scholarly journals First report of a tetracycline-inducible gene expression system for mollicutes

Microbiology ◽  
2010 ◽  
Vol 156 (1) ◽  
pp. 198-205 ◽  
Author(s):  
Marc Breton ◽  
Evelyne Sagné ◽  
Sybille Duret ◽  
Laure Béven ◽  
Christine Citti ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Inducible Promoter ◽  
High Level Expression ◽  
Spiroplasma Citri ◽  
High Level ◽  
Tetr Repressor ◽  
Promoter System ◽  
Inducible Gene

Inducible promoter systems are powerful tools for studying gene function in prokaryotes but have never been shown to function in mollicutes. In this study we evaluated the efficacy of the tetracycline-inducible promoter Pxyl/tetO2 from Bacillus subtilis in controlling gene expression in two mollicutes, the plant pathogen Spiroplasma citri and the animal pathogen Mycoplasma agalactiae. An S. citri plasmid carrying the spiralin gene under the control of the xyl/tetO2 tetracycline-inducible promoter and the TetR repressor gene under the control of a constitutive spiroplasmal promoter was introduced into the spiralin-less S. citri mutant GII3-9a3. In the absence of tetracycline, expression of TetR almost completely abolished expression of spiralin from the xyl/tetO2 promoter. Adding tetracycline (>50 ng ml−1) to the medium induced high-level expression of spiralin. Interestingly, inducible expression of spiralin was also detected in vivo: in S. citri-infected leafhoppers fed on tetracycline-containing medium and in S. citri-infected plants watered with tetracycline. A similar construct was introduced into the M. agalactiae chromosome through transposition. Tetracycline-induced expression of spiralin proved the TetR-Pxyl/tetO2 system to be functional in the ruminant pathogen, suggesting that this tetracycline-inducible promoter system might be of general use in mollicutes.

scholarly journals Development of a Sensitive Gene Expression Reporter System and an Inducible Promoter-Repressor System for Clostridium acetobutylicum

2003 ◽  
Vol 69 (8) ◽  
pp. 4985-4988 ◽  
Author(s):  
Laurence Girbal ◽  
Isabelle Mortier-Barrière ◽  
Frédéric Raynaud ◽  
Céline Rouanet ◽  
Christian Croux ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clostridium Acetobutylicum ◽  
Time Course ◽  
Specific Activity ◽  
Expression System ◽  
Regulatory System ◽  
Inducible Promoter ◽  
Reporter System ◽  
Inducible Gene

ABSTRACT A sensitive gene expression reporter system was developed for Clostridium acetobutylicum ATCC 824 by using a customized gusA expression cassette. In discontinuous cultures, time course profiles of β-glucuronidase specific activity reflected adequately in vivo dynamic up- and down-regulation of acidogenesis- and/or solventogenesis-associated promoter expression in C. acetobutylicum. Furthermore, a new inducible gene expression system was developed in C. acetobutylicum, based on the Staphylococcus xylosus xylose operon promoter-repressor regulatory system.

scholarly journals Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

Biology Open ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. bio055343 ◽  
Author(s):  
Daniel Chu ◽  
An Nguyen ◽  
Spenser S. Smith ◽  
Zuzana Vavrušová ◽  
Richard A. Schneider
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Inducible Promoter ◽  
In Ovo ◽  
Control Of Gene Expression ◽  
Promoter Construct ◽  
Avian Development ◽  
Spatiotemporal Control ◽  
Promoter System

ABSTRACTPrecisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

scholarly journals HOXA9 is required for survival in human MLL-rearranged acute leukemias

Blood ◽  
2009 ◽  
Vol 113 (11) ◽  
pp. 2375-2385 ◽  
Author(s):  
Joerg Faber ◽  
Andrei V. Krivtsov ◽  
Matthew C. Stubbs ◽  
Renee Wright ◽  
Tina N. Davis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiling ◽  
Therapeutic Option ◽  
Mixed Lineage Leukemia ◽  
High Level Expression ◽  
Acute Leukemias ◽  
Gene Expression Analyses ◽  
High Level ◽  
Novel Therapeutic

