Transfection Using DEAE‐Dextran

Tod Gulick1

1 Massachusetts General Hospital, Charlestown, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 20.4
DOI:  10.1002/0471143030.cb2004s19
Online Posting Date:  August, 2003
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Abstract

Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)‐dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE‐dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE‐dextran transfection, as well as two more specific protocols for typical experimental applications. The basic protocol is suitable for transfection of anchorage‐dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE‐dextran‐mediated transfection can be used.

     
 
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Table of Contents

  • Basic Protocol 1: General Procedure for DEAE‐Dextran Transfection
  • Alternate Protocol 1: Sample Experiment: Transfection to Test Promoter Function
  • Alternate Protocol 2: Sample Experiment: Transfection to Test Enzyme Structure/Activity Relationships
  • Support Protocol 1: Charcoal Stripping of Fetal Bovine Serum
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: General Procedure for DEAE‐Dextran Transfection

  Materials
  • Cells to be transfected and appropriate culture medium (e.g., complete DMEM; unit 1.1) with and without 10% FBS
  • 100 mM (1000×) chloroquine diphosphate in PBS, filter‐sterilized (store at 4°C)
  • Plasmid DNA(s), prepared by CsCl density‐gradient centrifugation or affinity chromatography
  • TE buffer ( appendix 2A)
  • 10 mg/ml DEAE‐dextran stock solution (see recipe)
  • 10% (v/v) dimethyl sulfoxide (DMSO) in PBS, filter‐sterilized (store up to 1 month at room temperature)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Appropriate‐sized tissue culture vessels (Table 20.4.1)
  • Inverted microscope
  • Additional reagents and equipment for mammalian cell culture (unit 1.1)

Alternate Protocol 1: Sample Experiment: Transfection to Test Promoter Function

  • CV‐1 cells (ATCC #CCL 70) growing in 100‐mm dish
  • Complete DMEM medium (unit 1.2) with and without 10% FBS
  • Complete DMEM medium (unit 1.2) with 10% charcoal‐treated FBS (see protocol 4)
  • Plasmid DNAs:
    • Control reporter plasmid (e.g., β‐galactosidase, secreted alkaline phosphatase, or growth hormone, driven by a viral promoter)
    • Four test promoter constructs (promoter/CAT or promoter/luciferase)
    • Expression plasmid with TR gene insert (pTR)
    • No‐insert expression plasmid (p[–])
  • Complete DMEM medium (unit 1.2) with 10% charcoal‐treated FBS (see protocol 4), supplemented with 10 nm thyroid hormone (T 3)
  • 12‐well tissue culture plates
  • 100‐ml tissue culture dishes
  • Additional reagents and equipment for trypsinizing and subculturing monolayer cells (unit 1.1) and analyzing reporter gene activity ( appendix 3A)

Alternate Protocol 2: Sample Experiment: Transfection to Test Enzyme Structure/Activity Relationships

  • COS cells (ATCC #1650) growing in 100‐mm dish
  • Complete DMEM medium (unit 1.2) with and without 10% FBS
  • Control plasmid containing reporter gene (e.g., luciferase, CAT, or secreted alkaline phosphatase)
  • CDM8 vectors containing gene for wild‐type enzyme and genes for four mutant enzymes
  • 100‐ml tissue culture dishes
  • Additional reagents and equipment for analyzing reporter gene activity ( appendix 3A) and analysis of recombinant proteins ( appendix 3A)

Support Protocol 1: Charcoal Stripping of Fetal Bovine Serum

  Materials
  • Fetal bovine serum (FBS), heat‐inactivated (unit 1.1)
  • Activated charcoal, acid‐washed (Sigma)
  • Ultracentrifuge with Beckman SW 28, JA‐20.1, or equivalent swinging‐bucket rotor
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Figures

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Literature Cited

Literature Cited
   Aruffo, A. and Seed, B. 1987. Molecular cloning of a CD28 cDNA by a high‐efficiency COS cell expression system. Proc. Natl. Acad. Sci. U.S.A. 84:8573‐8577.
   Fregeau, C.J. and Bleackley, R.C. 1991. Factors influencing transient expression in cytotoxic T cells following DEAE‐dextran‐mediated gene transfer. Somatic Cell Mol. Genet. 17:239‐257.
   Kluxen, F.‐W. and Lubbert, H. 1993. Maximal expression of recombinant cDNAs in COS cells for use in expression cloning. Anal. Biochem. 208:352‐356.
   Levesque, J.P., Sansilvestri, P., Hatzfeld, A., and Hatzfeld, J. 1991. DNA transfection in COS cells: A low‐cost serum‐free method compared to lipofection. Biotechniques 11:313‐318.
   Lopata, M.A., Cleveland, D.W., and Sollner‐Webb, B. 1984. High level transient expression of a chloramphenical acetyl transferase gene by DEAE‐dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment Nucl. Acids Res. 12:5707‐5717.
   Puchalski, R.B. and Fahl, W.E. 1992. Gene transfer by electroporation, lipofection, and DEAE‐dextran transfection: Compatibility with cell‐sorting by flow cytometry. Cytometry 13:23‐30.
   Sussman, D.J. and Milman, G. 1984. Short‐term, high‐efficiency expression of transfected DNA. Mol. Cell. Biol. 4:1641‐1643.
   Yang, Y.‐W. and Yang, J.‐C. 1997. Studies of DEAE‐dextran‐mediated gene transfer Biotechnol. Appl. Biochem. 25:47‐51.
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