scholarly journals The Steroid-Inducible pOp6/LhGR Gene Expression System is Fast, Sensitive and Does NOT Cause Plant Growth Defects in Rice (Oryza Sativa)

2021 ◽  
Author(s):  
Marketa Samalova ◽  
Ian Moore
Keyword(s):  
Transgene Expression ◽  
Expression System ◽  
Translation Efficiency ◽  
Crop Species ◽  
Potent Inducer ◽  
Growth Defects ◽  
Basic Plant ◽  
Inducible Systems ◽  
Negative Growth

Abstract Inducible systems for transgene expression activated by a chemical inducer or an inducer of non-plant origin are desirable tools for both basic plant research and biotechnology. Although, the technology has been widely exploited in model plants, it has not been optimised for use with the major monocotyledonous crop species, namely rice. We have adapted the dexamethasone-inducible pOp6/LhGR system for rice and shown that it is fast, sensitive and tightly regulated, with high levels of induction that remain stable over several generations. Most importantly, we have shown that the system does not cause negative growth defects in vitro or in soil grown plants. Interestingly in the process of testing, we found that another steroid, triamcinolone acetonide, is a more potent inducer in rice than dexamethasone. We present serious considerations for the construct design to avoid undesirable effects caused by the system in plants, leakiness and possible silencing, as well as simple steps how to maximize translation efficiency of a gene of interest. Finally, we compare the performance of the pOp6/LhGR system with other chemically inducible systems tested in rice in terms of the properties of an ideal inducible system.

scholarly journals The steroid-inducible pOp6/LhGR gene expression system is fast, sensitive and does NOT cause plant growth defects in rice (Oryza sativa)

2021 ◽  
Author(s):  
Marketa Samalova ◽  
Ian Moore
Keyword(s):  
Expression System ◽  
Optimal Solution ◽  
Translation Efficiency ◽  
Crop Species ◽  
Potent Inducer ◽  
Growth Defects ◽  
Basic Plant ◽  
Inducible Systems ◽  
Negative Growth

SummaryInducible systems for transgene expression activated by a chemical or an inducer of non-plant origin are desirable tools for both basic plant research as well as advanced biotechnological utilizations. Although, the technology has been widely exploited in model plant species, optimal solution is missing for the major monocotyledonous crop species – rice. Here, we characterise the inducible properties of the pOp6/LhGR adapted for rice as fast, sensitive, tight, with high levels of induction that remain stable over several generations. Most importantly, the system does not cause negative growth defects in vitro or in soil grown plants. We describe various methods of application and optimization of induction by finding out that another steroid, triamcinolone acetonide, is more potent inducer then dexamethasone in rice. We present serious considerations for the construct design to avoid undesirable effects caused by the system in plants, leakiness and possible silencing, as well as simple steps how to maximize translation efficiency of a gene of interest. Finally, we compare the performance of the pOp6/LhGR system with other chemically inducible systems tested in rice in terms of the properties of an ideal inducible system.Significance statementThe non-monocot codon-optimized version of the dexamethasone inducible pOp6/LhGR system does not cause severe developmental perturbations in rice plants!

scholarly journals The steroid-inducible pOp6/LhGR gene expression system is fast, sensitive and does not cause plant growth defects in rice (Oryza sativa)

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Marketa Samalova ◽  
Ian Moore
Keyword(s):  
Transgene Expression ◽  
Expression System ◽  
Translation Efficiency ◽  
Model Species ◽  
Potent Inducer ◽  
Growth Defects ◽  
Basic Plant ◽  
Inducible Systems ◽  
Negative Growth

AbstractInducible systems for transgene expression activated by a chemical inducer or an inducer of non-plant origin are desirable tools for both basic plant research and biotechnology. Although, the technology has been widely exploited in dicotyledonous model plants such as Arabidopsis, it has not been optimised for use with the monocotyledonous model species, namely rice. We have adapted the dexamethasone-inducible pOp6/LhGR system for rice and the results indicated that it is fast, sensitive and tightly regulated, with high levels of induction that remain stable over several generations. Most importantly, we have shown that the system does not cause negative growth defects in vitro or in soil grown plants. Interestingly in the process of testing, we found that another steroid, triamcinolone acetonide, is a more potent inducer in rice than dexamethasone. We present serious considerations for the construct design to avoid undesirable effects caused by the system in plants, leakiness and possible silencing, as well as simple steps to maximize translation efficiency of a gene of interest. Finally, we compare the performance of the pOp6/LhGR system with other chemically inducible systems tested in rice in terms of the properties of an ideal inducible system.

