Levels Of Transient Gene Expression Research Articles

Tombusvirus-based vector systems to permit over-expression of genes or that serve as sensors of antiviral RNA silencing in plants

A next generation Tomato bushy stunt virus (TBSV) coat protein gene replacement vector system is described that can be applied by either RNA inoculation or through agroinfiltration. A vector expressing GFP rapidly yields high levels of transient gene expression in inoculated leaves of various plant species, as illustrated for Nicotiana benthamiana, cowpea, tomato, pepper, and lettuce. A start-codon mutation to down-regulate the dose of the P19 silencing suppressor reduces GFP accumulation, whereas mutations that result in undetectable levels of P19 trigger rapid silencing of GFP. Compared to existing virus vectors the TBSV system has a unique combination of a very broad host range, rapid and high levels of replication and gene expression, and the ability to regulate its suppressor. These features are attractive for quick transient assays in numerous plant species for over-expression of genes of interest, or as a sensor to monitor the efficacy of antiviral RNA silencing.

Lipoic acid—an unique plant transformation enhancer

Including lipoic acid (LA) in culture media during Agrobacterium transformation processes of four crop species has significantly improved the transformation methods of the crops, even for previously recalcitrant genotypes. Plant transformation efficiency of soybean was significantly increased from 0.6% to 3.7% and tomato from 29.8% to 87.0%. Transformation efficiency was doubled from 2.8% to 5.7% in wheat. The frequency of glyphosate-resistant embryos had a significant increase from 41.4% to 61.2% in cotton. Regeneration of non-transgenic shoots under selection (“shoot escapes”) was significantly reduced in tomato from 91.5% to 46.2% while in soybean from 92.0% to 72.0% under optimal conditions. This study also demonstrated that the increase of transformation efficiency in tomato was accompanied by as much as a significant 2-fold reduction in severity of browning of Agrobacterium-infected plant tissues and up to a significant 3-fold increase in the percentage of explants with a high level of transient gene expression. LA application in plant transformation has enabled the resolution of three common problems in plant transformation: browning or necrosis of the transformed cells or tissues, difficulty in regenerating transformed cells or tissues, and shoot escapes, which severely limit the number of transgenic plants that can be regenerated.

Cross‐linked low molecular weight glycopeptide‐mediated gene delivery: Relationship between DNA metabolic stability and the level of transient gene expression in vivo

DNA co‐condensates were formed by reacting [125I]DNA with an admixture of a high‐mannose glycopeptide (Man9‐CWK18) and either of two poly(ethylene glycol) peptides (PEG‐VS‐CWK18 or PEG‐SS‐CWK18) followed by cross‐linking with 6–50 mol equiv of glutaraldehyde. [125I]DNA co‐condensates were administered intravenously in mice to determine the influence of peptide DNA formulation parameters on specific targeting to Kupffer cells. Optimal targeting to Kupffer cells required the combined use of 50 mol % Man9‐CWK18 and PEG‐CWK18 to mediate specific recognition by the mannose receptor to Kupffer cells. The cellular uptake of cross‐linked Man9‐CWK18/PEG‐CWK18 DNA co‐condensates was receptor mediated since Kupffer cell targeting was inhibited by pre‐administration of Man‐bovine serum albumin (BSA) but not BSA. An optimized formulation targeted 60% of the dose to the liver, with 80% of the liver‐targeted DNA localized to Kupffer cells. Cross‐linking with either 6, 15, or 50 mol equiv of glutaraldehyde led to a corresponding decrease in the metabolism rate of DNA in liver as measured by half‐live‐ of 4, 6, and 39 h, respectively. Tail vein dosing of 50 μg of DNA co‐condensates cross‐linked with 6 mol equiv of glutaraldehyde produced detectable levels of human α1‐antitrypsin in blood after 12 h, which peaked at day six and persisted for 10 days. The level of human α1‐antitrypsin was elevated two‐fold each day when dosing with DNA co‐condensates cross‐linked with 15 mol equiv of glutaraldehyde, revealing a correlation between the metabolic stability of the DNA in liver and level of gene expression. In addition to possessing greater metabolic stability, DNA co‐condensates cross‐linked with 50 mol equiv of glutaraldehyde, but lacking a targeting ligand, avoided rapid liver uptake and possessed a prolonged pharmacokinetic half‐life, providing insight into a means to target DNA condensates to peripheral tissues. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:2010–2022, 2001

