Introduction of antibody into viable cells using electroporation

Conditions for labelling an intracellular antigen, p21ras, using electroporation to introduce a fluorescent antibody, are described. Following labelling, cells were evaluated for p21ras associated fluorescence by flow cytometry. Electroporation, sorting, and cell handling parameters were varied to determine optimal conditions for cell viability. Cells were best held in serum containing growth medium both before and after electroporation, while antibody introduction during the electroporation phase was most efficient when carried out in a balanced saline solution. For maximum efficiency of antibody internalization, the antibody needed to be present during electroporation, and medium needed to be replaced several times in the first few hours after electroporation to ensure good cell survival.     

Introduction of impermeable molecules into pollen grains by electroporation

Summary Electroporation was used to introduce plasma membrane impermeable molecules into the cytoplasm of pollen grains ofLilium longiflorum. Ungerminated pollen grains were exposed to the fluorescent dye quin2 or FITC-labelled dextrans and electroporated with exponentially decaying voltage pulses of 250 to 2000 V/cm and time constants of 0.01 to 10 s. The number of electroporated pollen grains increased with the strength and duration of the voltage pulses, and with the osmolarity of the external medium. Optimal results were obtained with pulses of 1000 V/cm and 10 s time constant, and with 900 mM mannitol in the electroporation buffer. The size of the pores produced in the plasma membrane by electroporation allowed uptake of 40 kDa dextran but not 70 kDa dextran. The rate of germination of pollen grains was low immediately after electroporation, but increased with time in pollen growth medium. The conditions of electroporation reported here may be used to load genetic material into pollen grains for the production of transgenic plants.Abbreviations PGM pollen growth medium - FDA fluorescein diacetate - FITC fluorescein isothiocyanate     

Transformation of Escherichia coli with large DNA molecules by electroporation.

We have examined bacterial electroporation with a specific interest in the transformation of large DNA, i.e. molecules > 100 kb. We have used DNA from bacterial artificial chromosomes (BACs) ranging from 7 to 240 kb, as well as BAC ligation mixes containing a range o different sized molecules. The efficiency of electroporation with large DNA is strongly dependent on the strain of Escherichia coli used; strains which offer comparable efficiencies for 7 kb molecules differ in their uptake of 240 kb DNA by as much as 30-fold. Even with a host strain that transforms relatively well with large DNA, transformation efficiency drops dramatically with increasing size of the DNA. Molecules of 240 kb transform approximately 30-fold less well, on a molar basis, than molecules of 80 kb. Maximum transformation of large DNA occurs with different voltage gradients and with different time constants than are optimal for smaller DNA. This provides the opportunity to increase the yield of transformants which have taken up large DNA relative to the number incorporating smaller molecules. We have demonstrated that conditions may be selected which increase the average size of BAC clones generated by electroporation and compare the overall efficiency of each of the conditions tested.     

The loading of human erythrocytes with small molecules by electroporation

The technology for loading the cell with membrane-impermeable substances by means of electroporation consists of the following three stages: (i) the creation of pores permeable for the desired substance; (ii) the introduction of a substance into the cell cytosol; and (iii) the restoration of the membrane barrier function. In this paper, the experimental data on the loading of human erythrocytes with small molecules (molecular weight below 500 Da) is presented. The results obtained show that increasing the intensity of the electric field pulse increases the fraction of electroporated cells. The pores through which the molecules of ascorbic acid and mannitol (radius below 0.5 nm) can enter the erythrocytes appear when the field strength exceeds 2.5 kV/cm. The concentration of ascorbic acid inside the cells increases linearly. At 4 degrees C, the rate of ascorbic acid influx was constant for at least 4 hours. The original permeability of most of the cells towards ascorbic acid and mannitol was restored after about 6-7 min at 37 degrees C, and the characteristic time for complete resealing was about 20-40 min. The procedure described here can be used for loading cells with membrane-impermeable substances.     

Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation.

The fate of exogenous DNA introduced into Chlamydomonas reinhardtii by electroporation was analyzed. With single and double electrical pulses, plasmids as large as 14 kb were introduced into cells with and without intact cell walls. Within hours after introduction, exogenous plasmid DNA was associated with nuclei isolated from cells; several weeks after introduction, exogenous DNA was stably integrated into the Chlamydomonas genome. These studies establish electroporation as a method for introducing DNA, and potentially other molecules, into C. reinhardtii.     

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.     

Introduction of deoxyribonucleoside triphosphates into intact cells by electroporation

A simple method, employing high-voltage electric discharge (electroporation), was developed to introduce phosphorylated nucleosides into the cytoplasm of viable cells. HL-60 leukemia cells permeabilized by this technique remained viable and incorporated deoxyribonucleoside triphosphates into nuclear DNA. Furthermore, DNA synthesis was depressed for at least 24 h in HL-60 cells made permeable to 1-beta-D-arabinosylcytosine 5'-triphosphate by this methodology. Electroporation was found to be applicable to the permeabilization of a wide variety of cell lines in culture to nucleotides, suggesting that this methodology may be useful for the introduction into intact cells of a wide variety of molecules that are not normally transported effectively.     

