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Gene Expression System (gene + expression_system)
Selected AbstractsEfficient Gene Expression System Using the RTP801 Promoter in the Corpus Cavernosum of High-Cholesterol Diet-Induced Erectile Dysfunction Rats for Gene TherapyTHE JOURNAL OF SEXUAL MEDICINE, Issue 6 2008Minhyung Lee PhD ABSTRACT Introduction., The application of gene therapy for a nonlife-threatening disease, such as erectile dysfunction (ED), requires a higher safety level and more efficacious systems for gene transfer. Aim., To establish a novel technique for gene expression in a rat model of hypercholesterolemic ED that uses the RTP801 promoter, a hypoxia-inducible promoter. Methods., Two-month-old male Sprague,Dawley rats were fed a diet containing 4% cholesterol and 1% cholic acid, and age-matched control animals were fed a normal diet, for 3 months. Main Outcome Measures., Cavernous expression of hypoxia-inducible factor (HIF)-1, was evaluated by Western blot. After intracavernous injection of pSV-Luc or pRTP801-Luc, gene expression was evaluated by luciferase assay, and the gene expression area was evaluated by immunohistochemistry. Results., HIF-1, was up-regulated in the corpus cavernosum of hypercholesterolemic rats. Although pSV-Luc did not induce gene expression in either the control or the cholesterol group, pRTP801-Luc significantly induced gene expression in the cholesterol group and resulted in higher luciferase activity than did pSV-Luc up to 14 days after injection. Immunohistochemistry showed that the gene expression area was also greater in the pRTP801-Luc group than in the pSV-Luc group, but the difference was not as great as that in luciferase activity. This suggests that pRTP801-Luc exerts its effect mainly by inducing promoter activity under hypoxia, not by increasing the number of transfected cells. Conclusion., The RTP801 promoter-driven gene expression system increased gene expression in the corpus cavernosum tissue of rats with cholesterol-induced ED. This may be a useful system for the development of gene therapy in vasculogenic ED. Lee M, Ryu J-K, Piao S, Choi MJ, Kim HA, Zhang L-W, Shin H-Y, Jung HI, Kim I-H, Kim SW, and Suh J-K. Efficient gene expression system using the RTP801 promoter in the corpus cavernosum of high-cholesterol diet-induced erectile dysfunction rats for gene therapy. J Sex Med 2008;5:1355,1364. [source] Role of a highly conserved YPITP motif in 2-oxoacid:ferredoxin oxidoreductase.,FEBS JOURNAL, Issue 21 2001Heterologous expression of the gene from Sulfolobus sp. strain , characterization of the recombinant, variant enzymes 2-Oxoacid:ferredoxin oxidoreductase from Sulfolobus sp. strain 7, an aerobic and thermoacidophilic crenoarchaeon, catalyses the coenzyme A-dependent oxidative decarboxylation of pyruvate and 2-oxoglutarate, a cognate Zn-7Fe-ferredoxin serving as an electron acceptor. It comprises two subunits, a (632 amino acids) and b (305 amino acids). To further elucidate its structure and function, we constructed a gene expression system. The wild-type recombinant enzyme was indistinguishable from the natural one in every criterion investigated. A series of variants was constructed to elucidate the role of the YPITP-motif (residues 253,257) in subunit a, which is conserved universally in the 2-oxoacid:ferredoxin oxidoreductase (OFOR) family. Single amino-acid replacements at Y253 and P257 by other amino acids caused a drastic loss of enzyme activity. T256, the hydroxyl group of which has been proposed to be essential for binding of the 2-oxo group of the substrate in the Desulfovibrio africanus enzyme, was unexpectedly replaceable with Ala, the kcat and Km for 2-oxoglutarate being ,,33% and ,,51%, respectively, as compared with that of the wild-type enzyme. Replacement at other positions resulted in a significant decrease in the kcat of the reaction while the Km for 2-oxoacid was only slightly affected. Thus, the YPITP-motif is essential for the turnover of the reaction rather than the affinity toward 2-oxoacid. [source] Spatially patterned gene expression for guided neurite extensionJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2009Tiffany Houchin-Ray Abstract Axon pathfinding by localized expression of guidance molecules is critical for the proper development of the nervous system. In this report, we present a well-defined spatially patterned gene expression system to investigate neurite guidance in vitro. Nonviral gene delivery was patterned by combining substrate-mediated gene delivery with soft lithography techniques, and the amount of protein produced at the region of localized expression was varied by altering the vector concentration and the width of the pattern, highlighting the flexibility of the system. A neuronal coculture model was used to investigate responses to spatial patterns of nerve growth factor (NGF) expression. The soluble NGF gradient elicited a guidance cue, and the degree of guidance was governed by the distance a neuron was cultured from the pattern and the time between accessory cell and neuron seedings. A portion of the diffusible NGF bound to the culture surface in the extracellular space, and the surface-associated NGF supported neuron survival and neurite outgrowth. However, the surface-bound NGF gradient alone did not elicit a guidance signal, and in fact masked the guidance cue by soluble NGF gradients. Mathematical modeling