Leukemias that harbor translocations involving the mixed lineage leukemia gene (MLL) possess unique biologic characteristics and often have an unfavorable prognosis. Gene expression analyses demonstrate a distinct profile for MLL-rearranged leukemias with consistent high-level expression of select Homeobox genes, including HOXA9. Here, we investigated the effects of HOXA9 suppression in MLL-rearranged and MLL-germline leukemias using RNA interference. Gene expression profiling after HOXA9 suppression demonstrated co–down-regulation of a program highly expressed in human MLL-AML and murine MLL-leukemia stem cells, including HOXA10, MEIS1, PBX3, and MEF2C. We demonstrate that HOXA9 depletion in 17 human AML/ALL cell lines (7 MLL-rearranged, 10 MLL-germline) induces proliferation arrest and apoptosis specifically in MLL-rearranged cells (P = .007). Similarly, assessment of primary AMLs demonstrated that HOXA9 suppression induces apoptosis to a greater extent in MLL-rearranged samples (P = .01). Moreover, mice transplanted with HOXA9-depleted t(4;11) SEMK2 cells revealed a significantly lower leukemia burden, thus identifying a role for HOXA9 in leukemia survival in vivo. Our data indicate an important role for HOXA9 in human MLL-rearranged leukemias and suggest that targeting HOXA9 or downstream programs may be a novel therapeutic option.

scholarly journals Regulatable Arabinose-Inducible Gene Expression System with Consistent Control in All Cells of a Culture

2000 ◽  
Vol 182 (24) ◽  
pp. 7029-7034 ◽  
Author(s):  
Artem Khlebnikov ◽  
Øystein Risa ◽  
Tove Skaug ◽  
Trent A. Carrier ◽  
J. D. Keasling
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
High Capacity ◽  
Inducible Promoter ◽  
Transport Systems ◽  
Specific Expression ◽  
Homogeneous Population ◽  
E Coli ◽  
Transport Control ◽  
Inducible Gene

ABSTRACT The arabinose-inducible promoter PBAD is subject to all-or-none induction, in which intermediate concentrations of arabinose give rise to subpopulations of cells that are fully induced and uninduced. To construct a host-vector expression system with regulatable control in a homogeneous population of cells, thearaE gene of Escherichia coli was cloned into an RSF1010-derived plasmid under control of the isopropyl-β-d-thiogalactopyranoside-induciblePtac and Ptaclac promoters. This gene encodes the low-affinity, high-capacity arabinose transport protein and is controlled natively by an arabinose-inducible promoter. To detect the effect of arabinose-independentaraE expression on population homogeneity and cell-specific expression, the gfpuv gene was placed under control of the arabinose-inducible araBAD promoter (PBAD ) on the pMB1-derived plasmid pBAD24. The transporter and reporter plasmids were transformed into E. coli strains with native arabinose transport systems and strains deficient in one or both of the arabinose transport systems (araE and/or araFGH). The effects of the arabinose concentration and arabinose-independent transport control on population homogeneity were investigated in these strains using flow cytometry. The araE, and araE araFGHmutant strains harboring the transporter and reporter plasmids were uniformly induced across the population at all inducer concentrations, and the level of gene expression in individual cells varied with arabinose concentration. In contrast, the parent strain, which expressed the native araE and araFGH genes and harbored the transporter and reporter plasmids, exhibited all-or-none behavior. This work demonstrates the importance of including a transport gene that is controlled independently of the inducer to achieve regulatable and consistent induction in all cells of the culture.

scholarly journals Polychlorinated Biphenyl Rhizoremediation by Pseudomonas fluorescens F113 Derivatives, Using a Sinorhizobium meliloti nod System To Drive bph Gene Expression

2005 ◽  
Vol 71 (5) ◽  
pp. 2687-2694 ◽  
Author(s):  
Marta Villacieros ◽  
Clare Whelan ◽  
Martina Mackova ◽  
Jesper Molgaard ◽  
María Sánchez-Contreras ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Fluorescens ◽  
Sinorhizobium Meliloti ◽  
Polychlorinated Biphenyl ◽  
Expression System ◽  
Single Copy ◽  
Bacterial Strains ◽  
High Level Expression ◽  
High Level ◽  
Biodegradation Genes

