Optimized PEI‐based Transfection Method for Transient Transfection and Lentiviral Production

Shaozhe Yang1, Xiaoling Zhou2, Rongxiang Li1, Xiuhong Fu1, Pingnan Sun2

1 Reproductive and Genetic Center, The First Affiliated Hospital of Luohe Medical College, Luohe, 2 Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/cpch.25
Online Posting Date:  September, 2017
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Abstract

Polyethyleneimine (PEI), a cationic polymer vehicle, forms a complex with DNA which then can carry anionic nucleic acids into eukaryotic cells. PEI‐based transfection is widely used for transient transfection of plasmid DNA. The efficiency of PEI‐based transfection is affected by numerous factors, including the way the PEI/DNA complex is prepared, the ratio of PEI to DNA, the concentration of DNA, the storage conditions of PEI solutions, and more. Considering the major influencing factors, PEI‐based transfection has been optimized to improve its efficiency, reproducibility, and consistency. This protocol outlines the steps for ordinary transient transfection and lentiviral production using PEI. © 2017 by John Wiley & Sons, Inc.

Keywords: lentivirial production; polyethylenimine; transfection method; transient transfection

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Using PEI to Transiently Transfect Cells in a 12‐Well Plate
  • Basic Protocol 2: Transfecting 293T Cells in 10‐cm Dish to Produce Lentivirus
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Using PEI to Transiently Transfect Cells in a 12‐Well Plate

  Materials
  • Cell line: 293T cells (ATCC, cat. no. CRL‐11268); alternatively use Huh7 cells or Hep3B cells (also from ATCC)
  • Complete medium (see recipe)
  • Transfection medium (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Plasmid (see recipe for preparation): p‐LV‐EGFP
  • PEI solution (see recipe)
  • Radioimmunoprecipitation (RIPA) buffer (see recipe)
  • BCA protein assay kit (Thermo, cat. no. NCI3225CH)
  • 6× SDS‐PAGE sample loading buffer, (Beyotime, cat. no. P0015F)
  • Running buffer for SDS‐PAGE (see recipe)
  • Transfer buffer for immunoblotting (see recipe)
  • Non‐fat milk blocking buffer (see recipe)
  • Anti‐GFP antibody (from goat; Abcam, cat. no. Ab6673)
  • Anti‐β‐actin antibody (from mouse, ZSGB‐Bio, cat. no. ZM‐0001)
  • Antibody dilution buffer (see recipe)
  • 12‐well culture plate
  • Light microscope and fluorescence microscope
  • Safe‐Lock 1.5‐ml microcentrifuge tubes (Eppendorf)
  • Rocker
  • Refrigerated microcentrifuge
  • Nitrocellulose membrane for immunoblotting (Millipore, cat. no. HAHY00010)
  • 100°C water bath
  • Additional reagents and equipment for cell culture (Phelan & May, ), SDS‐PAGE (Gallagher, ), and transfer of proteins to nitrocellulose membrane by immunoblotting (western blotting; (Ni, Xu, & Gallagher, )

Basic Protocol 2: Transfecting 293T Cells in 10‐cm Dish to Produce Lentivirus

  Materials
  • 293T cells (ATCC, cat. no. CRL‐11268)
  • Complete medium (see recipe)
  • Transfection medium (see recipe)
  • Plasmids (see recipe for preparation):
    • p‐LV‐EGFP
    • pRSV‐REV
    • pMDLg/pRRE (gag/pol elements)
    • VSVL
  • PEI solution (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • PEG6000 (Sigma, cat. no. 81253)
  • Polybrene (Sigma, cat. no. H9268)
  • 1× 0.25% trypsin (Gibco, cat. no. 15050‐057)
  • 10‐cm dish
  • Light microscope and fluorescence microscope
  • 50‐ml conical centrifuge tubes (e.g., Corning Falcon)
  • Refrigerated centrifuge
  • 0.45‐µm PES membrane (ThermoFisher; cat. no. 124‐0045)
  • 12‐well culture plate
  • 5‐ml round‐bottom tubes (e.g., Corning Falcon)
  • 70‐µm cell strainer
  • Fluorescence‐activated cell sorting (FACS) instrument (Robinson et al., 2017)
  • Additional reagents and equipment for cell culture including counting cells with a hemacytometer (Phelan & May, ) and fluorescence‐activated cell sorting FACS; Robinson et al., 2017)
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Figures

Videos

Literature Cited

Literature Cited
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