High-velocity microprojectile mediated DNA delivery into Phaseolus vulgaris callus cells

We report the method for the establishment of rapidly growing callus cultures of Phaseolus vulgaris and the conditions required for efficient transformation using high velocity microprojectiles and high level of transient gene expression. Using hypocotyl explant and vertical culture on B5 medium with lmg/1 kinetin and 2 mg/1 2,4-D, we can recommend to get a rapidly growing callus from bean which is a good starting material to introduce foreign DNA into bean cells. The GeneBooster particle delivery system was used for the bombardment of bean callus and the Hgm resistance gene (Hgmr) was used as a selectable marker gene. 25mg/I hygromycin (Hgm) concentration was sufficient to kill the control callus. We used the standard physical factors, the appropriate pressure of N2 gas for the bombardment of the callus tissue, the shooting distance and the size of tungsten particles used as microprojectiles. Selective and nonselective tests were made by transferring the healthy green and white calluses, subcultured for 4 months on selective and nonselective medium. Several Hgm resistant calli had been obtained. Selective pressure was maintained over a period of 10 months.

Bean tissue culture and genetic transformation with Agrobacterium

In this paper we report the establishment methods of a rapidly growing callus culture of Phaseolus vulgaris bean as well as the conditions required for a high level of transient gene expression using Agrobacterium-mediated transformation. A vector is containing both the lindan-resistance gene as a selectable marker, and GUS gene as a screenable marker. By using hypocotyl explant and vertical culture on B5 medium supplemented with 1 mg/1 kinetin- and 2,4-D 2 mg/1 and subcultured every 3-4 weeks, we can recommend to get a good and much callus from bean. This will help in introducing foreign DNA into callus cells. One strain of Agrobacterium carrying plasmid as vector for introducing foreign DNA into plant cells was used. At different concentrations of lindan; 3, 4 and 4.5 mg/I, the transformed Maxidor callus survived and grew over a period of 6 month and subcultured every 3-4 weeks, but the control callus died. Callus were assayed for GUS activity to confirm the expression of the GUS gene using the histochemical assay test. The GUS gene was also correctly expressed in callus cultures grown on 4mg/I lindan-selected medium, the typical blue colour in the histochemical assay using the X-gluc as substrate. But the control, non-transformed callus was not able to grow in the presence of lindan, neither showed a positive reaction in the in vitro assays.

Transient gene expression in mammalian cells grown in serum-free suspension culture.

In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method). The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression levels. The precise ratio is dependent on the DNA concentration. For example, using 1 mug/ml DNA by the indirect method, the ratio of optimal PEI:DNA was about 10-13:1. However, the ratio increases to 33:1 for 0.1-0.2 mug/ml DNA. By testing several different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared to 1 mug/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1 (CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production of milligram amounts of recombinant proteins in 2-5 l spinner culture scale within 3-5 days. Fermentor scale experiments, however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium.

Cryopreserved callus: a source of protoplasts for rice transformation

We cryopreserved whole rice calli (Oryza sativa L cv Taipei 309) to investigate the ability of the surviving cells to regenerate plants and yield protoplasts competent for genetic transformation. Four out of six callus lines cryopreserved after four months in culture contained small sectors able to continue cell division and subsequently regenerate fertile plants. Both cryopreservation efficiency and regeneration ability decreased when using eight month old cultures. High yields of protoplasts were obtained from different cryopreserved callus lines. Protoplasts were transfected with chimeric genes consisting of the maize ubiquitin 1 promoter, first exon and first intron fused to the coding region of either the GUS or BAR marker genes. Levels of transient gene expression from both marker genes were similar to those previously obtained using protoplasts derived from callus that had not been frozen. Stable transformants were selected by their resistance to Bialaphos and could be identified with the pH indicator chlorophenol red. Southern blot analysis confirmed the integration of the BAR gene into the rice genome. Therefore, cryopreservation does not affect the ability of rice cells to integrate and express foreign genes.