Introduction of foreign DNA into Chlorella saccharophila by electroporation

Summary Plasmid, pBI221, was introduced into protoplasts of Chlorella saccharophila c-211-1a prepared from the cells in the stationary phase by electroporation. Transient expression of the introduced plasmid was observed under a field strength of between 600 and 900 V/cm, and a pulse duration of around 400 ms, where high membrane permeability to 70-kDa FITC-dextran was ascertained.     

Introduction of DNA into chick embryos by in ovo electroporation

Gene transfer by in ovo electroporation has been applied to the study of developmental biology, especially to central nervous system (CNS) development. Plasmids are injected into the neural tube of stage 10 chick embryos, and a 25-V 25-msec square pulse is applied five times. Since DNA moves toward the anode, the cathode side of the neural tube is transfected, and the cathode side is used as the control. Expression of translation product of the introduced DNA is observed 2 h after electroporation, peaks around 20 h after electroporation and then weakens. Expression is transient when plasmids are used as expression vectors, but they are very suitable for studying early developmental events (e.g., gene expression cascades or interactions). Misexpression of Pax-5 is shown as an example.     

Introduction of unlabeled proteins into living cells by electroporation and isolation of viable protein-loaded cells using dextran-fluorescein isothiocyanate as a marker for protein uptake.

Commonly, microinjection has been the method of choice for introducing proteins into living cells. Viable cells containing an introduced protein can be then identified providing that the protein is fluorochrome conjugated. This approach is applicable only for adherent cells, and the number of cells that can be analyzed is small. In this study, we have established that electroporation can be used to load proteins into large numbers of cells with high efficiency. Furthermore, we have developed a method for the isolation of protein-loaded cells using fluorescein isothiocyanate-dextran (dextran-FITC) as a molecular marker for protein uptake. The essential features of this method are that dextran-FITC is included in the electroporation medium and, thus, is cointroduced with the protein of interest. Purification of cells containing dextran-FITC using fluorescence-activated cell sorting yields a population which is composed almost entirely of cells containing the protein of interest.     

Introduction of plasmids into whole cells of Clostridium acetobutylicum by electroporation

Abstract A method is presented for the introduction of plasmids into Clostridium acetobutylicum ATCC 8052 by electroporation. A plasmid shuttle vector, pMTL500E, which contains the erythromycin resistance gene and replication machinery of plasmid pAMβ1, was constructed and introduced into C. acetobutylicum by electroporation. The vector was then used to introduce a 2.2 kb Cla I/ Sph I chromosomal fragment from C. pasteurianum into a leucine requiring mutant of C. acetobutylicum , SBA9, where complementation of auxotrophy was observed. Plasmid DNA indistinguishable from that introduced, on the basis of agarose gel electrophoresis, was observed in transformants containing either plasmid.     

Introduction of stable high-copy-number DNA into Chinese hamster ovary cells by electroporation

A new gene transfer protocol has been developed that introduces up to 800 copies of an expression vector into Chinese hamster ovary cells in a single step by electroporation. The DNA typically integrates in tandem repeats so that the restriction endonuclease site used to linearize the input DNA remains intact. This is likely due to ligation of vector DNA via cohesive ends prior to integration. This high-copy-number procedure is far more rapid than the conventional stepwise gene amplification method used to generate stable eukaryotic protein production cell lines. By employing the expression vector pJODtPA, in which the selectable marker dihydrofolate reductase (DHFR) and the human tissue plasminogen activator (tPA) casettes are separated by a spacer and an RNA polymerase II terminator, cell lines secreting as much as 24 pg/cell.day tPA were isolated following electroporation and a single methotrexate selection. Gene copies and expression levels are stable over long periods of growth. A single round of gene amplification was performed following the high-copy-number procedure to yield a clone having a tPA production level of 45 pg/cell.day.     

Introduction of phalloidin labelled with fluorescein isothiocyanate into living polymorphonuclear leukocytes by electroporation

To examine the mechanism by which polymorphonuclear leukocytes (PMNs) move, phalloidin labelled with fluorescein isothiocyanate was introduced into freshly sampled cells by use of an electric-cell fusion system. The best conditions for treatment were three pulses of direct current at 100 V for a pulse duration of 3 microseconds. The treated cells retained their usual motility when observed under a microscope, so the method was suitable for the analysis of motile living cells. We used the method to study PMNs during locomotion, spreading and phagocytosis. In locomotion, fluorescence first appeared at the head of the cell and shifted gradually along the cell margin from head to tail. In spreading, diffuse fluorescence around the marginal part of the cytoplasm was strongest near both the attachment sites and the perinuclear area of the cell and spots of fluorescence appeared in the cytoplasm. In phagocytosis, fluorescence developed from the attachment sites, spread to the entire phagocytizing area of the cytoplasm and disappeared when phagocytosis ended. Cells treated with cytochalasin B were randomly spotted with fluorescence. Freshly sampled cells had diffuse and scattered fluorescence, without the lines observed in fixed cells.     

