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In Vivo Imaging of Transiently Transgenized Mice with a Bovine Interleukin 8 (CXCL8) Promoter/Luciferase Reporter Construct  [PDF]
Fabio Franco Stellari, Valentina Franceschi, Antonio Capocefalo, Marcello Ronchei, Fabrizio Facchinetti, Gino Villetti, Gaetano Donofrio
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0039716
Abstract: One of the most remarkable properties of interleukin 8 (CXCL8/IL-8), a chemokine with known additional functions also in angiogenesis and tissue remodeling, is the variation of its expression levels. In healthy tissues, IL-8 is barely detectable, but it is rapidly induced by several folds in response to proinflammatory cytokines, bacterial or viral products, and cellular stress. Although mouse cells do not bear a clear homologous IL-8 gene, the murine transcriptional apparatus may well be capable of activating or repressing a heterologous IL-8 gene promoter driving a reporter gene. In order to induce a transient transgenic expression, mice were systemically injected with a bovine IL-8 promoter–luciferase construct. Subsequently mice were monitored for luciferase expression in the lung by in vivo bioluminescent image analysis over an extended period of time (up to 60 days). We demonstrate that the bovine IL-8 promoter–luciferase construct is transiently and robustly activated 3–5 hours after LPS and TNF-α instillation into the lung, peaking at 35 days after construct delivery. Bovine IL-8 promoter–luciferase activation correlates with white blood cell and neutrophil infiltration into the lung. This study demonstrates that a small experimental rodent model can be utilized for non-invasively monitoring, through a reporter gene system, the activation of an IL-8 promoter region derived from a larger size animal (bovine). This proof of principle study has the potential to be utilized also for studying primate IL-8 promoter regions.
High Level Constitutive Expression of Luciferase Reporter by lsd90 Promoter in Fission Yeast  [PDF]
Hemant Kumar Verma, Poonam Shukla, Md. Alfatah, Asheesh Kumar Khare, Udita Upadhyay, Kaliannan Ganesan, Jagmohan Singh
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0101201
Abstract: Because of a large number of molecular similarities with higher eukaryotes, the fission yeast Schizosaccharomyces pombe has been considered a potentially ideal host for expressing human proteins having therapeutic and pharmaceutical applications. However, efforts in this direction are hampered by lack of a strong promoter. Here, we report the isolation and characterization of a strong, constitutive promoter from S. pombe. A new expression vector was constructed by cloning the putative promoter region of the lsd90 gene (earlier reported to be strongly induced by heat stress) into a previously reported high copy number vector pJH5, which contained an ARS element corresponding to the mat2P flanking region and a truncated URA3m selectable marker. The resulting vector was used to study and compare the level of expression of the luciferase reporter with that achieved with the known vectors containing regulatable promoter nmt1 and the strong constitutive promoter adh1 in S. pombe and the methanol-inducible AOX1 promoter in Pichia pastoris. Following growth in standard media the new vector containing the putative lsd90 promoter provided constitutive expression of luciferase, at a level, which was 19-, 39- and 10-fold higher than that achieved with nmt1, adh1 and AOX1 promoters, respectively. These results indicate a great potential of the new lsd90 promoter-based vector for commercial scale expression of therapeutic proteins in S. pombe.
A Real Time Metridia Luciferase Based Non-Invasive Reporter Assay of Mammalian Cell Viability and Cytotoxicity via the β-actin Promoter and Enhancer  [PDF]
Shawn E. Lupold, Tamara Johnson, Wasim H. Chowdhury, Ronald Rodriguez
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0036535
Abstract: Secreted reporter molecules offer a means to evaluate biological processes in real time without the need to sacrifice samples at pre-determined endpoints. Here we have adapted the secreted bioluminescent reporter gene, Metridia luciferase, for use in a real-time viability assay for mammalian cells. The coding region of the marine copepod gene has been codon optimized for expression in human cells (hMLuc) and placed under the control of the human β-actin promoter and enhancer. Metridia luciferase activity of stably transfected cell models corresponded linearly with cell number over a 4-log dynamic range, detecting as few as 40 cells. When compared to standard endpoint viability assays, which measure the mitochondrial dehydrogenase reduction of tetrazolium salts, the hMLuc viability assay had a broader linear range of detection, was applicable to large tissue culture vessels, and allowed the same sample to be repeatedly measured over several days. Additional studies confirmed that MLuc activity was inhibited by serum, but demonstrated that assay activity remained linear and was measurable in the serum of mice bearing subcutaneous hMLuc-expressing tumors. In summary, these comparative studies demonstrate the value of humanized Metridia luciferase as an inexpensive and non-invasive method for analyzing viable cell number, growth, tumor volume, and therapeutic response in real time.
