Factors affecting transient gene expression in electroporated Glycine max protoplasts

Electroporation was used to evaluate parameters affecting transient gene expression in Glycine max protoplasts. Protoplast viability and reporter enzyme activity for chloramphenicol acetyl transferase (CAT) and ß-glucuronidase (GUS) depended on the field strength employed. Maximum CAT and GUS activity was obtained when a field strength of 500 V/cm at 1000 F and a protoplast concentration of 1–3 × 10⁶/ml was used. Transformation efficiencies up to approximately 1.6% GUS positive protoplasts were obtained. Transient gene expression increased with increasing plasmid DNA concentration and with the time after electroporation, reaching a maximum after 48 hr. Addition of polyethylene glycol at 5.6% and heat shock (5 rain at 45 °C) given to the protoplasts before adding DNA further enhanced the transformation efficiency. Under the optimized experimental conditions, CAT and GUS activity increased simultaneously, thereby indicating that the increased expression is caused by DNA uptake by more cells rather than greater DNA uptake by the same cells. Our results demonstrate that both GUS and CAT can be used as efficient screenable markers for transformation studies in soybean.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - PEG polyethylene glycol     

Transient gene expression and influence of promoters on foreign gene expression in Arabidopsis thaliana

Summary The influence of a variety of parameters was investigated on polyethylene glycol (PEG)-mediated transient nptII and gus gene expression in mesophyll protoplasts of Arabidopsis thaliana ecotype, Estland, in order to develop a suitable transient gene expression system. The investigation revealed that a combination of 20% PEG, incubation time of 15 min, 20–30 μg plasmid concentration per ml along with 50 μg carrier DNA m/l, and inclusion of calcium and magnesium ions during transfection followed by a culture period of 24 h registered maximum NPTII activity. Of the various promoters used for driving expression of the gus gene, the ubiquitin promoter from A. thaliana was the most efficient followed by 35S promoter of the CaMV and the actin promoter of rice. For comparison, similar studies in protoplasts of rice, wheat, and Brassica also revealed the differences in strength of these promoters. Arabidopsis ubiquitin promoter was the most effective in Brassica, and the rice actin1 promoter was the most effective in rice and wheat.     

PEG-mediated expression of GUS and CAT genes in protoplasts from embryogenic suspension cultures of Picea glauca

ß-Glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) were used as reporter proteins in protoplasts from embryogenic suspension cultures of Picea glauca (Moench) Voss (white spruce). Plasmid DNA enclosing chimeric GUS and CAT constructs, using the cauliflower mosaic virus 35S promoter, was introduced into Picea glauca protoplasts using polyethylene glycol (PEG). Transient expression was detected 12 to 40 h after PEG-mediated DNA delivery. Dose-response curves using covalently closed circular plasmid DNA, in the absence of carrier DNA, have been obtained for each of these reporter genes. Linearized plasmid DNA gave lower levels of expression than covalently closed circular plasmid DNA when assayed 40 h after PEG-mediated DNA transfer. The use of carrier DNA (herring sperm DNA), in combination with covalently closed circular plasmid DNA, increased the level of expression of GUS by about 50%. CAT expression was enhanced if PEG-mediated delivery was performed on ice rather than at room temperature. The highest level of expression for CAT, and the lowest signal-to-noise ratio, was found 24 h after PEG-mediated DNA transfer. Both GUS and CAT provided results that were quantifiable and can therefore be used as reporter genes in Picea glauca.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - CaMV cauliflower mosaic virus - NOS nopaline synthase - CCC covalently closed circular DNA - L linear DNA - PEG polyethylene glycol - HS herring sperm DNA - P protoplasts - PCM protoplast culture medium - MES morpholinoethane-sulfonic acid - Cm chloramphenicol - Ac acetylated - MUG 4-methyl umbelliferyl ß-D-glucuronide - TLC thin layer chromatography     

