Showing papers on "Electroporation published in 1989"
TL;DR: In this article, an efficient method for genetic transformation of lactococci by electroporation is presented, where highly competent cells for electrotransformation were obtained by growing cells in media containing high concentrations of glycine and 0.5 M sucrose as the osmotic stabilizers.
Abstract: An efficient method for genetic transformation of lactococci by electroporation is presented. Highly competent lactococci for electrotransformation were obtained by growing cells in media containing high concentrations of glycine and 0.5 M sucrose as the osmotic stabilizers. These cells could be stored at -85 degrees C without loss of competence. With Lactococcus lactis subsp. cremoris BC101, a transformation frequency of 5.7 x 10 transformants per mug of pIL253 DNA was obtained, which represents 5% of the surviving cells. All the lactococcal strains tested could be transformed by the present method.
1,030 citations
01 Jan 1989
TL;DR: Cells in Electric Fields: Physical and Practical Electronic Aspects of Electro Cell Fusion and Electroporation and External Electric Field-Induced Transmembrane Potentials in Biological Systems: Features, Effects, and Optical Monitoring.
Abstract: 1 Dielectrophoresis and Rotation of Cells.- 2 Cellular Spin Resonance.- 3 Dielectrophoresis: Behavior of Microorganisms and Effect of Electric Fields on Orientation Phenomena.- 4 The Relaxation Hysteresis of Membrane Electroporation.- 5 Electrical Breakdown of Lipid Bilayer Membranes: Phenomenology and Mechanism.- 6 Stochastic Model of Electric Field-Induced Membrane Pores.- 7 Theory of Electroporation.- 8 Leaks Induced by Electrical Breakdown in the Erythrocyte Membrane.- 9 Electroporation of Cell Membranes: Mechanisms and Applications.- 10 Electrofusion Kinetics: Studies Using Electron Microscopy and Fluorescence Contents Mixing.- 11 Electrofusion of Lipid Bilayers.- 12 Role of Proteases in Electrofusion of Mammalian Cells.- 13 Electrofusion of Mammalian Cells and Giant Unilamellar Vesicles.- 14 Cell Fusion and Cell Poration by Pulsed Radio-Frequency Electric Fields.- 15 The Mechanism of Electroporation and Electrofusion in Erythrocyte Membranes.- 16 Producing Monoclonal Antibodies by Electrofusion.- 17 Generation of Human Hybridomas by Electrofusion.- 18 Gaining Access to the Cytosol: Clues to the Control and Mechanisms of Exocytosis and Signal Transduction Coupling.- 19 Gene Transfer by Electroporation: A Practical Guide.- 20 Electropermeabilization and Electrosensitivity of Different Types of Mammalian Cells.- 21 Molecular Genetic Applications of Electroporation.- 22 Plant Gene Transfer Using Electrofusion and Electroporation.- 23 Electric Field-Induced Fusion and Cell Reconstitution with Preselected Single Protoplasts and Subprotoplasts of Higher Plants.- 24 Critical Evaluation of Electromediated Gene Transfer and Transient Expression in Plant Cells.- 25 Transformation Studies in Maize and Other Cereals.- 26 Cells in Electric Fields: Physical and Practical Electronic Aspects of Electro Cell Fusion and Electroporation.- 27 External Electric Field-Induced Transmembrane Potentials in Biological Systems: Features, Effects, and Optical Monitoring.
798 citations
TL;DR: The production of fertile transgenic rice plants obtained by introducing the bacterial hph gene, encoding hygromycin B resistance12 (Hmr), into protoplasts of Oryza sativa (L.) by electroporation is reported.
