Polyethylenimine (pei)

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PEI (Polyethylenimine) is a cationic polymer used as a transfection reagent in cell biology and molecular biology applications. It facilitates the delivery of nucleic acids, such as DNA or RNA, into cells for various experimental purposes.

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PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (7 citations) Polyethylenimine MAX (PEI “MAX”, (6 citations) Linear polyethylenimine (PEI (6 citations) Linear 25-kDa polyethylenimine (PEI) (4 citations) 25-kDa linear polyethylenimines (PEIs) (3 citations) Polyethylenimine (PEI) [linear poly(vinyl alcohol) (MW-25000)] (3 citations) Linear Polyethylenimine (PEI, MW 25,000) transfection reagent (2 citations) Polyethylenimine (PEI) 25kDa linear polymer (2 citations) Branched polyethylenimine (PEI) (2 citations) PEI MAX polyethylenimine reagent (2 citations)

1 522 protocols using polyethylenimine (pei)

Culturing and Transfecting Cell Lines

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Cell cultures were kept in a humidified incubator and maintained at 37°C and 5% CO₂. Cell lines used: Chinese Hamster Ovary (CHO-K1) cells (RRID: CVCL_0214, female) were grown in F12K media (Life Technologies, Carlsbad, CA, United States), 10% FBS (Fetal Bovine Serum) (Life Technologies), and penicillin/streptomycin; HEK293 cells (RRID: CVCL_0063, female) were grown in DMEM (Life Technologies), 10% FBS, and penicillin/streptomycin. Cells were transfected using polyethylenimine (PEI, Polysciences, Warrington, PA, United States) at a ratio of 10 mg PEI/1mg DNA for CHO cells, 4 mg PEI/1mg DNA for 293 cells used in the expression of Fc proteins, and 10 mg PEI/1mg DNA for 293 cells transfected for the synapse formation assay.
Neurons: Preparation of hippocampal rat cultures were previously described (52 (link),53 (link)). Briefly, P0 rat cortical glia were cultured to form monolayers on Poly-D-Lysine/collagen-coated coverslips in 24-well plates. P0 rat hippocampi from Sprague Dawley rats of both sexes (RRID: RGD_734476) were dissected one week later in cold 4-(2-hydroxyethyl)-1-piperazinee-thanesulfonic acid (HEPES)-buffered saline solution (Life Technologies), treated with enzymatic papain for 30 min, dissociated, and plated onto glial monolayers at a density of 5x10⁴ cells/well.

William N., Reissner C., Sargent R., Darlington T.M., DiBlasi E., Li Q.S., Keeshin B., Callor W.B., Ferris E., Jerominski L., Smith K.R., Christensen E.D., Gray D.M., Camp N.J., Missler M., Williams M.E, & Coon H. (2021). Neurexin 1 variants as risk factors for suicide death. Molecular Psychiatry, 26(12), 7436-7445.

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Cell Culture and Transfection Protocols

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Cell cultures were kept in a humidified incubator and maintained at 37° C and 5% CO₂. Cell lines used: Chinese Hamster Ovary (CHO-K1) cells (RRID: CVCL_0214, female) were grown in F12K media (Life Technologies, Carlsbad, CA, United States), 10% FBS (Fetal Bovine Serum) (Life Technologies), and penicillin/streptomycin; HEK293 cells (RRID: CVCL_0063, female) were grown in DMEM (Life Technologies), 10% FBS, and penicillin/streptomycin. Cells were transfected using polyethylenimine (PEI, Polysciences, Warrington, PA, United States) at a ratio of 10 mg PEI/1mg DNA for CHO cells, 4 mg PEI/1mg DNA for 293 cells used in the expression of Fc proteins, and 10 mg PEI/1mg DNA for 293 cells transfected for the synapse formation assay.
Neurons: preparation of hippocampal rat cultures were previously described [51 (link), 52 (link)]. Briefly, P0 rat cortical glia were cultured to form monolayers on Poly-D-Lysine/collagen-coated coverslips in 24-well plates. P0 rat hippocampi from Sprague Dawley rats of both sexes (RRID: RGD_734476) were dissected one week later in cold 4-(2-hydroxyethyl)-1-piperazinee-thanesulfonic acid (HEPES)-buffered saline solution (Life Technologies), treated with enzymatic papain for 30 min, dissociated, and plated onto glial monolayers at a density of 5 × 10⁴ cells/well.