scholarly journals A modular steroid-inducible gene expression system for use in rice

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Daniela Vlad ◽  
Basel Abu-Jamous ◽  
Peng Wang ◽  
Jane A. Langdale
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Rice Genome ◽  
Gus Activity ◽  
Target Sequence ◽  
Control Of Gene Expression ◽  
Growth Defects ◽  
Reporter Enzyme ◽  
Endogenous Genes

Abstract Background Chemically inducible systems that provide both spatial and temporal control of gene expression are essential tools, with many applications in plant biology, yet they have not been extensively tested in monocotyledonous species. Results Using Golden Gate modular cloning, we have created a monocot-optimized dexamethasone (DEX)-inducible pOp6/LhGR system and tested its efficacy in rice using the reporter enzyme β-glucuronidase (GUS). The system is tightly regulated and highly sensitive to DEX application, with 6 h of induction sufficient to induce high levels of GUS activity in transgenic callus. In seedlings, GUS activity was detectable in the root after in vitro application of just 0.01 μM DEX. However, transgenic plants manifested severe developmental perturbations when grown on higher concentrations of DEX. The direct cause of these growth defects is not known, but the rice genome contains sequences with high similarity to the LhGR target sequence lacO, suggesting non-specific activation of endogenous genes by DEX induction. These off-target effects can be minimized by quenching with isopropyl β-D-1-thiogalactopyranoside (IPTG). Conclusions Our results demonstrate that the system is suitable for general use in rice, when the method of DEX application and relevant controls are tailored appropriately for each specific application.

scholarly journals Comparative Analysis of Translation Efficiencies of Hepatitis C Virus 5′ Untranslated Regions among Intraindividual Quasispecies Present in Chronic Infection: Opposite Behaviors Depending on Cell Type

2000 ◽  
Vol 74 (22) ◽  
pp. 10827-10833 ◽  
Author(s):  
Julien Laporte ◽  
Isabelle Malet ◽  
Thibault Andrieu ◽  
Vincent Thibault ◽  
Jean-Jacques Toulme ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Expression System ◽  
Luciferase Expression ◽  
Hcv Rna ◽  
Translation Efficiency ◽  
Cell Type ◽  
Entry Site ◽  
Conserved Sequence

ABSTRACT Hepatitis C virus (HCV) RNA translation initiation is dependent on the presence of an internal ribosome entry site (IRES) that is found mostly in its 5′ untranslated region (5′ UTR). While exhibiting the most highly conserved sequence within the genome, the 5′ UTR accumulates small differences, which may be of biological and clinical importance. In this study, using a bicistronic dual luciferase expression system, we have examined the sequence of 5′ UTRs from quasispecies characterized in the serum of a patient chronically infected with HCV genotype 1a and its corresponding translational activity. Sequence heterogeneity between IRES elements led to important changes in their translation efficiency both in vitro and in different cell cultures lines, implying that interactions of RNA with related transacting factors may vary according to cell type. These data suggest that variants occasionally carried by the serum prior to reinfection could be selected toward different compartments of the same infected organism, thus favoring the hypothesis of HCV multiple tropism.

scholarly journals Optimization of a Modular Steroid-Inducible Gene Expression System for Use in Rice

2019 ◽  
Author(s):  
Daniela Vlad ◽  
Marketa Samalova ◽  
Basel Abu-Jamous ◽  
Peng Wang ◽  
Ian R. Moore ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Rice Genome ◽  
Gus Activity ◽  
Target Sequence ◽  
Control Of Gene Expression ◽  
Growth Defects ◽  
Reporter Enzyme ◽  
Endogenous Genes

SUMMARYChemically inducible systems that provide both spatial and temporal control of gene expression are essential tools, with many applications in plant biology. Using Golden Gate modular cloning, we have created a monocot-optimized dexamethasone (DEX)-inducible pOp6/LhGR system and tested its efficacy in rice using the reporter enzyme β-glucuronidase (GUS). The system is tightly regulated and highly sensitive to DEX application, with six hours of induction sufficient to induce high levels of GUS activity in transgenic callus. In seedlings, GUS activity was detectable in the root after in vitro application of just 0.01μM DEX. However, transgenic plants manifested severe developmental perturbations when grown on higher concentrations of DEX. The direct cause of these growth defects is not known, but the rice genome contains sequences with high similarity to the LhGR target sequence lacO, suggesting non-specific activation of endogenous genes by DEX induction. These off-target effects can be minimized by quenching with isopropyl β-D-1-thiogalactopyranoside (IPTG). The system is thus suitable for general use in rice, when the method of DEX application and relevant controls are tailored appropriately for each specific application.