Abstract: THE generation of transgenic plants using gene transfer techniques is important to both the investigation of gene regulation and the genetic engineering of crops1 The Ti plasmid of Agrobacterium tumefaciens is now routinely used to transform dicotyledonous plants2, and the transfer of foreign genes to unorganized tissue3–6and plants7,8 has been accomplished using direct DNA transfer methods9–11 A protocol for the easy and reproducible production of fertile transgenic cereals, however, has not yet been described We report here the production of fertile transgenic rice plants obtained by introducing the bacterial hph gene, encoding hygromycin B resistance12 (Hmr), into protoplasts of Oryza sativa (L) by electroporation The non-selectable gene encoding β-glucuronidase was also transferred with the hph gene and its expression was detected in the progeny of the stable transformant
653 citations
Patent•
30 Mar 1989TL;DR: An electrical process for enhancing and/or controlling transport of molecules across tissue such as human and animal skin is disclosed in this article, which involves the use of a high voltage, short duration electrical pulses on the tissue surface to produce electroporation.
Abstract: An electrical process for enhancing and/or controlling transport of molecules across tissue such as human and animal skin is disclosed. The process involves the use of a high voltage, short duration electrical pulses on the tissue surface to produce electroporation. Once this effect has occurred, concentration, pressure or temperature gradients, or iontopheresis can be used to move molecules across the tissue. The process can be repeatedly applied without producing undesirable tissue damage or can be used to purposely cause highly limited tissue damage for the purpose of providing a desired, relatively long term molecular transport pathway. The occurrence of the electroporation effect can be detected by monitoring the tissue for a reversible electrical breakdown, which, along with an enhanced tissue permeability, is the characteristic effect of electroporation.
327 citations
321 citations
300 citations
TL;DR: An electroporation method is developed which is more than three orders of magnitude more efficient than a similar method described recently, allowing the transfer of the binary plasmid vector pDG12Sa into A. tumefaciens strain LBA 4404 and preliminary experiments indicate, that this method is equally efficient in the transformation of A. rhizogenes strains.
Abstract: We developed an electroporation method which is more than three orders of magnitude more efficient than a similar method described recently (1), allowing the transfer of the binary plasmid vector pDG12Sa (2) into A. tumefaciens strain LBA 4404 (3). Preliminary experiments (data not shown) indicate, that this method is equally efficient in the transformation of A. tumefaciens and A. rhizogenes strains, both for binary and for cointegrative vectors. Bacteria are inoculated with 1/100 vol. of an overnight culture in 300 ml LB or YMB medium (GIBCO-BRL) resp. Incubation under shaking is continued until an OD^QQ of ca. 0.5 is reached. Bacteria are chilled on ice, harvested by centrifugation, washed three times in 10 ml of lmM HEPES pH7.0, once in 10% glycerol, finally suspended in 3 ml 10% glycerol, aliquoted (0.2 ml aliquots), shock frozen in a -70*C methanol bath and stored in a -70*C freezer. The final cell densities obtained are usually 2.5 5 x 10/ml. For electroporation, cells are thawed on ice and 200 ng plasmid DNA are added. 40 Ml of this suspension are placed in an electroporation cuvette with an electrode distance of 0.2 cm (Bio-Rad). A single electric pulse of 2.5kV initial voltage using the 25 nF capacitor (Gene Pulser, Bio-Rad) is applied immediately. After the pulse, cells are immediately transferred into 0.8 ml of SOC (GIBCO-BRL) or YMB medium and incubated at 28*C for one hour. Aliquots are then spread on LB (or YMB) plates containing 50jjg/ml kanamycin sulfate. After incubation at 28*C for 48 hours, colonies are counted and DNA extracted from randomly selected clones analyzed using restriction enzymes. Typically, a transformation efficiency of 1 1,5 x 10 colonies/jig plasmid is obtained. Transformation of fully expanded leaves or of sterile shoot cultures of Nicotiana clevelandii with Agrobacterium SP. transformed by either conventional methods or by electroporation showed no qualitative or quantitative difference.
245 citations
TL;DR: It is demonstrated for the first time that a minimum polyadenylated tail length of 25 adenylate residues is sufficient to substantially increase the expression and half-life of the reporter mRNA in plants.