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Establishing Stable Cell Lines via Transfection

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Cells were seeded at 2.5–3 × 10⁵ cells/well into a 6-well plate 24 h prior to transfection. Three hours prior to transfection, the medium was replaced with DMEM supplemented with 5% (v/v) FCS. Cells were transfected using linear polyethyleneimine (PEI; Polysciences Inc.) at a molar PEI nitrogen: DNA phosphorous ratio of 15:1, by adding preformed PEI/DNA complexes dropwise to the growth medium [34 (link)]. Successful transfection was confirmed 24 h later using an inverted epifluorescence microscope (Hg lamp, Carl Zeiss) and the medium was then replaced with DMEM supplemented with 10% (v/v) FCS. A further 24 h later, cells were detached (by trypsinisation) and transferred to T25 flasks with a fresh medium supplemented with 200 µg/ml Zeocin (ThermoFisherScientific) for a period of 2–3 weeks with periodic media changes until death of the non-transfected cells was observed and Zeocin resistant colonies of transfected cells had developed. Once healthy colonies were obtained, the cells were maintained at a lower Zeocin concentration (40 µg/ml).

Cox M.H., Kapoor P., Briggs D.A, & Kerr I.D. (2018). Residues contributing to drug transport by ABCG2 are localised to multiple drug-binding pockets. Biochemical Journal, 475(9), 1553-1567.

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Establishment of HEK293 Cell Lines Expressing DOPr and EPAC Biosensor

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HEK293
cells were cultured
in 100 mm Petri dishes (Sarstedt, Germany) at 37 °C and 5% CO₂ in the Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum, 2 mM l-glutamine,
and 100 unit mL^–1 penicillin–streptomycin.
For transient transfections of DOPr- and BRET-based biosensors constructs,
HEK293 cells were seeded at 3–3.5 × 10⁶ cells/100
mm Petri dish and were grown for 18–24 h before transfecting
with polyethylenimine (PEI) (Polysciences Inc., Warrington, PA, USA)
at a 3:1 PEI/DNA ratio as per the manufacturer’s instructions.
Monoclonal cell lines stably expressing DOPr and the EPAC biosensor
(hereafter referred to as EPAC DOPr HEK293 cells) were established
by first transfecting 6 μg of the pSig-Flag-DOPr DNA construct/100
mm Petri dish using Lipofectamine (Invitrogen), followed by a puromycin
selection (1 μg/mL). This stable cell line was subsequently
transfected with 3 μg of the EPAC biosensor, using Lipofectamine
(Invitrogen) for the transfection and hygromycin (50 μg/mL)
for selection.

Mansour A., Nagi K., Dallaire P., Lukasheva V., Le Gouill C., Bouvier M, & Pineyro G. (2021). Comprehensive Signaling Profiles Reveal Unsuspected Functional Selectivity of δ-Opioid Receptor Agonists and Allow the Identification of Ligands with the Greatest Potential for Inducing Cyclase Superactivation. ACS Pharmacology & Translational Science, 4(5), 1483-1498.

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Recombinant SARS-CoV-2 RBD Production

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The pCAGGs SARS-CoV2 RBD plasmid (provided by Florian Krammer) was used for recombinant RBD expression as previously described. FreeStyle 293F cells (ThermoFisher, R79007) were transiently transfected with a mixture of plasmid DNA diluted in PBS (0.67 μg total plasmid DNA per ml of culture) and Polyethylenimine (PEI) (Polysciences, Inc., 23966) at a DNA-to-PEI ratio of 1:3. At six days post-transfection, cultures were harvested by centrifugation at 4,000 x g for 20 min, and supernatant was incubated with Ni-NTA resin (Goldbio) for 2 hours at 4° C with gentle stirring. Resin was collected in columns by gravity flow, washed with 16 column volumes of wash buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl, 20 mM Imidazole) and eluted in 12mL elution buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl, 250 mM Imidazole). Eluates were concentrated and exchanged into storage buffer (50 mM Tris HCl pH 8.0, 250 mM NaCl) using an Amicon centrifugal units (EMD Millipore). Protein concentration was determined using an extinction coefficient (33350 M⁻¹cm⁻¹), estimated from amino acid sequence by Expasy online ProtParam, and was further analyzed by SDS-PAGE.

