Transfection Using DEAE‐Dextran

Tod Gulick1

1 Massachusetts General Hospital, Charlestown, Massachusetts
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Appendix 1D
DOI:  10.1002/0471142301.nsa01ds01
Online Posting Date:  May, 2001
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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., supplemented DMEM; appendix 2A) 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 (CPMB UNIT )
  • 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 1.0.1)
  • Inverted microscope
  • Additional reagents and equipment for mammalian cell culture (CPMB APPENDIX )
    Table 0.d.1   MaterialsSurface Areas of Commonly Used Tissue Culture Vessels and Corresponding Appropriate DEAE‐Dextran Transfection Medium Volumes

    Vessel Area (cm2) Appropriate vol. DEAE‐dextran medium a (ml)
    T175 flask 175
    T150 flask 150
    T75 flask 75
    T25 flask 25
    150‐mm dish 148 b 10
    100‐mm dish 55 b 4
    60‐mm dish 21 b 2
    35‐mm dish 8 b 1
    6‐well plate (35‐mm wells) 9.4 b 1
    12‐well plate (22‐mm wells) 3.8 b 0.5
    24‐well plate (15.5‐mm wells) 1.9 b 0.25

     aThese volumes are roughly a linear function of vessel surface area. To ensure that cells are completely covered by medium during the transfection, small wells require proportionately larger volumes due to annular sequestration of medium because of surface tension at the periphery.
     bCostar; other manufacturer products may deviate slightly.

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

  • CV‐1 cells (ATCC #CCL 70) growing in 100‐mm dish
  • Supplemented DMEM medium ( appendix 2A) with and without 10% FBS
  • Supplemented DMEM medium ( appendix 2A) 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; CPMB UNIT )
  •  Expression plasmid with TR gene insert (pTR)
  •  No‐insert expression plasmid (p[–])
  • Supplemented DMEM medium ( appendix 2A) 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 (CPMB APPENDIX ) and analyzing reporter gene activity (CPMB UNITS & )

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

  • COS cells (ATCC #1650) growing in 100‐mm dish
  • Supplemented DMEM medium ( appendix 2A) 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 (CPMB UNITS & ) and analysis of recombinant proteins (CPMB Chapter 10)

Support Protocol 1: Charcoal Stripping of Fetal Bovine Serum

  Materials
  • Fetal bovine serum (FBS), heat‐inactivated ( appendix 2A)
  • 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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