Abstract: In this paper we demonstrate that RNA sequences present upstream and downstream of a reporter gene coding region play an important role in determining the amount of protein produced from an mRNA. A translational enhancer, omega, derived from tobacco mosaic virus, when present at the 5'-end of beta-glucuronidase mRNA increased the efficiency of translation 16-fold to 18-fold in electroporated tobacco or carrot protoplasts, and threefold to 11-fold in maize or rice protoplasts. The presence of omega did not alter the half-life of the mRNA in vivo. We also demonstrate for the first time that a minimum polyadenylated tail length of 25 adenylate residues is sufficient to substantially increase the expression and half-life of the reporter mRNA in plants. When in vitro-produced mRNAs were synthesized such that extra sequence was added to the 3'-end of the poly(A) tail, however, the final level of expression was decreased up to 80%. Omega, the translational enhancer, and a poly(A) tail function independently of each other; their combined effect on translation, when both are present in an mRNA, is the multiplication of their individual effects. Histochemical analysis for the presence of beta-glucuronidase in tobacco established that virtually all viable cells receive mRNA during electroporation. Video image analysis of tobacco protoplasts electroporated with luciferase mRNA demonstrated that there is a wide range in the level of expression of this marker. Carrier RNA, when present during electroporation, had only a modest effect on increasing mRNA uptake. Reporter mRNA expression in electroporated protoplasts was directly proportional to the input mRNA up to at least 30 micrograms/ml.
229 citations
01 Jan 1989
TL;DR: The relative ease of obtaining transformed cells and, in certain species, fully developed, fertile transgenic plants at high frequencies have greatly facilitated the study of plant gene regulation and expression.
Abstract: The field of plant gene transfer technology has experienced a spectacular breakthrough during the recent years. From the early 1980s to date, cells of several plant species have been successfully transformed with a wide variety of techniques. The most frequently used method is to exploit the natural capability of Agrobacterium tumefaciens to transfer a segment of DNA into the genomes of most dicotyledonous plants. To overcome the difficulties for incorporating foreign DNA sequences into monocots via A. tumefaciens, direct DNA transfer methods (i.e. electroporation, microinjection, Ca2+ and PEG-mediated uptake of naked DNA) have been also developed. The relative ease of obtaining transformed cells and, in certain species, fully developed, fertile transgenic plants at high frequencies have greatly facilitated the study of plant gene regulation and expression.
193 citations
TL;DR: High-frequency electroporation of whole Corynebacterium glutamicum cells without enzymatic pretreatment was achieved under optimized conditions concerning growth stage, washing of cells, cell concentration and pulse parameter transformation efficiencies of far more than 10 transformants per microgram pWST4B plasmid DNA were reached.
Abstract: High-frequency electroporation of whole Corynebacterium glutamicum cells without enzymatic pretreatment was achieved. Under optimized conditions concerning growth stage, washing of cells, cell concentration and pulse parameters transformation efficiencies of far more than 107 transformants per μg pWST4B plasmid DNA were reached. Using electroporation, linearised and subsequently religated plasmid as well as chimeric ligase reaction products were directly introduced into C. glutamicum with reasonable efficiencies. Electrotransformation efficiency was reduced about 105-fold for plasmid DNA cycled through E. coli JM83. Restriction deficient mutants of C. glutamicum were isolated which could be efficiently transformed with foreign DNA.
168 citations
TL;DR: This protocol, based on high-voltage electro-transformation (electroporation) in the presence of polyethylene glycol, allows introduction of plasmid DNA in most of the Bacillus thuringiensis strains tested.
Abstract: A simple and reliable method of transforming Bacillus thuringiensis is described. This protocol, based on high-voltage electro-transformation (electroporation) in the presence of polyethylene glycol, allows introduction of plasmid DNA in most of the Bacillus thuringiensis strains tested. Efficiencies vary between 102 and 105 transformants per μg DNA, depending on the strain or the replicon used.
TL;DR: It is demonstrated that electroporation can be employed successfully for large scale transfer of antibodies into cultured mammalian cells for the study of cellular metabolism.
