Rapid optimization of electroporation conditions for plant cells,protoplasts, and pollen

The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfer experiments is critical to achieve high levels of gene expression in transformed plant cells. Two stains, trypan blue and fluorescein diacetate, have been applied to optimize electroporation conditions for three plant cell types, using different square wave and exponential wave electroporation devices. The different cell types included protoplasts from tobacco, a stable mixotrophic suspension cell culture from soybean with intact cell walls, and germinating pollen from alfalfa and tobacco. Successful electroporation of each of these cell types was obtained, even in the presence of an intact cell wall when conditions were optimized for the electroporation pulse. The optimal field strength for each of these cells differs, protoplasts having the lowest optimal pulse field strength, followed by suspension cells and finally germinating pollen requiring the strongest electroporation pulse. A rapid procedure is described for optimizing electroporation parameters using different types of cells from different plant sources.     

Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells

Transient expression of fluorescent fusion proteins in plant cells has dramatically facilitated our study of newly identified genes and proteins. This protocol details an in vivo transient expression system to study the subcellular localization and dynamic associations of plant proteins using protoplasts freshly prepared from Arabidopsis or tobacco BY-2 suspension cultured cells. The method relies on the transformation of DNA constructs into protoplasts via electroporation. The whole protocol is comprised of three major stages: protoplast generation and purification, transformation of DNA into protoplasts via electroporation and incubation of protoplasts for protein analysis. Similar to stably transformed cell lines, transformed protoplasts are compatible with protein localization studies, pharmaceutical drug treatment and western blot analysis. This protocol can be completed within 11-24 h from protoplast production to protein detection.     

Direct DNA transfer to plant cells

A range of somatic cell and molecular techniques are now available to supplement conventional plant breeding. The introduction and expression of foreign DNA has been used to modify basic aspects of physiology and development, to introduce commercially important characteristics such as herbicide and insect resistance into plants and to insert genes suitable as dominant selectable markers for somatic hybridisation. Several techniques for direct DNA delivery are available, ranging from uptake of DNA into isolated protoplasts mediated by chemical procedures or electroporation, to injection and the use of high-velocity particles to introduce DNA into intact tissues. Direct DNA uptake is applicable to both stable and transient gene expression studies and utilises a range of vectors, including those employed for gene cloning. Although the frequency of stable transformation is low, direct DNA uptake is applicable to those plants not amenable to Agrobacterium transformation, particularly monocotyledons.     

Biolistic plant transformation

The biolistic process represents a completely new approach to the problem of how to deliver DNA into intact cells and tissues. High velocity microprojectiles are used to carry DNA or other substances past cell walls and membranes. Because DNA is being 'shot' into cells, it represents a type of biological ballistics, hence the term "biolistics".
There are several fundamental advantages to the biolistic process over other plant transformation techniques. The biolistic process appears to be effective regardless of species or tissue type, it is a rapid and very simple procedure, and it should facilitate the direct transformation of totipotent tissues such as pollen, embryos, meristems and morphogenic cell cultures. In addition, the biolistic process appears to be uniquely suitable for organelle transformation.
The disadvantages of the biolistic process are that it requires special instrumentation, and is still in the early stages of its development. Consequently, delivery efficiencies are still not as high as can be achieved in highly optimized transformation systems such as electroporation or agrobacterial-infection of tobacco. Furthermore, potential users should be prepared to spend some time adapting existing protocols to their specific species or tissue of interest.     

Introduction and expression of DNA molecules in eukaryotic cells by electroporation

Exposing eukaryotic cells to brief, high voltage electrical fields can induce the uptake of exogenous materials, presumably through the transient formation of micropores in the cell membrane. This phenomenon has been exploited for the introduction of cloned DNA molecules into cells for permanent transformation or for transient expression and analysis of gene products. The magnitude and characteristics of the generated electrical field are critical for successful electroporation and simple, transient expression assays using indicator genes allow the calibration and optimization of electroporation conditions for a wide variety of eukaryotic cell types. These techniques may allow the genetic modification of a variety of host cells which cannot be easily transformed by other methods.     