Comparative analysis of free DNA delivery and expression into protoplasts of Panicum maximum Jacq. (Guinea grass) by electroporation and polyethylene glycol

Relative levels of gene expression were studied in protoplasts isolated from two cell lines of Panicum maximum following DNA delivery by electroporation and polyethylene glycol (PEG). Gene expression was evaluated by assaying for chloramphenicol acetyltransferase (CAT) activity expressed by the CaMV 35S promoter with a nopaline synthase 3' polyadenylation signal, approximately 48 hours after DNA delivery. The expression of the CAT gene was slightly higher in electroporated protoplasts in comparison to PEG mediated delivery. However, PEG treated protoplasts showed higher plating efficiency. The effect of different salts and the molecular weight of PEG used on gene expression was also studied.     

An improved method for the generation and transfection of protoplasts from Cucumis sativus Cotyledons

Summary A protocol was developed for the preparation of Cucumis sativus var Straight 8 protoplasts that incorporates a two-step Ficoll^® gradient and results in a high percentage of viable, debris-free protoplasts suitable for the transient expression of foreign genes. Polyethylene glycol and electroporation were compared for their effect on protoplast transfection with commonly used reporter genes. Using a polyethylene glycol method, cucumber protoplasts transfected with a plasmid containing the -glucuronidase gene showed high expression levels, while protoplasts transfected with a plasmid containing the chloramphenicol acetyl transferase gene showed levels of activity that were barely distinguishable from mock-transfected controls. Tomato ringspot virus genomic RNA was also transfected into the protoplasts, and the assembly of viral particles was confirmed.     

Efficient transient expression and transformation of PEG-mediated gene uptake into mesophyll protoplasts of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.)

PEG-mediated transformation was used for gene delivery and evaluation of various parameters affecting the transient expression of a gene for ß-glucuronidase (gus) in mesophyll protoplasts of Capsicum annuum. Transient expression was found to be dependent on PEG concentration and exposure time of plasmid DNA to protoplasts as well as the amount of plasmid DNA. Maximum GUS activity was obtained when protoplasts were applied to 40% concentration and molecular weight was 6,000 of PEG solution with 30 min of exposure time. Protoplasts of pepper were transformed with a vector, pCAMBIA::Ac, which contained a pCAMBIA1302 T-DNA vector carrying a maize transposable element, Ac (activator), a selection marker HPT (hygromycin phosphotransferase), and a GFP-coding region driven by the 35S promoter in the presence of PEG. Approximately 30% of the protoplasts expressed GFP. Visibly transformed colonies were obtained from protoplasts after 2 months of culture and GFP was expressed. Southern hybridization confirmed the presence of Ac in the pepper genome.     

Transient gene expression in aleurone protoplasts isolated from developing caryopses of barley and wheat

Methods have been developed for the isolation of aleurone protoplasts from developing caryopses of Hordeum vulgare and Triticum aestivum in order to study transient expression of introduced genes. Chimaeric gene constructs were introduced into aleurone protoplasts by polyethylene glycol (PEG). Transient expression directed by the 35S promoter from cauliflower mosaic virus (CaMV) of the reporter gene encoding chloramphenicol acetyl transferase (CAT) was detected in aleurone protoplasts from developing barley and wheat grains. Using a similar construct, CAT activity increased when the alcohol dehydrogenase intron 1 fragment from maize was ligated between the 35S promoter and the CAT coding region. The demonstration of transient expression in protoplasts from developing aleurone layers indicates that they may be useful for investigating tissue and developmental control of genes coding for cereal seed proteins.     

