Electroporative gene transfer (electrotransfection): A method for strain improvement of animal cells

Rüker, Florian; Liegl, W.; Mattanovich, Diethard; Reiter, Samantha; Himmler, Gottfried; Jungbauer, Alois; Katinger, Hermann

doi:10.1016/0302-4598(87)80030-2

Cited by 5 publications

(3 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Production and characterization of human MAbs 2F5 and iBI are described in detail elsewhere (3,4). Briefly, hybridomas were generated by a combined polyethylene glycol-electrofusion method (29). As HIV-1-sensitized cells, peripheral blood lymphocytes from asymptomatic seropositive volunteers were used.…”

Section: Methodsmentioning

confidence: 99%

A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1

Muster

Steindl

Purtscher

et al. 1993

J Virol

934

321

View full text Add to dashboard Cite

Vaccination against human immunodeficiency virus type 1 (HIV-1) requires an immunogen which will elicit a protective immunity against viruses that show a high degree of genetic polymorphism. Therefore, the identification of neutralizing epitopes which are shared by many strains would be useful. In previous studies, we established a human monoclonal antibody (2F5) that neutralizes a variety of laboratory strains and clinical isolates of HIV-1. In the present report, we define the amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) on the ectodomain of gp4l as the epitope recognized by this antibody. The sequence was found to be conserved in 72% of otherwise highly variable HIV-1 isolates. Escape mutants were not detected in cells infected with HIV-1 isolates MN and RF in the presence of antibody 2F5. Since sequence variability of neutralizing epitopes is considered to be a major obstacle to HIV-1 vaccine development, the conserved B-cell epitope described here is a promising candidate for inclusion in a vaccine against AIDS.

show abstract

Section: Methodsmentioning

confidence: 99%

A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1

Muster

Steindl

Purtscher

et al. 1993

J Virol

934

321

View full text Add to dashboard Cite

show abstract

“…22 Hybridomas were generated by a combined polyethylene glycol/electrofusion method. 23 Briefly, peripheral blood mononuclear cells (PBMCs) from naturally infected asymptomatic HIV-1-seropositive blood donors were used as HIV-1-sensitized cells. Lymphocytes were mixed with the heteromyeloma cell line CB-F7 in a ratio of 5:1 in medium containing 5% polyethylene glycol and were pulsed in plastic cuvettes (Bio-Rad, Richmond, CA Primary isolates WYG and WRE were obtained from two different AIDS patients.…”

Section: Monoclonal Antibody 2f5mentioning

confidence: 99%

A Broadly Neutralizing Human Monoclonal Antibody against gp41 of Human Immunodeficiency Virus Type 1

Purtscher¹,

Trkola²,

Grüber³

et al. 1994

AIDS Research and Human Retroviruses

333

264

View full text Add to dashboard Cite

We have established a hybridoma clone, designated 2F5, secreting a neutralizing human monoclonal antibody (MAb) specific for gp41 of human immunodeficiency virus type 1 (HIV-1). The epitope of MAb 2F5 was mapped to amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala on the ectodomain of gp41. In this study different in vitro test systems were used to characterize the neutralizing properties of MAb 2F5. In syncytium inhibition assays, fusion inhibition experiments, and neutralization assays on different HIV-susceptible cells (H9, U937, and peripheral blood mononuclear cells) MAb 2F5 showed broad-spectrum neutralizing capacity against HIV-1 laboratory isolates IIIB, MN, RF, and SF2. In addition, primary isolates from AIDS patients were also neutralized.

show abstract

“…Under appropriate conditions, the diffusion and exchange of intracellular and extracellular components can take place during the lifespan of the pore (7,32), and macromolecules such as DNA present in the suspending medium may enter into the treated cells. This technique has been shown to be extremely effective in enhancing the transfer of genetic information into mammalian cells (17,20,22,24,28), plant protoplasts (18,19,25,31), and nonprotoplasted (intact) yeast cells (10).…”

mentioning

confidence: 99%

Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation

McIntyre

Harlander

1989

Appl Environ Microbiol

View full text Add to dashboard Cite

To apply recombinant DNA techniques for genetic manipulation of the industrially important lactococci, an efficient and reliable high-frequency transformation system must be available. High-voltage electric pulses have been demonstrated to enhance uptake of DNA into protoplasts and intact cells of numerous gram-negative and gram-positive microorganisms. The objective of this study was to develop a system for electroporating intact cells of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) with a commercially available electroporation unit (BTX Transfector 100; BTX, Inc., San Diego, Calif.). Parameters which influenced the efficiency of transformation included growth phase and final concentration of cells, ionic strength of the suspending medium, concentration of plasmid DNA, and the amplitude and duration of the pulse. Washed suspensions of intact cells suspended in deionized distilled water were subjected to one high-voltage electric pulse varying in voltage (300 to 900 V corresponding to field strengths of 5 to 17 kV/cm) and duration (100 ,us to 1 s). Transformation efficiencies of 103 transformants per jig of DNA were obtained when dense suspensions (final concentration, 5 x 1010 CFU/ml) of stationary-phase cells were subjected to one pulse with a peak voltage of 900 V (field strength, 17 kV/cm) and a pulse duration of 5 ms in the presence of plasmid DNA. Dilution of porated cells in broth medium followed by an expression period of 2 h at 30°C was beneficial in enhancing transformation efficiencies. Plasmids ranging in size from 9.8 to 30.0 kilobase pairs could be transformed by this procedure. * Corresponding author. t Paper no. 16,316 of the contribution series of the Minnesota Agricultural Experiment Station.

show abstract

Electroporative gene transfer (electrotransfection): A method for strain improvement of animal cells

Cited by 5 publications

References 10 publications

A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1

A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1

A Broadly Neutralizing Human Monoclonal Antibody against gp41 of Human Immunodeficiency Virus Type 1

Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation

Contact Info

Product

Resources

About