TL;DR: The alterations in growth and differentiation of the cells appear to reflect the progressive oncogenic processes that result in cervical carcinoma in vivo.
Abstract: The human papillomaviruses (HPVs) are associated with specific benign and malignant lesions of the skin and mucosal epithelia. Cloned viral DNAs from HPV types 6b, 16, and 18 associated with different pathological manifestations of genital neoplasia in vivo were introduced into primary human cervical epithelial cells by electroporation. Cells transfected with HPV16 or HPV18 DNA acquired indefinite lifespans, distinct morphological alterations, and anchorage-independent growth (HPV18), and contain integrated transcriptionally active viral genomes. HPV6b or plasmid electroporated cells senesced at low passage. The alterations in growth and differentiation of the cells appear to reflect the progressive oncogenic processes that result in cervical carcinoma in vivo.
TL;DR: A simple and reproducible assay for DNA-mediated transfection in thetrypanosomatid protozoan Leptomonas seymouri has been developed and provides a method for defining transcriptional control elements in trypanosomes.
Abstract: A simple and reproducible assay for DNA-mediated transfection in the trypanosomatid protozoan Leptomonas seymouri has been developed. The assay is based on expression of the Escherichia coli chloramphenicol acetyl transferase (CAT) gene flanked by Leptomonas DNA fragments that are likely to contain necessary elements for gene expression in trypanosomes. After electroporation of cells in the presence of plasmid DNA, CAT activity was detected in crude cell lysates. No activity was detected when the orientation of the L. seymouri mini-exon sequence (placed upstream of the CAT gene) was reversed, or in additional control experiments. This system provides a method for defining transcriptional control elements in trypanosomes.
TL;DR: A reliable and efficient method has been developed for the electroporation-mediated transformation of Clostridium perfringens with plasmid DNA, and cells harvested early in the logarithmic stage of growth were transformed more efficiently than cells at other growth phases.
Patent•
20 Apr 1989TL;DR: An improved method, employing electroporation, for producing novel recombinant host cells characterized by stably integrated foreign DNA at high copy number was proposed in this paper, which is useful in the efficient, large-scale production of recombinant proteins and polypeptides.
Abstract: An improved method, employing electroporation, for producing novel recombinant host cells characterized by stably integrated foreign DNA at high copy number. These recombinant host cells are useful in the efficient, large-scale production of recombinant proteins and polypeptides.
TL;DR: Transfection by electroporation using square wave pulses, as opposed to exponentially decaying pulses, was found to be significantly increased by repetitive pulses, which improve the ability to obtain high efficiency gene transfer into many mammalian cell types.
TL;DR: Cell electroporation is used with great success to permeabilize Chinese hamster ovary cells for the introduction of restriction enzymes, resulting in extremely high frequencies (greater than 90%) of aberrant metaphase cells and also a dramatic decrease in cell survival, as measured by subsequent colony formation.
Abstract: Restriction endonucleases that make either blunt- or cohesive-end DNA double-strand breaks can induce chromosome aberrations. We have used cell electroporation with great success to permeabilize Chinese hamster ovary cells for the introduction of restriction enzymes. The introduction of restriction enzymes by this method resulted in extremely high frequencies (> 90%) of aberrant metaphase cells and also a dramatic decrease in cell survival, as measured by subsequent colony formation. Cell electroporation by itself caused no increase in aberrant chromosomes and had only a slight effect on cell survival.
TL;DR: Although pPH33 itself was non-transmissible by conjugation, it could be mobilized from Escherichia coli, using the transfer function of the IncP plasmid pRK2013, into P. haemolytica at a frequency of 0.3-2.2 x 10(-3) per recipient cell.