Ethylene and in vitro culture of potato: suppression of ethylene generation vastly improves protoplast yield,plating efficiency and transient expression of an alien gene

Ethylene release by potato shoots cultured in closed boxes was suppressed by the addition of silver thiosulfate to the culture medium. Shoots cultured in the presence of silver thiosulfate produced appreciably more tissue and the yield of protoplasts per unit tissue mass was vastly increased, resulting in an 8 fold increase of protoplast yield per shoot. Exposure of pricked leaves to macerating enzymes facilitated ethylene generation. Leaves of shoots which were previously cultured in silver thiosulfate containing medium generated much less ethylene than leaves from control shoots and this generation could be further reduced by the addition of acetylsalicylic acid during maceration. The capability of polyethylene glycol treated potato protoplasts to produce microcalli was vastly increased by the addition of silver thiosulfate during exposure of protoplasts to Ca(NO₃)₂ following the polyethylene glycol treatment. Similarly, when a plasmid (pCAP212) containing an expressible gene for chloramphenicol acetyltransferase was introduced into potato protoplasts through a polyethylene glycol treatment, the transient expression of acetyltransferase was very much increased by the addition of a short incubation of the protoplasts with silver thiosulfate.Abbreviations AOA (aminooxy)acetic acid - ASA acetylsalicylic acid - AVG aminoethoxyvinyl-glycine - CAT chloramphenicol acetyltransferase - MV methyl viologen - PEG polyethylene glycol - STS silver thiosulfate     

Polybrene-mediated transfection of cultured lepidopteran cells: Induction of aDrosophila heat shock promoter

Summary We have introduced hsp-cat plasmid DNA intoSpodoptera frugiperda (Lepidoptera: Noctuidae) cells by transfection with purified DNA (1 to 48 μg/ml) mixed with the polycation polybrene (100 μg/ml) in serum-free Grace's medium. The hsp-cat construct contains a gene coding for the bacterial enzyme chloramphenicol acetyltransferase (CAT), whose expression is controlled by a promoter derived from aDrosophila heat shock protein (hsp) gene. Expression of CAT activity in transfectedSpodoptera cells was induced by a 2-h heat shock at 43°C. The temperature of the heat shock was based on conditions that maximized the expression of endogenous heat shock protein genes in these cells. CAT activity was maximal in cells that were exposed to the heat shock 2 d after transfection; by 4 d, activity was diminished, and little activity was detectable after 6 d. Transfection frequencies, which varied with DNA concentration and ranged as high as 6000 per million cells, were determined using a histochemical staining procedure. This work was supported by grant 88-37263-4020 from the United States Department of Agriculture, Washington, DC, and by the University of Minnesota Experiment Station. This is contribution 17,543 from the University of Minnesota Experiment Station, St. Paul, MN.     

Chimeric gene expression using maize intron in cultured cells of breadwheat

High voltage electrical pulses were used to introduce the CAT reporter gene into cultured protoplasts of breadwheat,Triticum aestivum. Four DNA constructs harboring the CAT gene and the 35S or mannipine synthase promoter were tested for levels of CAT activity 40–45 hr after electroporation of protoplasts. One construct, containing a maize intron sequence between 35S and CAT sequences, conferred 30 to 185 fold greater CAT activity over the other three constructs. Data from these experiments suggest that a maize intron or sequences with similar effects may be required in DNA constructs for efficient heterologous gene expression in cultured cells of breadwheat.Abbreviations CAT Chloramphenicol acetyl transferase - NPT II neomycin phosphotransferase - 35S the 35S promoter of Cauliflower Mosaic Virus - PEG Polyethylene glycol - MES 2-[N-morpholino] ethanesulfonic acid     

Factors affecting transient gene expression in electroporated black spruce (Picea mariana) and jack pine (Pinus banksiana) protoplasts