Herrera N.G., Morano N.C., Celikgil A., Georgiev G.I., Malonis R.J., Lee J.H., Tong K., Vergnolle O., Massimi A.B., Yen L.Y., Noble A.J., Kopylov M., Bonanno J.B., Garrett-Thomson S.C., Hayes D.B., Bortz RH I.I.I., Wirchnianski A.S., Florez C., Laudermilch E., Haslwanter D., Fels J.M., Dieterle M.E., Jangra R.K., Barnhill J., Mengotto A., Kimmel D., Daily J.P., Pirofski L.A., Chandran K., Brenowitz M., Garforth S.J., Eng E.T., Lai J.R, & Almo S.C. (2020). Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis. bioRxiv.

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SARS-CoV-2 S Protein RBD Expression and Antibody Production

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The RBD (residues 319–529) of SARS-CoV-2 S protein (GenBank: MN975262.1) were subcloned into a pVRC vector with a C-terminal SBP-tag.
The CR3022 (GenBank: DQ168569 and DQ168570), B38 (27 (link)), and 0012C10 and 0012E4 antibodies (provided in-house) were produced in 293F suspension cells grown in FreeStyle 293 Expression media (Gibco, Thermo Fisher Scientific). Cells were transfected with Polyethylenimine (PEI; Polysciences) at 1 μg/μL in a ratio of 3 μg PEI to 1 μg DNA. Supernatants were harvested 5 days posttransfection, and antibody was purified using protein G magnetic beads (MilliporeSigma). For in vitro analysis, KZ52 (Mayflower Bioscience, 0260-001) was used as a negative control.

Atyeo C., Slein M.D., Fischinger S., Burke J., Schäfer A., Leist S.R., Kuzmina N.A., Mire C., Honko A., Johnson R., Storm N., Bernett M., Tong P., Zuo T., Lin J., Zuiani A., Linde C., Suscovich T., Wesemann D.R., Griffiths A., Desjarlais J.R., Juelg B.D., Goudsmit J., Bukreyev A., Baric R, & Alter G. (2021). Dissecting strategies to tune the therapeutic potential of SARS-CoV-2–specific monoclonal antibody CR3022. JCI Insight, 6(1), e143129.

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Visualizing NARS2 Mutant Localization

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Wild type (WT) human NARS2 cDNA (clone #Z7860) was obtained from GeneCopoeia and subcloned into pEGFP-N2 vector (Clontech) using InFusion cloning (Clontech). Stratagene QuikChange Lightning mutagenesis (Roche) was used to introduce the c.637G>T transversion and c.1142A>G transition into WT NARS2 sequence. All constructs were then sub-cloned in pcDNA3.1(+) (Invitrogen) vector and sequence verified. A HA tag was added at the C-terminal part of NARS cDNA using InFusion cloning.
Constructs were expressed in COS7 cells after transfection with PEI (Polysciences) using a 1:5 ratio (1μg cDNA/5μg PEI). Twenty-four hours post transfection, cells were incubated with 100nM Mito Tracker Red FM (Invitrogen) for 30 minutes, followed by fixation with 4% paraformaldehyde. Fixed cells were mounted with Fluorogel Mounting Medium (EMS) and imaged with a Zeiss LSM700 confocal microscope.

Simon M., Richard E.M., Wang X., Shahzad M., Huang V.H., Qaiser T.A., Potluri P., Mahl S.E., Davila A., Nazli S., Hancock S., Yu M., Gargus J., Chang R., Al-sheqaih N., Newman W.G., Abdenur J., Starr A., Hegde R., Dorn T., Busch A., Park E., Wu J., Schwenzer H., Flierl A., Florentz C., Sissler M., Khan S.N., Li R., Guan M.X., Friedman T.B., Wu D.K., Procaccio V., Riazuddin S., Wallace D.C., Ahmed Z.M., Huang T, & Riazuddin S. (2015). Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome. PLoS Genetics, 11(3), e1005097.