Highly efficient transformation of intact yeast-like conidium cells of Tremella fuciformis by electroporation

Tremella fuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. So it is a good recipient cell for exogenous gene expression. In this study, two expression vectors pGlg-gfp containing gpd-Gl promoter and gfp gene and pGlg-hph containing gpd-Gl promoter and hph gene were constructed. The lowest sensitive concentration of hygromycin for the blastospore was determined on three types of media. Our ex- periments showed that the lowest sensitive concentration of hygromycin for the blastospore was 5 μg/mL on MA medium. The intact blastospores were transformed with the expression vector pGlg-hph by electroporation. The putative transformants were obtained by the MA selective medium. Experi- mental results showed that the most effective parameters for the electroporation of intact blastospores were obtained by using STM buffer, 1.0×108 cells/mL of blastospores, 200 μL in transformation volume, 6 μg plasmid, 2.0 kV/cm of electric pulse voltage, stillness culturing on MB liquid medium for 48 h after electroporation. In these transformation conditions, the efficiency reached 277 colonies/μg DNA. Co-transformation of plasmid pGlg-gfp and pGlg-hph with ratio of 1:1 was performed by electroporation with the optimal parameters. The putative co-transformants were obtained by the MA selective medium. Eight randomly selected colonies from the vast putative co-transformants were analyzed by PCR de- tection and Southern blotting. The experiments showed that the gfp was integrated into the genomes of three transformants. The co-transformation efficiency was 37.5%. Green fluorescence was observed under laser scanning confocal microscope in these gfp positive transformants. This indicates that the exogenous gfp can be expressed effectively in the yeast-like conidia of T. fuciformis.     

Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells

This report describes the delivery of plasmid DNA containing either the β-glucuronidase (GUS) or the green fluorescent protein (GFP) reporter gene into intact plant cells of bamboo callus, lilium scales, and Nicotiana benthamiana suspension culture cells. By first plasmolyzing the tissues or cells with 0.4 m sucrose in the presence of plasmid DNA, electroporation effectively delivers plasmid DNA into the intact plant cells. Transient expression of the GUS gene, as revealed by histochemical assays, showed the presence of blue-staining areas in the electroporated tissues. A short exposure of cells to 2% DMSO (dimethyl sulfoxide) prior to plasmolysis elevated the level of transient GUS activity. When plasmid DNA containing a synthetic GFP gene was used, a strong green fluorescence was observed in N. benthamiana suspension culture cells that were subjected to plasmolysis and electroporation. These results suggest that plasmolysis brings the plasmid DNA into the void space that is in close vicinity to the plasmalemma, allowing electroporation to efficiently deliver the plasmid DNA into intact plant cells. Received: 15 June 1998 / Revision received: 18 August 1998 / Accepted: 28 August 1998     

Transformation of Intact Aspergillus niger by Electroporation

A new rapid transformation system for Aspergillus niger that uses electroporation to render intact germinating conidia permeable to DNA is described. The transformant colonies appeared earlier than transformants obtained by the protoplast-forming method. Without pretreatment of the conidia the transformation frequencies were 1.2 colonies per μg of integrative vector and 100 colonies per μg of plasmid DNA. When the conidia were treated with a dilute solution of fungal cell wall lytic enzyme, the frequency of transformation was increased by approx. 2-fold when using two vectors. Southern blot analysis of genomic DNA and restriction endonuclease-digested DNA from a random sample of transformants showed homologous and nonhomologous integration of the integrative vector into the genome, as is also observed with the protoplast-forming method. In transformation with the plasmid vector, the transformant DNA was shown to be mostly maintained in free form with minimal integration into the chromosome when transformed by either intact electroporation or the conventional method.     

Uptake of Al across the plasma membrane of plant cells

Measurements of intracellular, cytosolic Al are plagued with technical difficulties. An accurate quantification of Al uptake into the cytosol relies on the effectiveness of the methods that desorb Al bound to the cell wall. However, published desorption methods are not completely effective in removing cell wall Al. Using giant algal cells of Chara corallina, where a physical separation of the cell wall and the cytosol can be achieved surgically, it was shown that up to 99.99% of the total cellular Al accumulates in the cell wall. Even when 95% of total Al present in intact cells was desorbed, still over 20 times more Al was left in the cell wall than in the cytosol. Therefore, without physical separation of the cell wall and the cytosol, minute amounts of cytosolic Al need to be measured in the considerably larger background of the cell wall Al. Consequently, up to several orders of magnitude lower uptake rates of Al were measured across the plasma membrane of intact Chara cells in comparison to currently available values on higher plant cells (Triticum aestivum, t Glycine max, Phaseolus vulgaris), where at least some of the cell wall Al was attributed to the intracellular, cytosolic Al. Uptake of Al across the plasma membrane of Chara cells occurs without a delay at a very low rate that is directly proportional to Al concentration in the uptake medium. Moreover, residual Al left in the cell wall after desorption can be taken up into the cytosol of Chara cells during subsequent growth in the artificial pond water. For measuring Al uptake into roots of higher plants, the Secondary Ion Mass Spectrometry is the best available technique because it appears to overestimate the cytosolic Al to the lower extent than any other currently used analytical method for determination of Al.     