Summary Methods were developed for transient gene expression in protoplasts of black spruce (Picea mariana) and jack pine (Pinus banksiana). Protoplasts were isolated from embryogenic suspension cultures of black spruce and from non-embryogenic suspensions of jack pine. Using electroporation, transient expression of the chloramphenicol acetyltransferase (CAT) gene was assayed and shown to be affected by the cell line used, by voltage, temperature, and by the plasmid concentration and conformation. Increasing the plasmid DNA concentration (0–150g ml^–1) resulted in higher levels of transient CAT expression. In jack pine, linearized plasmid gave 2.5 times higher levels of CAT enzyme activity than circular. Optimal voltage varied for each cell line of the two species within the range 200–350 V cm^–1 (960 F). A heat shock treatment of protoplasts for 5 min at 45 °C resulted in enhanced CAT gene expression for both species.NRCC No. 30491     

Electroporation and PEG delivery of DNA into maize microspores

Summary The ability to deliver and detect reporter gene activity in maize microspores was tested. Tested expression vectors contained the chloramphenicol acetyl transferase (CAT) gene and one of the following promoter-intron combinations: 1) cauliflower mosaic virus (CaMV 35S), 2) CaMV 35S + maize alcohol dehydrogenase 1 intron 6 (Adh1-I6), 3) maize alcohol dehydrogenase 1 + intron 1 (Adh1-I1), or 4) maize ubiquitin 1 + intron 1 (Ubiq 1-I1) promoter + intron. The expression vectors were delivered into maize microspores using electroporation or polyethylene glycol (PEG). Both methods were effective for delivering free DNA into microspores. Although all four promoters were active in maize protoplasts, only two promoters were active in maize microspores. The CaMV 35S and the Adh1 promoters did not promote gene expression in maize microspore. The CaMV 35S + Adh1-I6 and Ubiq1-I1 promoters produced high levels of CAT activity in maize microspores.     

Genetic transformation of cauliflower (Brassica oleracea var. botrytis) by direct DNA uptake into mesophyll protoplasts

Mesophyll protoplasts of Brassica oleracea var. botrytis were successfully transformed using polyethylene glycol (PEG). The success of plant transformation depended on both gene transfer and plant regeneration. Parameters, such as PEG and vector concentrations and heat shock conditions were tested in experiments on transient expression of the β -glucuronidase (EC 3.2.1.31) gene and the most suitable conditions for DNA uptake were determined. Two antibiotic resistance marker genes for neomycin phosphotransferase (EC 2.7.1.95) and hygromycin phosphotransferase (EC 2.7.1.104), and three vector plasmids with different lengths were used to obtain stable transformants.     

Liposome-mediated introduction of the chloramphenicol acetyl transferase (CAT) gene and its expression in tobacco protoplasts

The expression plasmid vector pUC8CaMVCAT, containing the chloramphenicol acetyl transferase (CAT) gene, was encapsulated in large unilamellar vesicles (LUV) and introduced into tobacco protoplasts derived from either cell suspension culture or leaf mesophyll. Treatment with liposomes took place in a buffer containing either NaCl or CaCl₂, but no polyethylene glycol. The presence of polylysine in the incubation buffer increased the adsorption of liposomes to protoplasts but decreased the efficiency of CAT gene expression.The expression of the introduced CAT gene could be monitored for at least seven days, following the treatment (about 25% acetylation at day 3 as well as at day 7). Plasmid DNA sequences could be detected, apparently unmodified, for at least nine days in the plant cells, though unintegrated in the host genome.     

Optimization of polyethylene glycol mediated transient gene expression in pea protoplasts

We have investigated factors influencing polyethylene glycol mediated DNA uptake and ß-glucuronidase expression in pea (Pisum sativum L.) protoplasts. It was found that for optimal \-glucuronidase expression the molecular weight and concentration of polyethylene glycol should be 4000 and 20%, respectively. The amount of plasmid DNA should be 25 g per 5×10⁵ protoplasts in each treatment, and the concentration of Mg²⁺ in the transformation buffer should be 15 mM. The optimized protocol was applicable to all four pea cultivars tested.Abbreviations FDA fluorescein diacetate - GUS ß-glucuronidase - MU 4-methylumbelliferone - MUG 4-methyl umbelliferyl glucuronide - MW molecular weight - PEG polyethylene glycol - X-Gluc 5-bromo-4-chloro-3-indolyl glucuronide     