Abstract: Summary: Three broad-host-range plasmids (pRK290, pSa4 and pKT230) and one native Pasteurella haemolytica plasmid (pPH33) were used in transformation experiments with P. haemolytica strains T179 (serotype Al), Y216 (serotype A2)and its capsular-deficient variant Y216/NS1. No transformants were detected with either heat-shock or freeze-thaw techniques. However, by electroporation, all P. haemolytica strains were transformed by pPH33 but not by pRK290 or pSa4. The highest frequency obtained was 91 × 104 transformants per μg of pPH33 DNA with P. haemolytica strain Y216/NS1. Although pPH33 itself was non-transmissible by conjugation, it could be mobilized from Escherichia coli, using the transfer function of the IncP plasmid pRK2013, into P. haemolytica at a frequency of 0·3-2·2 × 10−3 per recipient cell.
TL;DR: There is disclosed a procedure for DNA-mediated transformation of Saccharomyces cerevisiae by electroporation utilizing lithium acetate and dithiothreitol to weaken cell wall structure resulting in increased transformation efficiency.
Abstract: A method for introducing heterologous DNA into Saccharomyces cerevisiae rapidly and efficiently by electroporation was developed. Transformant colonies appeared somewhat sooner than by the LiCl or spheroplast transformation method, and the time spent in manipulation was much less than for these two methods. The pores in the cell membrane formed by the high voltage of electroporation were resealed within 6 to 7 min after electroporation. At a capacitance of 25 microF, the optimum voltage was 2.0 to 2.25 kV/cm. Log-phase cells concentrated to 10 to 20 units at an optical density of 600 nm in 200 microliters of fresh rich medium and electroporated at 2.25 kV/cm in the presence of 0.1 microgram of supercoiled plasmid DNA will yield 1,000 to 4,500 colonies per microgram of DNA.
TL;DR: The filamentous cyanobacterium Anabaena sp.
Abstract: The filamentous cyanobacterium Anabaena sp. strain M131 was transformed with the shuttle vector pRL6 by electroporation. Optimum conditions for electroporation required relatively high field strengths with short time constants. Restriction significantly lowered the efficiency of transformation. A plasmid containing a single unmodified AvaII restriction site transformed cells with about 100-fold-lower efficiency than did the same plasmid with a modified restriction site.
TL;DR: Protoplasts from adult leaves of lettuce are amenable to direct transformation under the same general conditions as those employed for tobacco protoplasts and provide new opportunities in some specific strategies for lettuce breeding.
Abstract: Transgenic plants of lettuce (Lactuca sativa) obtained through electroporation of protoplasts
TL;DR: Direct DNA uptake is applicable to those plants not amenable to Agrobacterium transformation, particularly monocotyledons, and utilises a range of vectors, including those employed for gene cloning.
Abstract: 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.
TL;DR: Electroporation is a very efficient method for introducing foreign DNA into human lymphoid cell lines by introducing stably the p40 gene of human T-cell leukemia virus type I (HTLV-I) and high frequencies of stable transformations were obtained.
Abstract: Conditions were developed for stable introduction of foreign DNA into human lymphoid cell lines by electroporation. To introduce stably the p40 gene of human T-cell leukemia virus type I (HTLV-I) into the human lymphoid cell line Jurkat, the p40 expressing plasmid, pMAXRHneo-1, which carries the neo resistant gene, was transfected into Jurkat cells at a voltage of 2500 V and capacitance of 21.7 microF, and stable transformants were screened for neo (G418) resistance. The frequency of transformants was more than one per 2 x 10(5) cells used initially. Clones that were resistant to G418 were shown to have the p40 gene integrated into the host genome and to express mRNA and protein from the introduced plasmid. Expression of p40 in the transformed Jurkat cells was also confirmed by testing the trans-activating effect of HTLV-I enhancer by p40. High frequencies of stable transformations of 10(-4) to 10(-6) were also reproducibly obtained by electroporation of the human T cell lines HSB-2 and TALL-1, a human B cell line Raji, a human monocytic cell line U937, and a human erythroleukemia cell line K562. These results demonstrate that electroporation is a very efficient method for introducing foreign DNA into human lymphoid cell lines.
