Electroporation for the efficient transfection of mammalian cells with DNA

Chu, Gilbert; Hayakawa, Hiroshi; Berg, Paul

doi:10.1093/nar/15.3.1311

Cited by 836 publications

(378 citation statements)

References 27 publications

Supporting

Mentioning

365

Contrasting

Unclassified

Order By: Relevance

“…These cells were transfected by electroporation using a Biorad Gene Pulser (Chu et al, 1987). Cells to be stimulated with 10 ng/ml epidermal growth factor (EGF) or 50 nM phorbol 12-myristate 13-acetate (PMA) were ®rst grown overnight in low serum-containing medium (DMEM with 0.5% calf serum).…”

Section: Cell Culturementioning

confidence: 99%

Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms

et al. 1998

View full text Add to dashboard Cite

We have uniformly examined the regulatory steps required by oncogenic Ras, Src, EGF and phorbol 12-myristate 13-acetate (PMA) to activate Raf-1. Speci®-cally, we determined the role of Ras binding and the phosphorylation of serines 338/339, tyrosines 340/341 and the activation loop (491 ± 508) in response to these stimuli in COS-7 cells. An intact Ras binding domain was found to be essential for Raf-1 kinase activation by each stimulus, including PMA. Brief treatment of COS-7 cells with PMA was found to rapidly promote accumulation of the active, GTP-bound form of Ras. Furthermore, loss of the serine 338/339 and tyrosine 340/341 phosphorylation sites also blocked Raf-1 activation by all stimuli tested. Loss of the serine 497 and serine 499 PKCa phosphorylation sites failed to signi®cantly reduce Raf-1 activation by any stimulus including PMA. Alanine substitution of all other potential phosphorylation sites within the Raf-1 activation loop had little or no e ect on kinase regulation by Ras[V12] or vSrc although some mutants were less responsive to PMA. These results suggest that in mammalian cells, Raf-1 can be regulated by a variety of di erent stimuli through a common mechanism involving association with Ras-GTP and multiple phosphorylations of the amino-terminal region of the catalytic domain. Phosphorylation of the activation loop does not appear to be a signi®cant mechanism of Raf-1 kinase regulation in COS-7 cells.

show abstract

Section: Cell Culturementioning

confidence: 99%

Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms

et al. 1998

View full text Add to dashboard Cite

show abstract

“…9 To overcome these problems, non-viral methods have been developed, [10][11][12][13][14] and one such method is electrotransfection (or electro-gene transfer). [15][16][17][18][19][20][21] Comparing with viralbased methods, electrotransfection does not require packing of genes into vectors and is less immunogenic. The technique relies on delivery of a sequence of electric pulses to target cells or tissues placed between electrodes, which leads to the entry of cell-impermeable molecules, including plasmid DNA (pDNA).…”

Section: Introductionmentioning

confidence: 99%

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

et al. 2017

View full text Add to dashboard Cite

“…The lower transfection efficiencies reported by other authors using various electroinjection protocols (Potter et al, 1984;Toneguzzo et al, 1986) may in part be attributed to Iack of attention to one or more of the aforementioned considerations. Only Chu et al (1987) have reported stable transfection rates as high as 1%, but this was achieved in a variety of adherent monolayer cells by applying very long duration pulses (7 ms) of relatively low voltage and using DNA concentrations of 20 JLg/ml with 500 JLg/ml carrier DNA. Such long pulse durations would be expected to result in irreversible membrane breakdown and to result in the death of many otherwise viable cells.…”

Section: Discussionmentioning

confidence: 99%

Increased efficiency of transfection of murine hybridoma cells with DNA by electropermeabilization

Stopper

Zimmermann

Neil

1988

Journal of Immunological Methods

View full text Add to dashboard Cite

Dispase-treated murine hybridoma cells (SP2/0-Ag14) were transfected with the G418 resistance gene bearing plasmid pSV2-neo by electropermeabilization with a high degree of efficiency. The cells were subjected to intermittent multiple high-voltage short duration (5 p.s) DC pulses at intervals of 1 min in a weakly conducting medium followed by selection in G418-containing medium. The transfection medium, temperature, pulse duration, and voltage were empirically determined by preliminary electropermeabilization experiments. Increasing the number of pulses resulted in a higher percentage of transfected cells, but a decrease in the number of viable cells, with the optimal transfectant yield resulting when five pulses of 10 kV jcm were administered. This method allows the rapid and efficient injection of DNA into mammalian cells, and permits the rapid production of stable, drug resistant hybridoma celllines for use in subsequent fusion experiments.Key words: DNA transfection; Electropermeabilization; Eukaryotic cell; Hybridoma; Drug resistance IntroducnonThe injection of foreign DNA into eukaryotic cells, and its subsequent expression in these cells is a powerful research tool with wide application in cellular and molecular biology. Using this technique, it is possible to rapidly render otherwise susceptible cell lines resistant to antibiotics by injection and subsequent genomic integration and expression of bacterial antibiotic resistance genes. One important use for such drug resistant cells is the production of 'fusomas' or hybrid hybridomas (Milstein et al., 1983;Semenenko et al.;1985, Correspondence to: G.A. Neil, Ludwig Institute for Cancer Research, Toronto M4Y 1 M4, Canada.• This work was supported by grants from the BMFT, the DFLVR, the Deutsche Forschungsgemeinschaft (SFB 176), and the Medical Research Council of Canada. Suresh et al., 1986) which may then be conveniently selected after somati~ cell hybridization in appropriate antibiotic-containing medium (Lanzavecchia et al., 1987). A number of such drug resistance genes, incorporated in weil characterized plasmid vectors are now readily available and functional in mammalian cells after integration.We have recently refined and optimized the methodology for insertion of the plasmid vector containing the bacterial drug resistance gene, neomycin phosphotransferase (pSV2-neo) (Southem and Berg, 1982) into the non-secreting mammalian hybridoma cell line, SP2/0-Agl4 (Sp2/0) by means of electropermeabilization, thus en_. abling the selection of transfectants in growth medium supplemented with the antibiotic G418.The crucial step in this method is the production of reversible electrical breakdown of the cell

show abstract

Electroporation for the efficient transfection of mammalian cells with DNA

Cited by 836 publications

References 27 publications

Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms

Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms

Involvement of a Rac1-Dependent Macropinocytosis Pathway in Plasmid DNA Delivery by Electrotransfection

Increased efficiency of transfection of murine hybridoma cells with DNA by electropermeabilization

Contact Info

Product

Resources

About