Introduction of foreign genes into silkworm eggs by electroporation and its application in transgenic vector test

Up to now, the most practical method for introducingforeign gene into insect eggs is the microinjection as thatwas utilized in Drosophila melanogaster [1–3]. The dis-covery of transposable elements including: P, Minos,Hermes, mariner, hobo, piggyBac, etc…     

Membrane fusion as a mechanism of simian virus 40 entry into different cellular compartments.

Permissive and nonpermissive simian virus 40 (SV40)-infected cells were ultrastructurally analyzed. Viral particles were found in the cytoplasm, rough endoplasmic reticulum, nuclear envelope, lysosomes, and mitochondria. Upon entering the cell the virion obtains a tight membrane envelope. It seems to be either released from the envelope upon fusion with other membranes of the cell or aggregated into tubular membrane specializations upon fusion with other membrane-enveloped particles. Reconstructed morphological sequences and the finding of SV40 in different spaces of the cell suggest that entry of SV40 into the different compartments and eventually into the site of replication is facilitated by its capacity for being enveloped by a variety of membranes (notably the cell membrane and the nuclear membrane) and the sequential fusion and fission of these membranes.     

Introduction of cell markers into germ layer tissues of the mouse gastrula by whole embryo electroporation

We have optimized the technique of electroporation for introducing genetic markers into cells of the gastrulating mouse embryo to follow cell fates, tissue movement, and lineage differentiation. Using a plate-needle electrode combination and specific route of plasmid delivery, labeling could be targeted to discrete regions of the epiblast or the endoderm of the late gastrula. Among the various types of fluorescent and chromogenic reporter constructs tested, those driven by CMV promoter are efficient and strong expression can be detected as soon as 2-3 h after electroporation. The efficacy of marking cell lineages by CRE-mediated activation of reporters proved to be inefficient for tracking cell lineages due to an obligatory 8-9-h lag from the electroporation of constructs to the expression of reporter. This significant time lag also raises concern of the temporal precision at which tissue- or stage-specific knock-out or activation of genetic activity may be achieved by the Cre-loxP mechanism.     

Efficiency of cellular delivery of antisense peptide nucleic acid by electroporation depends on charge and electroporation geometry

Electroporation is potentially a very powerful technique for both in vitro cellular and in vivo drug delivery, particularly relating to oligonucleotides and their analogs for genetic therapy. Using a sensitive and quantitative HeLa cell luciferase RNA interference mRNA splice correction assay with a functional luciferase readout, we demonstrate that parameters such as peptide nucleic acid (PNA) charge and the method of electroporation have dramatic influence on the efficiency of productive delivery. In a suspended cell electroporation system (cuvettes), a positively charged PNA (+8) was most efficiently transferred, whereas charge neutral PNA was more effective in a microtiter plate electrotransfer system for monolayer cells. Surprisingly, a negatively charged (-23) PNA did not show appreciable activity in either system. Findings from the functional assay were corroborated by pulse parameter variations, polymerase chain reaction, and confocal microscopy. In conclusion, we have found that the charge of PNA and electroporation system combination greatly influences the transfer efficiency, thereby illustrating the complexity of the electroporation mechanism.     

Introduction of proteins into living bacterial cells: distribution of labeled HU protein in Escherichia coli.

Growing bacterial cells forming division septa have sites near the septa that are sensitive to EDTA shock. Cells treated with EDTA incorporate proteins and other molecules from the surrounding medium, probably via vesiclelike lesions at the septa that are induced by EDTA. The amount of protein taken up is proportional to the protein concentration in the permeabilization medium. Incorporated molecules equilibrate throughout the cytoplasm, and those with affinity for DNA bind to the nucleoid. Conditions that promote the viability of permeabilized cells and help to avoid otherwise irreversible effects of EDTA are defined. Procedures for selecting cells that have incorporated protein and for studying the distribution of the protein and its effects in growing-dividing cells are described. The procedure may have several applications to molecular and cellular biology; however, we describe here the localization in living cells of the histonelike protein HU. Fluorescence microscopy of cells containing different amounts of fluorescein-labeled HU (varied from approximately 10(3) to 10(5) molecules per cell) showed that the HU concentrates in the nucleoid and is uniformly distributed throughout this structure. Control experiments demonstrated that unlabeled interior parts of the nucleoid can be resolved when labeled proteins that do not bind DNA or enter the nucleoid are introduced into living cells. It was concluded that in vivo added HU binds primarily DNA and that there are no intrinsic restrictions on major regions of the nucleoid to which the added HU protein may bind.