Electroporation of Haemophilus influenzae is effective for transformation of plasmid but not chromosomal DNA.

Electroporation of plasmid and chromosomal DNAs were tested in Haemophilus influenzae because of an interest in introducing DNA into mutants that are deficient in competence for transformation. The initial experiments were designed to investigate and optimize conditions for electroporation of H. influenzae. Plasmid DNA was introduced into the competence proficient strain Rd and its competence-deficient uptake mutants com-52, com-59, and com-88, and the recombination deficient mutant rec1. Plasmid DNA could also be electroporated into the non-transforming strains Ra, Rc, Re and Rf. Plasmid DNA without sequences that are involved in tight binding (uptake) of DNA by competent cells of H. influenzae Rd was electroporated into both competent and non-competent cells. Competent cells were several orders of magnitude less efficient than non-competent cells for electroporation of plasmid DNAs. Electroporation of H. influenzae chromosomal DNA was not successful. Low levels of integration of chromosomal markers were observed following electroporation and these could be ascribed to transformation. The treatment of cells with DNasel following electroporation separated the effects due to electroporation from those due to transformation. The DNasel treatment did not affect the efficiency of plasmid incorporation, but severely restricted effects due to natural DNA transformation.     

Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism

Transformation (i.e., genetic modification of a cell by the incorporation of exogenous DNA) is indispensable for manipulating fungi. Here, we review the transformation methods for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris and Aspergillus species and discuss some common modifications to improve transformation efficiency. We also present a model of the mechanism underlying S. cerevisiae transformation, based on recent reports and the mechanism of transfection in mammalian systems. This model predicts that DNA attaches to the cell wall and enters the cell via endocytotic membrane invagination, although how DNA reaches the nucleus is unknown. Polyethylene glycol is indispensable for successful transformation of intact cells and the attachment of DNA and also possibly acts on the membrane to increase the transformation efficiency. Both lithium acetate and heat shock, which enhance the transformation efficiency of intact cells but not that of spheroplasts, probably help DNA to pass through the cell wall.     

Flow cytometric quantification of electroporation-mediated uptake of macromolecules into plant protoplasts

Summary Flow cytometry was used to provide a rapid and accurate assessment of electroporation-induced uptake of macromolecules into plant protoplasts. Rice protoplasts were electroporated in the presence of fluorescein isothiocyanate-conjugated dextran (FITC-dextran). After washing, the protoplasts were resuspended in a solution containing propidium iodide which intercalates with DNA, but which is excluded by an intact plasma membrane. Electroporation in the presence of FITC-dextran gave rise to populations of protoplasts that fluoresced green or yellow due to the presence of non-conjugated FITC. Non-viable protoplasts fluoresced red because of their inability to exclude propidium iodide molecules. Flow cytometry was used to resolve and quantify these protoplast populations and thus identify optimal conditions for macromolecule uptake. A direct relationship was observed between FITC-dextran uptake and transient gene expression following plasmid uptake. Thus, simultaneous electroporation of protoplasts with foreign DNA and FITC-dextran followed by fluorescence activated cell sorting may permit partial selection of transformed cells and so reduce the need for a selectable marker.Abbreviations ADC analogue to digital converter - CAT chloramphenicol acetyl transferase (enzyme) - cat chloramphenicol acetyl transferase (gene) - CPW solution cell and protoplast wash solution - DC direct current - EF electrofusion - FALS forward angle light scatter - FITC fluorescein isothiocyanate - FITC-dextran fluorescein isothiocyanate conjugated dextran - PI propidium iodide - PMT photomultipliertube - TLC thin layer chromatography     

Transient and stable electrotransformations of intact black Mexican sweet maize cells are obtained after preplasmolysis