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Production of HIV-1 and VSV-G Pseudotyped Viruses

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Polyethylenimine (PEI) stock solution (1 mg/ml) was prepared by dissolving 500 mg PEI (Polysciences) into 500 ml sterilized water after adjusting pH to 4.5 with HCl and filtrating the solution through 0.22 μM membrane. To produce HIV-1, a total of 4 × 10⁵ 293T cells in 2 ml culture medium were seeded in each well of 6-well culture plates. 3 μg total proviral DNA was diluted into 200 μl serum-free DMEM medium, and mixed with 9 μg PEI. In order to produce VSV-G-pseudotyped viruses, 0.5 μg VSV-G expression vector was added to the HIV-1 proviral DNA in this procedure. After 15 min incubation at room temperature, these tranfection reagents were added directly into the culture of each well. After 48 hours, viruses were collected from the supernatants and viral production was measured by p24^Gag ELISA.

Zhang X., Zhou T., Frabutt D.A, & Zheng Y.H. (2016). HIV-1 Vpr increases Env expression by preventing Env from endoplasmic reticulum-associated protein degradation (ERAD). Virology, 496, 194-202.

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Optimized PEI-based Transfection of HEK-293 Cells

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Human embryonic kidney cells (HEK-293, CRL-1573; ATCC) were cultured in DMEM (catalog. no. C11995500BT; Gibco) containing 10% (v/v) FBS (catalog. no. 16000-044; Gibco) and 1% (v/v) penicillin/streptomycin antibiotic solution (catalog. no. ST488-1/ST488-2; Beyotime Inc.). The cells were cultured in a humidified atmosphere at 37°C and 5% CO₂, and testing for mycoplasma and bacterial contamination was done routinely. Transfection was performed using polyethyleneimine (PEI, molecular weight 40,000; catalog no. 24765; Polysciences) according to an optimized PEI-based protocol [45 (link)]. Briefly, 1.2 × 10⁴ cells were plated into a 96-well cell culture plate 18 h before transfection and then cotransfected with pFR1 and pFR2/pFR3 at a ratio of 1 : 1 (w/w) for 6 h with 25 μL PEI (stock solution 1 mg/mL in ddH₂O) and DNA mixture (PEI and DNA at a mass ratio of 3 : 1). Cell concentration and viability were determined with Countess II (Life Technologies, USA).

Liu Z., Zhou Y., Qu X., Xu L., Zou Y., Shan Y., Shao J., Wang C., Liu Y., Xue J., Jiang D., Fan Y., Li Z, & Ye H. (2022). A Self-Powered Optogenetic System for Implantable Blood Glucose Control. Research, 2022, 9864734.

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Fluorescent V(D)J Recombination Assay

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The fluorescent V(D)J substrates (diagramed in Figure 10, top panels) and V(D)J assays have recently been described (Neal et al., 2016 (link)). These substrates (derived from pECFP-N1, Clontech) contain the SV40 origin of replication, and are thus efficiently replicated episomally in all primate cell strains. Briefly, extrachromosomal fluorescent V(D)J assays were performed on cells plated at 20–40% confluency into 24-well plates in complete medium. Cells were transfected with 0.125 µg substrate, 0.25 µg RAG1 and 0.25 µg RAG2 per well using polyethylenimine (PEI, Polysciences) using 2 µL of PEI at 1 μg/mL per 1 µg of DNA. In experiments with additional expression plasmids, 0.25 µg of the expression plasmid or vector control was included. Cells were harvested 72 hr after transfection and analyzed for CFP and RFP expression by flow cytometry. The percentage of recombination was calculated as the percentage of cells expressing CFP divided by the percentage expressing RFP. Data presented represent at least three independent experiments, which each includes triplicate transfections.

Normanno D., Négrel A., de Melo A.J., Betzi S., Meek K, & Modesti M. (2017). Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining. eLife, 6, e22900.

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