Application of the Dual-Luciferase Reporter Assay to the Analysis of Mouse Kit and Yy1 Promoter Activation Regulation by Sohlh1
Shengyang Li,Bing Du,Guangbin Luo,Wei Yan,Zhihong Zheng
Journal of Animal and Veterinary Advances , 2012, DOI: 10.3923/javaa.2012.2523.2528
Abstract: Kit is significantly downregulated in Sohlh1 deletion ovaries and Sohlh1 was over-expressed in Yy1 cKO ovaries and whether Sohlh1 regulates kit and Yy1 transcription is unknown. Here, researchers examine the promoter transcription activation of Kit and Yy1 regulated by Sohlh1 using dual-luciferase reporter assays. The Kit responsiveness of the Sohlh1 gene has been validated and provided evidence of Sohlh1 transcriptional activation of the Kit gene promoter in the mouse ovary. In contrast, Sohlh1 has little effect on the essential activity of Yy1 promoter. Researchers identifid -1769 to -297 as the transcriptional activitation region of Yy1 promoter and revealed strong inhibitor in -297 to +135 region where maybe G-quadruplexes motifs exist. Researchers found two Yy1 binding Sites in Sohlh1 promoter and surmise that Yy1 negatively regulates Sohlh1 in mouse ovary.
Non-Homologous End Joining Plays a Key Role in Transgene Concatemer Formation in Transgenic Zebrafish Embryos
Jun Dai, Xiaojuan Cui, Zuoyan Zhu, Wei Hu
International Journal of Biological Sciences , 2010,
Abstract: This study focused on concatemer formation and integration pattern of transgenes in zebrafish embryos. A reporter plasmid based on enhanced green fluorescent protein (eGFP) driven by Cytomegalovirus (CMV) promoter, pCMV-pax6in-eGFP, was constructed to reflect transgene behavior in the host environment. After removal of the insertion fragment by double digestion with various combinations of restriction enzymes, linearized pCMV-pax6in-eGFP vectors were generated with different combinations of 5′-protruding, 3′-protruding, and blunt ends that were microinjected into zebrafish embryos. Repair of double-strand breaks (DSBs) was monitored by GFP expression following religation of the reporter gene. One-hundred-and-ninety-seven DNA fragments were amplified from GFP-positive embryos and sequenced to analyze the repair characteristics of different DSB end combinations. DSBs involving blunt and asymmetric protruding ends were repaired efficiently by direct ligation of blunt ends, ligation after blunting and fill-in, or removed by cutting. Repair of DSBs with symmetric 3′-3′ protrusions was less efficient and utilized template-directed repair. The results suggest that non-homologous end joining (NHEJ) was the principal mechanism of exogenous gene concatemer formation and integration of transgenes into the genome of transgenic zebrafish.
Tissue-specific expression of GFP reporter gene in germline driven by GATA-2 promoter and enhancers in zebrafish
Anming Meng,Shuo Lin
Chinese Science Bulletin , 2000, DOI: 10.1007/BF02884898
Abstract: GATA-2, a transcription factor, is expressed in several types of blood cells and in the central nervous system (CNS), and regulates the differentiation of these cells. We have obtained five zebrafish transgenic germlines that carry and express the green fluorescent protein (GFP) gene ligated to various 5′ flanking sequences of zebrafish GATA-2 gene. The spatial pattern of GFP expression varies, mainly depending on which regulatory sequence is used, among the germlines. In some of the germlines, the expression of GFP is restricted to the CNS and the enveloping layer (EVL) cells, while in some other lines GFP is observed only in the CNS. It is noted that the intensity of GFP in the transgenic fish remain unchanged after a six-generation passage of the transgenes. The transgenic fish could find its uses in the future in generating tissue-specific, even cellspecific mutant fish and in functional study of related genes through transgenesis.