of NGF diffusion was used to predict the concentration gradients, and both the absolute and fractional gradients capable of guiding neurites produced by patterned gene expression differed substantially from the values obtained with existing engineered protein gradients. Spatially patterned gene expression provides a versatile tool to investigate the factors that may promote neurite guidance. © 2008 Wiley-Liss, Inc. [source] Efficient Gene Expression System Using the RTP801 Promoter in the Corpus Cavernosum of High-Cholesterol Diet-Induced Erectile Dysfunction Rats for Gene TherapyTHE JOURNAL OF SEXUAL MEDICINE, Issue 6 2008Minhyung Lee PhD ABSTRACT Introduction., The application of gene therapy for a nonlife-threatening disease, such as erectile dysfunction (ED), requires a higher safety level and more efficacious systems for gene transfer. Aim., To establish a novel technique for gene expression in a rat model of hypercholesterolemic ED that uses the RTP801 promoter, a hypoxia-inducible promoter. Methods., Two-month-old male Sprague,Dawley rats were fed a diet containing 4% cholesterol and 1% cholic acid, and age-matched control animals were fed a normal diet, for 3 months. Main Outcome Measures., Cavernous expression of hypoxia-inducible factor (HIF)-1, was evaluated by Western blot. After intracavernous injection of pSV-Luc or pRTP801-Luc, gene expression was evaluated by luciferase assay, and the gene expression area was evaluated by immunohistochemistry. Results., HIF-1, was up-regulated in the corpus cavernosum of hypercholesterolemic rats. Although pSV-Luc did not induce gene expression in either the control or the cholesterol group, pRTP801-Luc significantly induced gene expression in the cholesterol group and resulted in higher luciferase activity than did pSV-Luc up to 14 days after injection. Immunohistochemistry showed that the gene expression area was also greater in the pRTP801-Luc group than in the pSV-Luc group, but the difference was not as great as that in luciferase activity. This suggests that pRTP801-Luc exerts its effect mainly by inducing promoter activity under hypoxia, not by increasing the number of transfected cells. Conclusion., The RTP801 promoter-driven gene expression system increased gene expression in the corpus cavernosum tissue of rats with cholesterol-induced ED. This may be a useful system for the development of gene therapy in vasculogenic ED. Lee M, Ryu J-K, Piao S, Choi MJ, Kim HA, Zhang L-W, Shin H-Y, Jung HI, Kim I-H, Kim SW, and Suh J-K. Efficient gene expression system using the RTP801 promoter in the corpus cavernosum of high-cholesterol diet-induced erectile dysfunction rats for gene therapy. J Sex Med 2008;5:1355,1364. [source] pOp6/LhGR: a stringently regulated and highly responsive dexamethasone-inducible gene expression system for tobaccoTHE PLANT JOURNAL, Issue 6 2005Marketa Samalova Summary We describe pOp/LhGR, a dexamethasone-inducible derivative of the pOp/LhG4 transcription activation system, and its use in tobacco to regulate expression of uidA (encoding , -glucuronidase; GUS) and the cytokinin-biosnythetic gene ipt. The pOp/LhGR system exhibited stringent regulation and strong induced phenotypes in soil and tissue culture. In conjunction with an improved target promoter, pOp6, that carries six copies of an optimized lac operator sequence the pOp6/LhGR system directed induced GUS activities that exceeded those obtained with pOp/LhG4 or the CaMV 35S promoter but without increased uninduced activity. A single dose of dexamethasone was sufficient to direct cytotoxic levels of ipt expression in soil-grown plants although uninduced plants grew normally throughout a complete life cycle. In vitro, induced transcripts were detectable within an hour of dexamethasone application and 1 nm dexamethasone was sufficient for half maximal induction of GUS activity. Various methods of dexamethasone application were successfully applied under tissue culture and greenhouse conditions. We observed no inhibitory effects of dexamethasone or LhGR on plant development even with the highest concentrations of inducer, although tobacco seedlings were adversely affected by ethanol used as a solvent for dexamethasone stock solutions. The pOp/LhGR system provides a highly sensitive, efficient, and tightly regulated chemically inducible transgene expression system for tobacco plants. [source] Understanding heterologous protein overproduction under the T7 promoter: A practical exerciseBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 3 2002Evangelos Christodoulou Abstract Because various genome projects have been advanced many genes are known, and large amounts of proteins are required to elucidate their function. Most biomolecular research laboratories have a need to overexpress a certain gene, or a part of it, in eukaryotic or prokaryotic expression systems. It is therefore important for young students to become familiar with the technology of heterologous gene expression systems. Gene expression in eukaryotic cells is rather complicated and costly and is therefore not ideally suited to exercises for students. The goal of this paper is to describe an experimental example of a well known and broadly used prokaryotic system, the pET system, that works under the strong T7 promoter. The clones described in this paper are suitable for the practical exercise and are available upon request. [source] | ||||||||||