A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications

Raymond, Céline; Tom, Roseanne; Perret, Sylvie; Moussouami, Pascal; L’Abbé, Denis; St-Laurent, Gilles; Durocher, Yves

doi:10.1016/j.ymeth.2011.04.002

Cited by 74 publications

(60 citation statements)

References 37 publications

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“…Cotransfections of Ig heavy‐chain and matching Ig light‐chain expression vectors (20 μ g each/flask) were performed using linear polyethylenimine (PEI) as a transfection reagent (Polysciences, Inc.)25. In brief, 1:3 concentration ratio of plasmids:PEI were mixed in 500 μ L Opti‐MEM‐reduced serum media (Invitrogen), 30 min at RT, and subsequently incubated on 90% confluent cells in a total volume of 20 mL/flask for 24 h. The transfection medium was replaced with antibody‐harvesting medium containing RPMI 1640 supplemented with 1× Nutridoma‐SP (Roche), 100 μ g/mL streptomycin, 100 U/mL penicillin, and cultured.…”

Section: Methodsmentioning

confidence: 99%

NMDAR encephalitis: passive transfer from man to mouse by a recombinant antibody

Malviya

Barman

Golombeck

et al. 2017

Ann Clin Transl Neurol

112

106

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ObjectiveAutoimmune encephalitis is most frequently associated with anti‐NMDAR autoantibodies. Their pathogenic relevance has been suggested by passive transfer of patients' cerebrospinal fluid (CSF) in mice in vivo. We aimed to analyze the intrathecal plasma cell repertoire, identify autoantibody‐producing clones, and characterize their antibody signatures in recombinant form.MethodsPatients with recent onset typical anti‐NMDAR encephalitis were subjected to flow cytometry analysis of the peripheral and intrathecal immune response before, during, and after immunotherapy. Recombinant human monoclonal antibodies (rhuMab) were cloned and expressed from matching immunoglobulin heavy‐ (IgH) and light‐chain (IgL) amplicons of clonally expanded intrathecal plasma cells (cePc) and tested for their pathogenic relevance.ResultsIntrathecal accumulation of B and plasma cells corresponded to the clinical course. The presence of cePc with hypermutated antigen receptors indicated an antigen‐driven intrathecal immune response. Consistently, a single recombinant human GluN1‐specific monoclonal antibody, rebuilt from intrathecal cePc, was sufficient to reproduce NMDAR epitope specificity in vitro. After intraventricular infusion in mice, it accumulated in the hippocampus, decreased synaptic NMDAR density, and caused severe reversible memory impairment, a key pathogenic feature of the human disease, in vivo.InterpretationA CNS‐specific humoral immune response is present in anti‐NMDAR encephalitis specifically targeting the GluN1 subunit of the NMDAR. Using reverse genetics, we recovered the typical intrathecal antibody signature in recombinant form, and proved its pathogenic relevance by passive transfer of disease symptoms from man to mouse, providing the critical link between intrathecal immune response and the pathogenesis of anti‐NMDAR encephalitis as a humorally mediated autoimmune disease.

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Section: Methodsmentioning

confidence: 99%

NMDAR encephalitis: passive transfer from man to mouse by a recombinant antibody

Malviya

Barman

Golombeck

et al. 2017

Ann Clin Transl Neurol

112

106

View full text Add to dashboard Cite

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“…Cells were grown in either DMEM (HEK) or Ham's F-12 (CHO) media supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (Invitrogen). Briefly, 2.7 g of DNA was applied to a 75% confluent 6-well plate (Corning) using the polycation polyethylenimine transfection method (41). When BRI2-A␤ constructs were co-transfected with either wild-type PSEN1 or the mutants M139V or ⌬exon9 into CHO cells, 1.35 g of each DNA were used.…”

Section: Methodsmentioning

confidence: 99%

γ-Secretase Processing and Effects of γ-Secretase Inhibitors and Modulators on Long Aβ Peptides in Cells

Ran

Cruz

Ladd

et al. 2014

Journal of Biological Chemistry

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Background: A␤ peptides are generated by stepwise cleavage of the amyloid precursor protein.Results: A␤s longer than A␤1-48 are efficiently cleaved by ␥-secretase in cells with different response to inhibitors (GSIs) and modulators (GSMs). Conclusion:The initial ␥-secretase cleavage does not precisely define subsequent product lines. Significance: Understanding how different species of A␤ are generated and modulated by small molecules has broad therapeutic implications.

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“…HEK-293F cells (Invitrogen) were maintained in Freestyle 293 Expression Medium (Invitrogen) in spinner flasks at 37°C and 5% v/v CO 2 . Transfections utilized linear 25-kDa polyethyleneimine (PEI; Polysciences) as described previously (41). Briefly, plasmid and PEI (26 and 52 g, respectively) were added to 2.6 ml of phosphate-buffered saline (PBS), pH 7.2.…”

Section: Methodsmentioning

confidence: 99%

The Crystal Structure of Six-transmembrane Epithelial Antigen of the Prostate 4 (Steap4), a Ferri/Cuprireductase, Suggests a Novel Interdomain Flavin-binding Site

Gauss

Kleven

Sendamarai

et al. 2013

Journal of Biological Chemistry

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Background:In-depth biochemical and structural analyses of Steap4 are lacking. Results: Crystallographic and kinetic characterization of Steap4 metalloreductase and flavin-dependent NADPH oxidase activities are reported. Conclusion: Physiologically relevant ferric/cupric reductase and flavin-dependent NADPH oxidase activities are observed at neutral and endosomal pH; the N-terminal tail is dispensable for these activities. Significance: Metalloreductase activity should be considered in Steap4-associated insulin resistance.

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A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications

Cited by 74 publications

References 37 publications

NMDAR encephalitis: passive transfer from man to mouse by a recombinant antibody

NMDAR encephalitis: passive transfer from man to mouse by a recombinant antibody

γ-Secretase Processing and Effects of γ-Secretase Inhibitors and Modulators on Long Aβ Peptides in Cells

The Crystal Structure of Six-transmembrane Epithelial Antigen of the Prostate 4 (Steap4), a Ferri/Cuprireductase, Suggests a Novel Interdomain Flavin-binding Site

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