Summary When interested in plant cell transformation, the cell wall is often considered as a barrier to DNA transfer, which is only overcome by wounding or wall degrading enzymes. In this work, we demonstrate that cell plasmolysis before electropulsation is an efficient approach to DNA delivery into intact plant cells. Using such a method, transient expression (-glucuronidase and chloramphenicol acetyltransferase) and stable expression (phosphinotricin acetyltransferase) of exogenous genes are obtained in intact black Mexican sweet maize cells.Abbreviations BMS cells Black Mexican Sweet cells - GUS -glucuronidase - CAT chloramphenicol acetyltransferase - PAT Phosphinotricin acetyltransferase - MS Murashige and Skoog - PCV packed cell volume - 4-MU and 4-MUG 4-methylumbelliferone and 4-methylumbelliferyl-glucuronide - BSA bovine serum albumin - TTC triphenyl tetrazolium chloride     

Direct gene transfer to protoplasts: Fate of the transferred genes

Direct gene transfer to protoplasts is one of several methods developed for the production of transgenic plants. This method utilizes the efficient uptake of DNA from the surrounding medium by protoplasts (cell wall-less plant cells). Where a suitable protoplast system exists large numbers of transformant clones can be efficiently produced and often regenerated to normal fertile plants. This review concentrates on the fate of the DNA which is taken up into the protoplasts. Particular emphasis is given to the factors which can influence the integration and form of the transferred DNA, the expression of transferred genes, and the inheritance in further generations of those genes. The information available suggests (1) that DNA is taken up by a large proportion of the cells in a transformation mixture, (2) that this DNA forms complexes sometimes involving carrier DNA, (3) that fewer cells actually take up DNA into the nucleus, and (4) that the complex may be rearranged and/or amplified and then integrated into the genome. If the DNA is arranged in such a way that a gene can be expressed it does so in a normal manner and is stably inherited both mitotically and meiotically.     

Optimization of electroporation for transfection of mammalian cell lines

Electroporation can be a highly efficient method for introducing DNA molecules into cultured cells for transient expression of genes or for permanent genetic modification. However, effective transformation by electroporation requires careful optimization of electric field strength and pulse characteristics. We have used the transient expression of the firefly luciferase gene as a rapid and sensitive indicator of gene expression to describe the effects on transfection efficiency of altering electroporation field strength and shape. Using the luciferase assay, we investigated the correlation of cell viability with optimal transfection efficiency and determined the optimal parameters for a number of phenotypically distinct mammalian cell lines derived from the nervous and immune systems. The efficiency of electroporation under optimal conditions was compared with that obtained using DEAE-dextran or calcium phosphate-mediated transformation. Transfection by electroporation using square wave pulses, as opposed to exponentially decaying pulses, was found to be significantly increased by repetitive pulses. These methods improve the ability to obtain high efficiency gene transfer into many mammalian cell types.     

The Role of Cell Wall Revealed by the Visualization of Saccharomyces cerevisiae Transformation

Transformation is an indispensable method for the manipulation of Saccharomyces cerevisiae cell. The spf1 cell, in which the gene encoding an endoplasmic reticulum-located P-type ATPase is deleted, has been known to show the high-transformation phenotype. In this study, fluorescent microscopic observation of transformation process of S. cerevisiae using plasmid DNA labelled with fluorescent DNA probe, YOYO-1, suggested that the spf1 cell absorbed more plasmid DNA on cellular surface than did the wild-type cell and the unwashed cell did more plasmid DNA than the washed cell. The amounts of the absorbed DNA correlated with the transformation efficiency (number of transformants per μg plasmid DNA) and frequency (transformation efficiency per viable cell number). The high-transformation phenotype of spf1 cell and the effect of heat shock, which effectively induces the transformation of intact cell, disappeared upon cell wall digestion. Electron microscopic observation of the transformation process using negatively charged Nanogold as a mimic of plasmid DNA supported the result obtained using YOYO-1 and implied that plasmid DNA enters into cell together with membrane structure. These data strongly suggest that during the transformation of intact cell, plasmid DNA is initially absorbed on the cell wall, passes through the cell wall with the aid of heat shock, reaches to the membrane, and enters into the cell together with the membrane structure and that the capacity of the cell wall to absorb DNA is at least one of the determinants of transformation efficiency and frequency.     

Transformation ofHaemophilus influenzae following electroporation with plasmid and chromosomal DNA

Naturally transformable species, such asHaemophilus influenzae, have evolved systems for the efficient uptake and integration of DNA from the surrounding environment. We compared this competence-dependent DNA uptake system to electroporation, which has been widely used in the past few years to introduce DNA into cells, for transformingHaemophilus influenzae. Electroporation improved transformation efficiency when noncompetent cells were used and when DNA lackingHaemophilus-specific uptake sequences was used for transformation of competent cells. An increase in plasmid-to-chromosome recombination was seen when plasmid DNA containing chromosomal inserts was introduced into competent cells by electroporation, as observed previously for natural transformation.     

