Transfection Using DEAE‐Dextran

Richard F Selden1

1 Transkaryotic Therapies, Cambridge, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.14
DOI:  10.1002/0471142735.im1014s03
Online Posting Date:  May, 2001
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Abstract

Two protocols for DEAE‐dextran transfection of cells are provided in this unit. The describes a procedure used to transfect adherent cells and the first presents a method used to transfect suspension cells. If an increase in transfection efficiency is needed, cells can be treated with chloroquine as described in the second .

     
 
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Table of Contents

  • Transfection Using DEAE‐Dextran
  • Basic Protocol 1: Transfection of Adherent Cells
  • Alternate Protocol 1: Transfection of Suspension Cells
  • Alternate Protocol 2: Chloroquine Treatment of Cells
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Transfection of Adherent Cells

  Materials
  • Mouse L cell fibroblasts or eukaryotic cells of choice
  • Plasmid DNA (CsCl purified; unit 10.3)
  • recipeTris‐buffered saline (TBS)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Complete Dulbeccos minimum essential medium containing 10% NuSerum (complete DMEM‐10 NS)
  • recipe10 mg/ml DEAE‐dextran in TBS, 37°C
  • Complete DMEM‐10 ( appendix 2A)
  • 10% (v/v) dimethyl sulfoxide (DMSO) in PBS, filter sterilized and stored at room temperature
  • 10‐cm tissue culture dishes
  • Additional reagents and equipment for ethanol precipitation (unit 10.1)

Alternate Protocol 1: Transfection of Suspension Cells

  Additional Materials
  • recipeSuspension TBS (STBS) solution, 37°C
  • Lymphoid cell line or eukaryotic cells of choice
  • recipe10 mg/ml DEAE‐dextran in STBS solution
  • Complete medium without serum (appropriate for the cell line)
  • Sterile 50‐ml polypropylene tube
  • Beckman JS‐4.2 rotor or equivalent
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Figures

Videos

Literature Cited

Literature Cited
   Fujita, T., Shubiya, H., Ohashi, T., Yamanishi, K., and Taniguchi, T. 1986. Regulation of human interleukin‐2 gene: Functional DNA sequences in the 5′ flanking region for the gene expression in activated T lymphocytes. Cell 46:401‐407.
   Lopata, M.A., Cleveland, D.W., and Sollner‐Webb, B. 1984. High‐level expression of a chloramphenicol acetyltransferase gene by DEAE‐dextran‐mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucl. Acids Res. 12:5707.
   Reeves, R., Gorman, C., and Howard, B. 1985. Minichromosome assembly of nonintegrated plasmid DNA transfected into mammalian cells. Nucl. Acids Res. 13:3599.
   Selden, R.F., Burke‐Howie, K., Rowe, M.E., Goodman, H.M., and Moore, D.D. 1986. Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol. Cell. Biol. 6:3173‐3179.
   Sussman, D.J. and Milman, G. 1984. Short‐term, high‐efficiency expression of transfected DNA Mol. Cell. Biol 4:1641.
Key Reference
   Lopata et al., 1984. See above.
  This paper presents a carefully controlled study of several of the factors that affect the efficiency of DEAE‐dextran transfection. It also demonstrates the value of a DMSO or glycerol shock for obtaining maximal expression of a transfected gene.
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