstantinos I; Barda Angeliki K; Kerenidi Theodora; Loules Gedeon; Kalala Fani; Zamanakou Maria; Speletas Matthaios; Karanikas Vaios; Germenis Anastasios E

    2008-01-01

    Abstract Objective Transcription factor forkhead box protein 3 (Foxp3) specifically characterizes the thymically derived naturally occurring regulatory T cells (Tregs). Limited evidence indicates that it is also expressed, albeit to a lesser extent, in tissues other than thymus and spleen, while, very recently, it was shown that Foxp3 is expressed by pancreatic carcinoma. This study was scheduled to investigate whether expression of Foxp3 transcripts and mature protein occurs constitutively i...

  9. Facial Expression Recognition from World Wild Web

    OpenAIRE

    Mollahosseini, Ali; Hassani, Behzad; Salvador, Michelle J.; Abdollahi, Hojjat; Chan, David; Mahoor, Mohammad H.

    2016-01-01

    Recognizing facial expression in a wild setting has remained a challenging task in computer vision. The World Wide Web is a good source of facial images which most of them are captured in uncontrolled conditions. In fact, the Internet is a Word Wild Web of facial images with expressions. This paper presents the results of a new study on collecting, annotating, and analyzing wild facial expressions from the web. Three search engines were queried using 1250 emotion related keywords in six diffe...

  10. PLANEX: the plant co-expression database

    OpenAIRE

    Yim, Won Cheol; Yu, Yongbin; Song, Kitae; Jang, Cheol Seong; Lee, Byung-Moo

    2013-01-01

    Background The PLAnt co-EXpression database (PLANEX) is a new internet-based database for plant gene analysis. PLANEX (http://planex.plantbioinformatics.org) contains publicly available GeneChip data obtained from the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (NCBI). PLANEX is a genome-wide co-expression database, which allows for the functional identification of genes from a wide variety of experimental designs. It can be used for the characterization...

  11. What Can Spontaneous Facial Expression Tell Us?

    OpenAIRE

    Yang, Songfan

    2014-01-01

    Facial expression plays a significant role in human communication. It is considered the single most important cue in the psychology of emotion. Facial expression is taken as a universally understood signal, which triggers a discrete categorical basic emotion, including joy, sadness, fear, surprise, anger, and disgust. Thus, automatic analysis of emotion from images of human facial expression has been an interesting and challenging problem for the past 30 years. Aiming towards the applications...

  12. The Commitment Function of Angry Facial Expressions

    OpenAIRE

    Reed, Lawrence Ian; DeScioli, Peter; Pinker, Steven

    2014-01-01

    What function do facial expressions have? We tested the hypothesis that some expressions serve as honest signals of subjective commitments—in particular, that angry faces increase the effectiveness of threats. In an ultimatum game, proposers decided how much money to offer a responder while seeing a film clip depicting an angry or a neutral facial expression, together with a written threat that was either inherently credible (a 50-50 split) or less credible (a demand for 70% of the money). Pr...

  13. Dimensions of emotion in expressive musical performance.

    Science.gov (United States)

    Vines, Bradley W; Krumhansl, Carol L; Wanderley, Marcelo M; Dalca, Ioana M; Levitin, Daniel J

    2005-12-01

    This paper explores the dimensions of emotion conveyed by music. Participants rated emotion terms after seeing and/or hearing recordings of clarinet performances that varied in expressive content. A factor analysis revealed four independent dimensions of emotion. Changes to the clarinetists' expressive intentions did not significantly affect emotions conveyed by sound. It was largely through the visual modality that expressive intentions influenced the experience for observers. PMID:16597804

  14. Universals and Cultural Variations in Emotional Expression

    OpenAIRE

    Cordaro, Daniel

    2014-01-01

    One of the most fascinating characteristics of emotions is that they have universal expressive patterns. These expressions, which are encoded through multiple bodily channels, allow us to identify distinct emotions in ourselves and others. After groundbreaking theorizing by Charles Darwin and early empirical work by Ekman and Izard, further research in emotion science revealed evidence in single cultures for more emotional expressions above and beyond the well-studied set comprising of anger,...

  15. FACIAL EXPRESSION RECOGNITION BASED ON EDGE DETECTION

    OpenAIRE

    Chen, Xiaoming; Cheng, Wushan

    2015-01-01

    Relational Over the last two decades, the advances in computer vision and pattern recognition power have opened the door to new opportunity of automatic facial expression recognition system[1]. This paper use Canny edge detection method for facial expression recognition. Image color space transformation in the first place and then to identify and locate human face .Next pick up the edge of eyes and mouth's features extraction. Last we judge the facial expressions after compared wi...