Microprojectile-mediated DNA delivery to the Salicaceae family

A transient gene expression system was developed for the Salicaceae family using microprojectile-mediated DNA delivery (Biolistic™) to cell suspensions. Using Populus nigra × Populus maximowiczii line NM1, 10 variables were optimized. Optimum transient gene expression under the 10 conditions tested was obtained with a 7- to 9-day-old cell suspension. Highest transient levels were observed with samples positioned 13.5 cm from the stopping plate and bombarded with 1.6-μm gold particles coated with 1 μg of DNA precipitated with CaCl2. Assaying for β-glucuronidase gene expression was performed 1 day after bombardment. The fate of the microparticles in the bombarded cells was studied, showing that 58.9% of cells expressing the β-glucuronidase gene had microparticles located in the nucleus or its vicinity and the remaining cells had microparticles in the cytoplasm. Cell suspensions from five different lines (P. nigra × P. maximowiczii lines NM1 and NM6, Populus deltoides × P. nigra line DN106, Populus tremula × Populus alba line 7171-B4, and Salix alba sanguined line SA-2) yielded transient gene expression. The relative strengths of β-glucuronidase expression in the lines tested were NM1 = 7171-B4 > NM6 > DN106 > SA-2. Six plasmid constructions were also tested in line NM1 for transient β-glucuronidase gene expression. The x-glucuronide histochemical assay did not reveal any differences, but the methyl umbelliferone glucuronide fluorescent assay yielded the following relative levels of transient gene expression with the different promoter sequences: 35S-35S-AMV enhancer = 35S-AMV enhancer > 35S-35S = 35S-35S-AMV enhancer with the β-glucuronidase – neomycin phosphotransferase fusion > 35S. Four transgenic cell lines of P. nigra × P. maximowiczii were characterized for kanamycin resistance and neomycin phosphotransferase II gene activity. Polymerase chain reaction and Southern hybridization analyses indicated the presence of the β-glucuronidase and neomycin phosphotransferase II genes in the genome of three of these transgenic lines. Key words: microprojectile, particle bombardment, Salicaceae, poplar, willow.

Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.

Transient gene expression in vegetative shoot apical meristems of wheat after ballistic microtargeting

A method has been established that allows the targeted delivery of DNA‐carrying gold particles to vegetative shoot apical meristems of cereal species. Meristems of 8‐ to 10‐day‐old seedlings of wheat (Triticum aestivum), rice (Oryza sativa) and sorghum (Sorghum bicolor) were manually exposed by removal of the coleoptile and the first three to four leaves. Using ballistic microtargeting, equal‐sized gold particles of different diameters ranging from 1.0 to 2.0 µm were propelled to meristems by pulses of compressed nitrogen ranging from 9 to 13 MPa. When accelerated by 13 MPa, particles of 1.4 µm or larger penetrated to cells of L2 in 80% of the bombarded wheat meristems. Expression vectors containing either the gene for β‐glucuronidase (gusA) or genes regulating the anthocyanin biosynthesis (Bperu and C1 from maize) were delivered to wheat meristems. The level of transient gene expression was dependent on the osmotic pressure of the MS‐based medium that was used to mount the explants for shooting: an increase of the sucrose concentration from 2 to 10% in the medium resulted in an increase of transient gene expression from 5 to 25% of the bombarded meristems. Although particles of 1.4 µm were efficiently delivered to L1 and L2, transient gene expression occurred predominantly in the L1 layer. In contrast, up to 10% of the bombarded meristems had expressing cells in L2 when particles of 2.0 µm were used. Ten days after bombardment, GUS‐positive sectors in meristems and in leaf primordia were observed. When destructive GUS detection was omitted, between 80 and 90% of the bombarded ex‐plants recovered in vitro on basal MS medium and then regenerated to fertile plants in the greenhouse.

Particle Bombardment-Mediated Gene Transfer and Expression in Rat Brain Tissues

We have previously demonstrated that the particle bombardment method for gene transfer (Accell) provides a new means for transfection of various cell types in culture. In this study we evaluate its application to rat brain systems. Using a luciferase (luc) gene as a reporter, we obtained high levels of transient gene expression in primary cultures of fetal brain tissue. Reduced but significant levels were also detected in adult brain primary cultures. Both neuron and glial cells were transfected using this technique. The transient gene expression level obtained with Accell was at least 100-fold higher than that obtained with three other gene transfer methods. The relative strengths of four cellular and seven viral promoters were also evaluated in these cultures. In vivo gene expression was studied using freshly excised and bombarded fetal brain tissues which were immediately transplanted into caudate or intracortical brain tissues of adult host animals. Assays showed that luciferase activity was present in transplants for up to two months following gene transfer. In vitro and in vivo expression of a rat tyrosine hydroxylase (TH) gene, a candidate gene for treatment of Parkinson's disease, was also detected in this rat brain system. Our results suggest that the particle bombardment gene transfer technology can be employed as an effective method for ex vivo gene transfer into brain tissues.