ABSTRACT Rhizoremediation of organic chemicals requires high-level expression of biodegradation genes in bacterial strains that are excellent rhizosphere colonizers. Pseudomonas fluorescens F113 is a biocontrol strain that was shown to be an excellent colonizer of numerous plant rhizospheres, including alfalfa. Although a derivative of F113 expressing polychlorinated biphenyl (PCB) biodegradation genes (F113pcb) has been reported previously, this strain shows a low level of bph gene expression, limiting its rhizoremediation potential. Here, a high-level expression system was designed from rhizobial nod gene regulatory relays. Nod promoters were tested in strain F113 by using β-galactosidase transcriptional fusions. This analysis showed that nodbox 4 from Sinorhizobium meliloti has a high level of expression in F113 that is dependent on an intact nodD1 gene. A transcriptional fusion of a nodbox cassette containing the nodD1 gene and nodbox 4 fused to a gfp gene was expressed in the alfalfa rhizosphere. The bph operon from Burkholderia sp. strain LB400 was cloned under the control of the nodbox cassette and was inserted as a single copy into the genome of F113, generating strain F113L::1180. This new genetically modified strain has a high level of BphC activity and grows on biphenyl as a sole carbon and energy source at a growth rate that is more than three times higher than that of F113pcb. Degradation of PCBs 3, 4, 5, 17, and 25 was also much faster in F113L::1180 than in F113pcb. Finally, the modified strain cometabolized PCB congeners present in Delor103 better than strain LB400, the donor of the bph genes used.

Characterization of a pH-inducible promoter system for high-level expression of recombinant proteins inEscherichia coli

1995 ◽  
Vol 47 (2) ◽  
pp. 186-192 ◽  
Author(s):  
Chih-Hsiung Chou ◽  
Aristos A. Aristidou ◽  
Shi-Yuan Meng ◽  
George N. Bennett ◽  
Ka-Yiu San
Keyword(s):  
Recombinant Proteins ◽  
Inducible Promoter ◽  
Inescherichia Coli ◽  
High Level Expression ◽  
High Level ◽  
Promoter System

scholarly journals A toolkit to generate inducible and interconvertible Drosophila transgenes

2020 ◽  
Author(s):  
Franz Wendler ◽  
Sangbin Park ◽  
Claire Hill ◽  
Alessia Galasso ◽  
Kathleen R. Chang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binary Systems ◽  
Expression System ◽  
Fruit Fly ◽  
Regulatory Elements ◽  
Conditional Gene Expression ◽  
Dna Elements ◽  
Independent Site ◽  
Inducible Gene

ABSTRACTThe existence of three independent binary systems for conditional gene expression (Gal4/UAS; LexA/LexAop; QF/QUAST) has greatly expanded versatile genetic analyses in the fruit fly Drosophila melanogaster; however, the experimental application of these tools is limited by the need to generate multiple collections of non-interchangeable transgenic fly strains for each inducible gene expression system. To address this practical limitation, we developed a multipurpose modular vector that contains the regulatory elements from all three binary systems, enabling Gal4-, LexA- or QF-dependent expression of transgenes. Our methods also incorporate DNA elements that facilitate independent site-specific recombination and elimination of regulatory UAS, LexAop or QUAST modules with spatial and temporal control, thus offering unprecedented possibilities and logistical advantages for in vivo genetic modulation and efficient interconversion of transgenic fly lines.

scholarly journals Stable integration of an optimized inducible promoter system enables spatiotemporal control of gene expression throughout avian development

2020 ◽  
Author(s):  
Daniel Chu ◽  
An Nguyen ◽  
Spenser S. Smith ◽  
Zuzana Vavrušová ◽  
Richard A. Schneider
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Inducible Promoter ◽  
In Ovo ◽  
Control Of Gene Expression ◽  
Promoter Construct ◽  
Avian Development ◽  
Spatiotemporal Control ◽  
Promoter System

AbstractPrecisely altering gene expression is critical for understanding molecular processes of embryogenesis. Although some tools exist for transgene misexpression in developing chick embryos, we have refined and advanced them by simplifying and optimizing constructs for spatiotemporal control. To maintain expression over the entire course of embryonic development we use an enhanced piggyBac transposon system that efficiently integrates sequences into the host genome. We also incorporate a DNA targeting sequence to direct plasmid translocation into the nucleus and a D4Z4 insulator sequence to prevent epigenetic silencing. We designed these constructs to minimize their size and maximize cellular uptake, and to simplify usage by placing all of the integrating sequences on a single plasmid. Following electroporation of stage HH8.5 embryos, our tetracycline-inducible promoter construct produces robust transgene expression in the presence of doxycycline at any point during embryonic development in ovo or in culture. Moreover, expression levels can be modulated by titrating doxycycline concentrations and spatial control can be achieved using beads or gels. Thus, we have generated a novel, sensitive, tunable, and stable inducible-promoter system for high-resolution gene manipulation in vivo.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

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