  16. Regulation of immunoglobulin gene rearrangement and expression.

    Science.gov (United States)

    Taussig, M J; Sims, M J; Krawinkel, U

    1989-05-01

    The molecular genetic events leading to Ig expression and their control formed the topic of a recent EMBO workshop. This report by Michael Taussig, Martin Sims and Ulrich Krawinkel discusses contributions dealing with genes expressed in early pre-B cells, the mechanism of rearrangement, aberrant rearrangements seen in B cells of SCID mice, the feedback control of rearrangement as studied in transgenic mice, the control of Ig expression at the transcriptional and post-transcriptional levels, and class switching. PMID:2787158

  17. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy (Davis, CA); Bachkirova, Elena (Davis, CA); Rey, Michael (Davis, CA)

    2012-05-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  18. Molecular fingerprint of neuropeptide S-producing neurons in the mouse brain

    DEFF Research Database (Denmark)

    Liu, Xiaobin; Zeng, Joanne; Zhou, Anni;

    2011-01-01

    , it is critical to identify transmitter systems that control NPS release and transmitters that are co-released with NPS. For this purpose, we generated several lines of transgenic mice that express enhanced green-fluorescent protein (EGFP) under control of the endogenous NPS precursor promoter. NPS....../EGFP-transgenic mice show anatomically correct and overlapping expression of both NPS and EGFP. A total number of ∼500 NPS/EGFP-positive neurons are present in the mouse brain, located in the pericoerulear region and the Kölliker-Fuse nucleus. NPS and transgene expression is first detectable around E14, indicating a...... incoming neurotransmission, controlling neuronal activity of NPS-producing neurons. Stress-induced functional activation of NPS-producing neurons was detected by staining for the immediate-early gene c-fos, thus supporting earlier findings that NPS might be part of the brain stress response network....

  19. Transcriptional stochasticity in gene expression.

    Science.gov (United States)

    Lipniacki, Tomasz; Paszek, Pawel; Marciniak-Czochra, Anna; Brasier, Allan R; Kimmel, Marek

    2006-01-21

    Due to the small number of copies of molecular species involved, such as DNA, mRNA and regulatory proteins, gene expression is a stochastic phenomenon. In eukaryotic cells, the stochastic effects primarily originate in regulation of gene activity. Transcription can be initiated by a single transcription factor binding to a specific regulatory site in the target gene. Stochasticity of transcription factor binding and dissociation is then amplified by transcription and translation, since target gene activation results in a burst of mRNA molecules, and each mRNA copy serves as a template for translating numerous protein molecules. In the present paper, we explore a mathematical approach to stochastic modeling. In this approach, the ordinary differential equations with a stochastic component for mRNA and protein levels in a single cells yield a system of first-order partial differential equations (PDEs) for two-dimensional probability density functions (pdf). We consider the following examples: Regulation of a single auto-repressing gene, and regulation of a system of two mutual repressors and of an activator-repressor system. The resulting PDEs are approximated by a system of many ordinary equations, which are then numerically solved. PMID:16039671

  20. Heterologous expression in transgenic mosquitoes

    Institute of Scientific and Technical Information of China (English)

    Santhosh P K; Yu hua Deng; Weidong Gu; Xiaoguang Chen

    2010-01-01

    Arthropod-borne diseases such as malaria and dengue virus afflict billions of people worldwide imposing major economic and social burdens. Control of such pathogens is mainly performed by vector management and treatment of affected individuals with drugs. The failure of these conventional approaches due to emergence of insecticide-resistant insects and drug-resistant parasites demonstrate the need of novel and efficacious control strategies to combat these diseases. Genetic modification(GM) of mosquito vectors to impair their ability to be infected and transmit pathogens has emerged as a new strategy to reduce transmission of many vector-borne diseases and deliver public health gains. Several advances in developing transgenic mosquitoes unable to transmit pathogens have gained support, some of them attempt to manipulate the naturally occurring endogenous refractory mechanisms, while others initiate the identification of an exogenous foreign gene which disrupt the pathogen development in insect vectors. Heterologous expression of transgenes under a native or heterologous promoter is important for the screening and effecting of the transgenic mosquitoes. The effect of the transgene on mosquito fitness is a crucial parameter influencing the success of this transgenic approach. This review examines these two aspects and describes the basic research work that has been accomplished towards understanding the complex relation between the parasite and its vector and focuses on recent advances and perspectives towards construction of transgenic mosquitoes refractory to vector-borne disease transmission.