TL;DR: The usefulness of the electroporation method for the introduction of cloned toxin genes, in either their native or modified form, into a variety of host strains is demonstrated.
Abstract: Electroporation was used as a method to transform intact cells of Bacillus thuringiensis and B. cereus. With our optimized method a range of plasmid vectors could be transformed into strains of B. thuringiensis at frequencies of up to 107 transformants/μg DNA. This high frequency allows cloning experiments to be bone directly in B. thuringiensis. A bifunctional vector capable of replicating in Escherichia coli and in Bacillus spp. was constructed. The kurhd1 protoxin gene was cloned into this shuttle vector to produce plasmid pXI93, then transformed into B. thuringiensis HDl cryB and B. cereus 569K. The cloned protoxin gene was expressed in sporulating cultures of both strain HD1 cryB (pXI93) and 569K (pXI93), producing crystal protein active in biotests against larvae of Heliothis virescens. This demonstrates the usefulness of the electroporation method for the introduction of cloned toxin genes, in either their native or modified form, into a variety of host strains.
TL;DR: Electroporation; Molecular transport; Number of molecules; Flow cytometry; Yeast
TL;DR: Although the method was primarily developed for transient expression it was also found to improve stable expression, and should have wide applicability, particularly in studies seeking to identify DNA sequences that lead to inhibition of DNA synthesis and proliferation in human fibroblasts and other cells refractory to transfection.
Abstract: To achieve high transfection efficiency in human fibroblasts with good preservation of proliferative capacity we developed an electroporation procedure that combines two distinct modalities: use of recipient cells synchronized in the late G2/mitotic phase of the cell cycle and treatment of cells post-electroporation with 5 mM butyrate. This combination enabled reduction of plasmid DNA concentration and electroporation voltage, both associated with cytotoxicity, while greatly enhancing transfection efficiencies. Although the method was primarily developed for transient expression it was also found to improve stable expression. This procedure should have wide applicability, particularly in studies seeking to identify DNA sequences that lead to inhibition of DNA synthesis and proliferation in human fibroblasts and other cells refractory to transfection.
TL;DR: Using a colorimetric reaction, the GUS enzyme was found to be localized in vascular tissues, demonstrating the similar expression of the roIC gene promoter in monocots and dicots.
Abstract: Transgenic rice plants were obtained from protoplasts treated with two plasmids by electroporation. Primary transformants were selected on the basis of resistance to hygromycin, conferred by one of the co-transferred plasmids. Out of 26 hygromycin-resistant plants 2 showed the reporter gene activity due to another plasmid possessing a chimeric gene consisting of the promoter (about 900 by upstream non-coding region) of the ORF12 gene (roIC of the Ri plasmid and the coding region for β-glucuronidase (GUS). Using a colorimetric reaction, the GUS enzyme was found to be localized in vascular tissues, demonstrating the similar expression of the roIC gene promoter in monocots and dicots (Sugaya et al. 1989; Schmulling et al. 1989).
TL;DR: Very successful transformation of plasmids into Corynebacterium glutamicum, Brevibacterium lactofermentum and several other species of these coryneform bacteria is reported.
Abstract: High Frequency Transformation of Whole Cells of Amino Acid Producing Coryneform Bacteria Using High Voltage Electroporation
TL;DR: Transformation of untreated vegetative cells of Bacillus cereus 569 with plasmid pC194 by high-voltage electroporation resulted in a maximum of 2 x 10(-5) transformants per viable cell, obtaining transformant colonies in 14 to 24 h.
Abstract: Transformation of untreated vegetative cells of Bacillus cereus 569 with plasmid pC194 (1.8 megadaltons) by high-voltage electroporation resulted in a maximum of 2 x 10(-5) transformants per viable cell. Transformation of a 130-megadalton plasmid occurred at a comparable frequency. The method was simple, rapid, and yielded transformant colonies in 14 to 24 h. Transformation was obtained with unpurified total plasmid DNA.