Transient uidA gene expression in electroporated cotyledonary protoplasts of Pinus nigra ssp. salzmannii and in bombarded cotyledons

2000 ◽  
Vol 30 (3) ◽  
pp. 448-455 ◽  
Author(s):  
Marian López ◽  
Jaime M Humara ◽  
Roberto Rodríguez ◽  
Ricardo J Ordás
Keyword(s):  
Gene Expression ◽  
Transient Gene Expression ◽  
Gene Promoter ◽  
Pinus Nigra ◽  
Cauliflower Mosaic Virus ◽  
Promoter Strength ◽  
Uida Gene ◽  
Uida Expression ◽  
Polyubiquitin Gene ◽  
Exponential Pulse

We have developed a method for the routine isolation of Pinus nigra Arn. ssp. salzmannii (Dunal) Franco protoplasts. The optimized electroporation conditions for uidA gene (gene for beta-glucuronidase) expression in protoplasts from cotyledons excised from 8-day-old seedlings were determined using an exponential pulse wave generator for gene transfer. The protoplasts were electroporated with plasmids containing the chimeric uidA gene under the control of several promoters, and in parallel, cotyledons were bombarded with the same constructs using a biolistic gun. Both techniques confirmed that gene expression was higher when controlled by the sunflower polyubiquitin gene promoter than by the cauliflower mosaic virus CaMV35S promoter, whereas the rice actin and maize alcohol dehydrogenase promoters resulted in lower uidA expression levels, as determined fluorometrically. In this study, the electroporation procedure has been more effective than the particle bombardment procedure to determine the promoter strength on transient gene expression in P. nigra.

scholarly journals Transient expression of a GUS reporter gene from cauliflower mosaic virus replacement vectors in the presence and absence of helper virus

2001 ◽  
Vol 82 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Rita Viaplana ◽  
David S. Turner ◽  
Simon N. Covey
Keyword(s):  
Gene Expression ◽  
Mosaic Virus ◽  
Transient Gene Expression ◽  
Helper Virus ◽  
Gus Activity ◽  
Cauliflower Mosaic Virus ◽  
Primer Binding Site ◽  
Wild Type Virus ◽  
Foreign Gene ◽  
Wild Type

Vectors based upon the genome of cauliflower mosaic virus (CaMV) have only a limited capacity for replicating foreign DNA in plants. A helper virus system has been developed to complement CaMV constructs capable of carrying a large foreign gene (glucuronidase; GUS). GUS replaced part or all of the non-essential CaMV gene II and the essential genes III, IV and V. This construct was co-inoculated mechanically with wild-type CaMV helper virus onto Brassica rapa leaves to promote GUS vector complementation. After 1 week, blue foci of GUS activity were observed in the centres of the local lesions. Leaves inoculated with the GUS construct in the absence of helper virus showed randomly distributed foci of GUS activity that were generally smaller than the lesion-associated GUS foci. Inoculation with a simple non-replicating CaMV 35S promoter–GUS construct also produced small GUS foci. Co-inoculation of helper virus with CaMV gene replacement vectors in which replication was prevented by moving the primer-binding site or by deletion of an essential splice acceptor produced only small, randomly distributed GUS activity foci, demonstrating that the lesion-associated foci were produced by gene expression from replicating constructs. These experiments show that CaMV genes III–V can be complemented by wild-type virus and replacement gene vectors can be used for transient gene expression studies with CaMV constructs that distinguish gene expression associated with a replicating vector from that associated with a non-replicating vector.

Transient expression of foreign chimeric genes in the gymnosperm hybrid larch following electroporation

1991 ◽  
Vol 69 (8) ◽  
pp. 1731-1736 ◽  
Author(s):  
Pierre J. Charest ◽  
Yvonne Devantier ◽  
Christine Ward ◽  
Catherine Jones ◽  
Ulriche Schaffer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Expression System ◽  
Transient Gene Expression ◽  
Inducible Promoter ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Hybrid Larch ◽  
Naked Plasmid ◽  
Angiosperm Species

A transient gene expression system using electroporation and naked plasmid DNA has been developed for hybrid larch (Larix × eurolepis). The β-glucuronidase, neomycin phosphotransferase II, and chloramphenicol acetyltransferase genes were used effectively, but the latter was found to be the most useful. Electroporation conditions were comparable with protocols developed for other conifer and angiosperm species. Of the parameters tested the optimum conditions were 300 V, 150 μF, and 300 μg/mL pCaMVCN DNA. The 35S promoter of cauliflower mosaic virus yielded a stronger level of transient expression than the nopaline synthase promoter, which is consistent with other studies. A construct with the wound-inducible promoter of the potato proteinase IIK gene and the chloramphenicol acetyl transferase reporter coding sequence did not yield to any transient gene expression, even after induction with acetylsalicylic acid and exposure to ultraviolet radiation. Key words: larch, Larix × eurolepis, electroporation, transient gene expression.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

scholarly journals Gene Expression Space Shapes the Bioprocess Trade-Offs among Titer, Yield and Productivity

2021 ◽  
Vol 11 (13) ◽  
pp. 5859
Author(s):  
Fernando N. Santos-Navarro ◽  
Yadira Boada ◽  
Alejandro Vignoni ◽  
Jesús Picó
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Performance Metrics ◽  
Cell Mass ◽  
Gene Copy ◽  
Substrate Uptake ◽  
Promoter Strength ◽  
Expression Levels ◽  
Trade Offs ◽  
Wide Range

Optimal gene expression is central for the development of both bacterial expression systems for heterologous protein production, and microbial cell factories for industrial metabolite production. Our goal is to fulfill industry-level overproduction demands optimally, as measured by the following key performance metrics: titer, productivity rate, and yield (TRY). Here we use a multiscale model incorporating the dynamics of (i) the cell population in the bioreactor, (ii) the substrate uptake and (iii) the interaction between the cell host and expression of the protein of interest. Our model predicts cell growth rate and cell mass distribution between enzymes of interest and host enzymes as a function of substrate uptake and the following main lab-accessible gene expression-related characteristics: promoter strength, gene copy number and ribosome binding site strength. We evaluated the differential roles of gene transcription and translation in shaping TRY trade-offs for a wide range of expression levels and the sensitivity of the TRY space to variations in substrate availability. Our results show that, at low expression levels, gene transcription mainly defined TRY, and gene translation had a limited effect; whereas, at high expression levels, TRY depended on the product of both, in agreement with experiments in the literature.

Poly(ethyleneimine)-Mediated Large-Scale Transient Gene Expression: Influence of Molecular Weight, Polydispersity and N -Propionyl Groups

2012 ◽  
Vol 12 (5) ◽  
pp. 628-636 ◽  
Author(s):  
Zuzana Kadlecova ◽  
Sophie Nallet ◽  
David L. Hacker ◽  
Lucia Baldi ◽  
Harm-Anton Klok ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Weight ◽  
Large Scale ◽  
Transient Gene Expression

scholarly journals Mild Hypothermia Improves Transient Gene Expression Yields Several Fold in Chinese Hamster Ovary Cells

2008 ◽  
Vol 24 (2) ◽  
pp. 458-465 ◽  
Author(s):  
S. Wulhfard ◽  
S. Tissot ◽  
S. Bouchet ◽  
J. Cevey ◽  
M. DeJesus ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chinese Hamster Ovary ◽  
Mild Hypothermia ◽  
Chinese Hamster Ovary Cells ◽  
Transient Gene Expression ◽  
Chinese Hamster ◽  
Ovary Cells
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