Microprojectile-DNA delivery in conifer species: factors affecting assessment of transient gene expression using the ?-glucuronidase reporter gene

The Biolistic(®) microprojectile DNA-delivery method was used to test the usefulness in conifers of eight gene constructs based on the 35S promoter, the AMV translational enhancer, and gene fusion between the P-glucuronidase and the neomycin phosphotransferase II genes. The evaluation was done with embryogenic cells of Picea glauca, where the relative strengths of the promoters were 35S-35S-AMVE>35S-AMVE>35S-35S>35S as evaluated by transient gene expression. The fusion gene of GUS and NPT II gave lower levels of transient gene expression than the unfused GUS gene as detected by X-GLU histochemical assays. Experiments comparing the EM promoter of wheat and the 35S-35S-AMVE promoter (with and without fusion between GUS and NPT II) were done in Picea rubens, P. mariana, P. glauca, and Larix x eurolepis. The unfused gene with the 35S-35S-AMVE promoter gave higher levels of transient gene expression than the fused GUS-NPT II gene. The fluorescent MUG assay was more sensitive than the histochemical X-GLU assay to detect the activity of the β-glucuronidase gene.

Microprojectile-mediated DNA delivery in haploid and diploid embryogenic cells of Larix spp.

A particle bombardment protocol using the Dupont Biolistic™ particle delivery system and the β-glucuronidase reporter gene was developed for embryogenic cell lines of Larix. Comparison of four sizes of tungsten microprojectiles showed the highest transient gene expression with 1.11 μm diameter particles. The highest β-glucuronidase transient gene activity was in cells bombarded 5 and 6 days after subculturing to fresh media. Comparison of 22 different cell lines of haploid and diploid Larixdecidua, Larixleptolepis, LarixXleptoeuropae, and LarixXeurolepis showed variation among the cell lines in β-glucuronidase gene activity, embryogenesis, and cell growth. In diploid lines, the levels of transient gene expression were higher with youngest lines; however, this trend was not distinguishable in haploid lines

Hybrid genes in the analysis of transformation conditions: II. Transient expression vs stable transformation—analysis of parameters influencing gene expression levels and transformation efficiency

Reports on direct gene transfer have dealt with either the obtention of stable transformants and transgenic plants, or described the use of reporter genes to analyse different aspects of gene expression in plant protoplasts and conditions for their use in transient gene expression assays.In this paper we present comparisons between several transformation techniques, show species‐specific differences in efficiencies of stable transformants and in the levels of transient gene expression, and report on the identification of major parameters responsible for DNA uptake as judged from transient chloramphenicol acetyl transferase (CAT) expression levels and from efficiencies of transformation based on kanamycin‐resistance. The described procedures have been simplified, optimized and standardized and should allow routine use with a great variety of plant species.

Transient Gene Expression in Maize, Rice, and Wheat Cells Using an Airgun Apparatus

An airgun apparatus has been constructed for transient gene expression studies of monocots. This device utilizes compressed air from a commercial airgun to propel macroprojectile and DNA-coated tungsten particles. The beta-glucuronidase (GUS) reporter gene was used to monitor transient expression in three distinct cell types of maize (Zea mays), rice (Oryza sativa), and wheat (Triticum aestivum). The highest level of GUS activity in cultured maize cells was observed when distance between stopping plate and target cells was adjusted to 4.3 centimeters. Efficiency of transformation was estimated to be 4.4 x 10(-3). In a partial vacuum of 700 millimeters Hg, velocity of macroprojectile was measured at 520 meters per second with a 6% reduction in velocity at atmospheric pressure. A polyethylene film placed in the breech before firing contributed to a 12% increase in muzzle velocity. A 700 millimeters Hg level of vacuum was necessary for maximum number of transfornants. GUS expression was also detected in wheat leaf base tissue of microdissected shoot apices. High levels of transient gene expression were also observed in hard, compact embryogenic callus of rice. These results show that the airgun apparatus is a convenient, safe, and low-cost device for rapid transient gene expression studies in cereals.