Generation of FGF reporter transgenic zebrafish and their utility in chemical screens
Gabriela A Molina, Simon C Watkins, Michael Tsang
BMC Developmental Biology , 2007, DOI: 10.1186/1471-213x-7-62
Abstract: Expression of Dual Specificity Phosphatase 6 (dusp6, also known as Mkp3) is controlled by FGF signalling throughout development. The Dusp6 promoter was isolated from zebrafish and used to drive expression of destabilized green fluorescent protein (d2EGFP) in transgenic embryos (Tg(Dusp6:d2EGFP)). Expression of d2EGFP is initiated as early as 4 hours post-fertilization (hpf) within the future dorsal region of the embryo, where fgf3 and fgf8 are initially expressed. At later stages, d2EGFP is detected within structures that correlate with the expression of Fgf ligands and their receptors. This includes the mid-hindbrain boundary (MHB), pharyngeal endoderm, otic vesicle, hindbrain, and Kupffer's vesicle. The expression of d2EGFP is under the control of FGF signalling as treatment with FGF Receptor (FGFR) inhibitors results in the suppression of d2EGFP expression. In a pilot screen of commercially available small molecules we have evaluated the effectiveness of the transgenic lines to identify specific FGF inhibitors within the class of indolinones. These compounds were counter screened with the transgenic line Tg(Fli1:EGFP)y1, that serves as an indirect read-out for Vascular Endothelial Growth Factor (VEGF) signalling in order to determine the specificity between related receptor tyrosine kinases (RTKs). From these assays it is possible to determine the specificity of these indolinones towards specific RTK signalling pathways. This has enabled the identification of compounds that can block specifically the VEGFR or the FGFR signalling pathway.The generation of transgenic reporter zebrafish lines has allowed direct visualization of FGF signalling within the developing embryo. These FGF reporter transgenic lines provide a tool to screen for specific compounds that can distinguish between two conserved members of the RTK family.The complex process of embryogenesis is directed by the regulation of signalling pathways that are achieved in part by the activity of a variety o
Evaluation of a novel luciferase reporter construct: a positive control plasmid for reporter gene assay
T Tencomnao, V Rakkhitawatthana, K Sukhontasing
African Journal of Biotechnology , 2008,
Abstract: Reporter gene technology has been increasingly important in the post-genomic era to explain human complexity and diversity. The pGL3-Basic vector has been prevalently used as a tool for analyzing cisacting elements critical for transcriptional mechanisms. In this work, we constructed and evaluated the pGL3-Basic plasmid containing the cytomegalovirus (CMV) enhancer/promoter aiming to establish a positive control of pGL3-Basic vector. Using a human melanoma cell line UACC-903 for transient transfection, the novel luciferase reporter construct, pGL3-CMV, showed an extremely high transcriptional activity approximately 4,260-fold greater than that of pGL3-Basic, indicating its qualification as a positive control for luciferase reporter gene assays.
The Effect of Linear Versus Circular Vector on Enhanced Green Fluorescent Protein (EGFP) Expression in Transgenic Zebrafish (Danio rerio)
Aygul Ekici,Digdem Aktoprakligil,Metin Timur,Tolga Akkoc,Haydar Bagis
Journal of Animal and Veterinary Advances , 2012, DOI: 10.3923/javaa.2010.1232.1236
Abstract: Green Fluorescent Protein (GFP) has been used as an indicator of transgene expression in living cells and organisms. In this study, a transgene construct containing the Cyto-Megalo-Virus (CMV) promoter sequences, SV40 polyA signal and the Enhanced Green Fluorescent Protein reporter gene (EGFP) was microinjected into the cytoplasm of one-cell zebrafish embryos. About 65 ng μL-1 circular and linearized pEGFP-N1 DNA was used in microinjection. Transgenic founders were detected by Polymerase Chain Reaction (PCR), Slot and Southern blots and Reverse-Transcriptase PCR (RT-PCR). EGFP gene expression was detected by inverted fluorescence microscope in F0 transgenic zebrafish larvae. About 54 and 25 F0 transgenic zebrafish were obtained after microinjection of linearized and circular gene constructs, respectively. This is the first study for generation of transgenic zebrafish via cytoplasmic DNA microinjection in Turkey. In conclusion, these results indicate that the gene expression efficiency of circular form was higher than the linearized form in F0 transgenic zebrafish larvae.
小鼠巨噬细胞活化标志物IL-12/IL-10启动子荧光素酶报告基因模型的构建及鉴定 Construction and Identification of Luciferase Reporter Gene Containing Murine IL-12 Promoter and IL-10 Promoter  [PDF]
张帆,叶鹏,邓君健,冯静,沈秉正,宋金春
- , 2017,
Abstract: 目的:构建pGL3-IL-12p40promoter和pGL3-IL-10promoter两个荧光素酶报告基因载体,并对其进行生物活性鉴定。方法:通过提取小鼠T细胞基因组DNA,分别PCR扩增IL-12p40和IL-10基因的启动子序列,与pGL3-Basic连接成重组体pGL3-IL-12p40promoter和pGL3-IL-10promoter,通过转化扩增,筛选出阳性克隆,并通过酶切、测序及生物学活性检测鉴定构建好的荧光素酶报告基因载体。结果:成功构建了pGL3-IL-12p40promoter和pGL3-IL-10promoter两个荧光素酶报告基因载体,分别在IFN-γ或IL-4的诱导下,能启动细胞内荧光素酶的表达。结论:pGL3-IL-12p40promoter和pGL3-IL-10promoter的荧光素酶报告基因载体的成功构建为研究IL-12和IL-10蛋白的表达调控机制和以IL-12/IL-10为检测指标的巨噬细胞活化状态的判断,以其为靶标的抗肿瘤药物的快速高通量筛选提供了重要的研究工具
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