Electroporation: parameters affecting transfer of DNA into mammalian cells

Electroporation, the reversible breakdown of cell membranes caused by a high-voltage discharge, is a rapid, simple, and efficient method for introducing DNA into mammalian cells. An instrument for electroporation which permits the high-voltage discharge waveform to be varied with respect to rise time, peak voltage, and fall time is described. The uptake and expression of SV40 DNA following electroporation of two cell types, a human carcinoma-derived cell line, HEp-2, and a human lymphoblastoid cell line, 721, depended on the peak voltage and the fall time of the voltage discharge. The electronic parameters which produced optimum DNA transfer, however, differed for the two cell types. DNA as large as 150 kb was introduced into cells by electroporation. Cells can be electroporated in either phosphate-buffered saline or culture medium containing fetal bovine serum, and the efficiency of DNA transfer does not vary with cell densities from 10(6) to 2 X 10(7)/0.5 ml. Exposing the cells to multiple voltage discharges did not improve DNA transfer. DNA has been introduced by electroporation into all cell types tested, including human carcinoma-derived cell lines, human lymphoblastoid cell lines, human fibroblast strains, and primary human lymphocytes. To obtain maximal DNA transfer by this method, however, one must optimize the peak voltage and fall time of the discharge waveform for each cell type.     

Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression

The development of inexpensive and effective approaches to transiently decrease gene expression in vivo would be useful for the study of physiological processes in living animals. DNAzymes are a novel class of DNA oligonucleotides that can catalytically cleave target mRNAs and thereby reduce protein production. However, current methods for their delivery in vivo are limited and inefficient. In this study, we show that electroporation can be used to deliver DNAzymes to the intact mesenteric vasculature of rats. With the use of PKC-epsilon as a target, a set of wild-type and mutant control DNAzymes was designed and shown to reduce both PKC-epsilon mRNA and protein levels in cultured smooth muscle cells in a specific manner. The wild-type DNAzyme reduced PKC-epsilon protein levels by 70% at 24 h in two different cell lines without decreasing the levels of the five other PKC isoforms tested. When delivered to the intact vasculature using electroporation, the DNAzyme reduced PKC-epsilon protein levels by >60% without affecting these other PKC isoforms. Electroporation was required for oligonucleotide transfer and was able to deliver the DNAzymes to multiple cell layers in the vessel wall. Protein levels were reduced maximally by 24 h postelectroporation and returned to normal by 48 h. These results suggest that electroporation can be used to deliver DNAzymes and other DNA oligonucleotides to the vasculature in vivo and can decrease gene expression for a window of time that can be used for experimental studies.     

Plant transformation by microinjection techniques

Several techniques have been developed for introducing cloned genes into plant cells. Vectorless delivery systems such as PEG-mediated direct DNA uptake (e.g. Pasz-kowski et al. 1984), electroporation (e.g. Shillito et al. 1985), and fusion of protoplasts with liposomes (Deshayes et al. 1985) are routinely used in many experiments (see several chapters of this issue). A wide range of plant species, dicotyledonous as well as monocotyledonous, has been transformed by these vectorless DNA transfer systems. However, the availability of an efficient protoplast regeneration system is a prerequisite for the application of these techniques. For cells with intact cell walls and tissue explants the biological delivery system of virulent Agrobacterium species has been routinely used (for review see Fraley et al. 1986). However, the host range of Agrobacterium restricts the plant species, which can be transformed using this vector system. In addition, all these methods depend on selection systems for recovery of transformants. Therefore a selection system has to be established first for plant species to be transformed. The microinjection technique is a direct physical approach, and therefore host-range independent, for introducing substances under microscopical control into defined cells without damaging them. These two facts differentiate this technique from other physical approaches, such as biolistic transformation and macroinjection (see chapters in this issue). In these other techniques, damaging of cells and random manipulation of cells without optical control cannot be avoided so far. In recent years microinjection technology found its application in plant sciences, whereas this technique has earlier been well established for transformation of animal tissue culture cells (Capecchi 1980) and the production of transgenic animals (Brin-ster et al. 1981, Rusconi and Schaffner 1981). Furthermore, different parameters affecting the DNA transfer via microinjection, such as the nature of microinjected DNA, and cell cycle stage, etc, have been investigated extensively in animal cells (Folger et al. 1982, Wong and Capecchi 1985), while analogous experiments on plant cells are still lacking.