Establishment of an in vitro transgene expression system in epimastigotes of Trypanosoma congolense

2009 ◽  
Vol 58 (1) ◽  
pp. 110-113 ◽  
Author(s):  
Tatsuya Sakurai ◽  
Miho Tanaka ◽  
Shin-ichiro Kawazu ◽  
Noboru Inoue
Keyword(s):  
Transgene Expression ◽  
Expression System ◽  
Trypanosoma Congolense

scholarly journals Nucleotide Variability and Translation Efficiency of the 5′ Untranslated Region of Hepatitis A Virus: Update from Clinical Isolates Associated with Mild and Severe Hepatitis

2010 ◽  
Vol 84 (19) ◽  
pp. 10139-10147 ◽  
Author(s):  
Vincent Mackiewicz ◽  
Anne Cammas ◽  
Delphine Desbois ◽  
Eric Marchadier ◽  
Sandra Pierredon ◽  
...  
Keyword(s):  
Hepatitis A ◽  
Clinical Presentation ◽  
Hepatitis A Virus ◽  
Expression System ◽  
Luciferase Expression ◽  
Translation Efficiency ◽  
Entry Site ◽  
Severe Hepatitis ◽  
Nucleotide Variability

ABSTRACT Mutations in the internal ribosome entry site (IRES) of hepatitis A virus (HAV) have been associated with enhanced in vitro replication and viral attenuation in animal models. To address the possible role of IRES variability in clinical presentation, IRES sequences were obtained from HAV isolates associated with benign (n = 8) or severe (n = 4) hepatitis. IRES activity was assessed using a bicistronic dual-luciferase expression system in adenocarcinoma (HeLa) and hepatoma (HuH7) cell lines. Activity was higher in HuH7 than in HeLa cells, except for an infrequently isolated genotype IIA strain. Though globally low, significant variation in IRES-dependent translation efficiency was observed between field isolates, reflecting the low but significant genetic variability of this region (94.2% ± 0.5% nucleotide identity). No mutation was exclusive of benign or severe hepatitis, and variations in IRES activity were not associated with a clinical phenotype, indirectly supporting the preponderance of host factors in determining the clinical presentation.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

scholarly journals In Vitro Reconstitution of an NADPH-Dependent Superoxide Reduction Pathway from Pyrococcus furiosus

2005 ◽  
Vol 71 (3) ◽  
pp. 1522-1530 ◽  
Author(s):  
Amy M. Grunden ◽  
Francis E. Jenney ◽  
Kesen Ma ◽  
Mikyoung Ji ◽  
Michael V. Weinberg ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Pyrococcus Furiosus ◽  
Expression System ◽  
Anaerobic Bacteria ◽  
Flavin Mononucleotide ◽  
Superoxide Reductase ◽  
Recombinant Enzymes ◽  
Significant Similarity ◽  
Midpoint Potential

ABSTRACT A scheme for the detoxification of superoxide in Pyrococcus furiosus has been previously proposed in which superoxide reductase (SOR) reduces (rather than dismutates) superoxide to hydrogen peroxide by using electrons from reduced rubredoxin (Rd). Rd is reduced with electrons from NAD(P)H by the enzyme NAD(P)H:rubredoxin oxidoreductase (NROR). The goal of the present work was to reconstitute this pathway in vitro using recombinant enzymes. While recombinant forms of SOR and Rd are available, the gene encoding P. furiosus NROR (PF1197) was found to be exceedingly toxic to Escherichia coli, and an active recombinant form (rNROR) was obtained via a fusion protein expression system, which produced an inactive form of NROR until cleavage. This allowed the complete pathway from NAD(P)H to the reduction of SOR via NROR and Rd to be reconstituted in vitro using recombinant proteins. rNROR is a 39.9-kDa protein whose sequence contains both flavin adenine dinucleotide (FAD)- and NAD(P)H-binding motifs, and it shares significant similarity with known and putative Rd-dependent oxidoreductases from several anaerobic bacteria, both mesophilic and hyperthermophilic. FAD was shown to be essential for activity in reconstitution assays and could not be replaced by flavin mononucleotide (FMN). The bound FAD has a midpoint potential of −173 mV at 23°C (−193 mV at 80°C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80°C) and low (23°C) temperatures, consistent with its proposed role in the superoxide reduction pathway. This is the first demonstration of in vitro superoxide reduction to hydrogen peroxide using NAD(P)H as the electron donor in an SOR-mediated pathway.

Sign in / Sign up

Export Citation Format

Share Document