Preparation of leaf mesophyll protoplasts for transient gene expression in Brachypodium distachyon

Transient gene expression systems using protoplasts have been widely used for rapid functional characterization of genes in many plant species. Brachypodium distachyon has recently been employed as a model plant for studies on biofuel grass species and grass crops because of its small genome size, short life-span, and availability of efficient transformation systems. Here, we report the an efficient protocol for the preparation of leaf mesophyll protoplasts from Brachypodium seedlings. We also modified the polyethylene glycol (PEG)-mediated transformation procedure to optimize experimental conditions, such as duration of enzyme digestion, PEG incubation time, and plasmid DNA concentration and size. The green fluorescence protein (GFP)- and ??-glucuronidase (GUS)-coding genes were used as reporters to evaluate the feasibility of this transient expression system. We found that the yield of viable protoplasts was highest after 3 h of enzymatic digestion. Viability of enzyme-digested protoplasts was moderately maintained up to 24 h in Mmg preincubation solution. In addition, the highest transient expression of reporter genes was obtained when protoplasts were transformed with 20 ??g of plasmid DNA and incubated for 16 h. Together with the recent completion of the Brachypodium genome sequence, the Brachypodium transient expression system using leaf mesophyll protoplasts can be widely used for cellular, molecular, and biochemical studies of genes involved in carbon metabolism and signaling pathways mediating intrinsic and environmental cues.     

Protoplast-mediated gene transfer into human leukemia (K562) cells

We have examined the usefulness of a protoplast fusion technique as a tool to transfer cloned genes into hematopoietic cells. Protoplasts carrying cloned plasmids, which would express specific markers when successfully transfected into human cells, were prepared and fused with human leukemic cell line K562 cells using polyethylene glycol as a fusogenic factor. As a result, K562 cells fused with protoplasts containing a plasmid pSV2-cat constructed to code for chloramphenicol acetyltransferase (CAT) expressed CAT activity efficiently. K562 cells were also readily transformed to geneticin-(G418) resistant cells following fusion with protoplasts carrying a plasmid pSV2-neo-SV-gpt, which confers the resistance of mammalian cells to G418 and mycophenolic acid. It was also demonstrated that the plasmid genome was stably integrated into the chromosomal DNA of G418-resistant K562 cells. Our results proved that protoplast fusion could be used to study the specific expression and the biologic activities of cloned genes in human hematopoietic cells.     

A Comparison of Methods for Plasmid Delivery into Plant Protoplasts

Three methods of plasmid delivery to mesophyll protoplasts ofNicotiana tabacum cv. Xanthi have been evaluated. Specifically;a) chemically stimulated uptake of isolated plasmid, b) deliveryof plasmid encapsulated in liposomes, and c) fusion of plasmid-containingspheroplasts, were combined with divalent cation (Ca²⁺ and Mg²⁺)or polyalcohol [polyethylene glycol (PEG) and polyvinyl alcohol(PVA)] treatments. The quantity and quality of plasmid associatedwith intact protoplasts, was assessed by DNA-DNA blot hybridisationanalysis, following stringent washing to separate intact protoplastsfrom non-viable protoplasts and debris. Treatments which increasedassociation of plasmid with protoplasts decreased protoplastviability. Optimum association of plasmid with protoplasts,in the context of acceptable loss of viability, was achievedwhen protoplasts were interacted with either naked plasmid orliposomeencapsulated DNA in the presence of 15% w/v PEG 6000,or with Escherichia coli spheroplasts containing chloramphenicol-amplifiedplasmid in the presence of 25% w/v PEG 6000. Divalent cationsdid not stimulate significant plasmid delivery without unacceptableloss of protoplast viability. Strategies to further increasethe efficiency of plasmid delivery are discussed. (Received June 21, 1984; Accepted August 20, 1984)