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Calcium Phosphate Transfection

Robert E. Kingston1,  Claudia A. Chen2,  Hiroto Okayama3

1Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
2National Institute of Mental Health, Bethesda, Maryland
3Osaka University, Osaka, Japan



Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
Unit 20.3
DOI: 
10.1002/0471143030.cb2003s19
Online Posting Date: 
August, 2003
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Abstract

This unit presents two methods of calcium phosphate-based eukaryotic cell transfection that can be used for both transient and stable transfections. In these protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. A HEPES-buffered solution is used to form a calcium phosphate precipitate that is directly layered onto the cells. For some cells, shocking the cells with glycerol or DMSO improves transfection efficiency. In the alternate high-efficiency method, a BES-buffered system is used that allows the precipitate to form gradually in the medium and then drop onto the cells. While the alternate method is particularly efficient for stable transformation of cells with circular plasmid DNA, both protocols yield similar results for transformation with linear plasmid or genomic DNA, or for transient expression.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Transfection Using Calcium Phosphate–DNA Precipitate Formed in HEPES
  • Support Protocol: Glycerol/DMSO Shock of Mammalian Cells
  • Alternate Protocol: High-Efficiency Transfection Using Calcium Phosphate–DNA Precipitate Formed in BES
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol: Transfection Using Calcium Phosphate–DNA Precipitate Formed in HEPES

 Materials
  • Exponentially growing eukaryotic cells (e.g., HeLa, BALB/c 3T3, NIH 3T3, CHO, or rat embryo fibroblasts)
  • Complete medium (depending on cell line used)
  • CsCl-purified plasmid DNA (10 to 50 µg per transfection)
  • 2.5 M CaCl2 (see recipe)
  • 2× HEPES-buffered saline (HeBS; see recipe)
  • PBS (appendix 2A)
  • 10-cm tissue culture dishes
  • 15-ml conical tube
  • Additional reagents and equipment for ethanol precipitation (appendix 3A) and mammalian cell tissue culture (unit 1.1)

NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.


Support Protocol: Glycerol/DMSO Shock of Mammalian Cells

 Additional Materials (also see Basic Protocol)
  • 10% (v/v) glycerol solution or DMSO in complete medium, sterile
  • PBS (appendix 2A), sterile

Replace step of the Basic Protocol with the following:

Alternate Protocol: High-Efficiency Transfection Using Calcium Phosphate–DNA Precipitate Formed in BES

 Materials
  • Exponentially growing mammalian cells (see Critical Parameters)
  • Complete medium: Dulbecco modified Eagle medium containing 10% (v/v) fetal bovine serum (FBS)
  • CsCl-purified plasmid DNA
  • TE buffer, pH 7.4 (appendix 2A)
  • 2.5 M CaCl2 (see recipe)
  • 2× BES-buffered solution (BBS; see recipe)
  • PBS (appendix 2A)
  • Selection medium (appendix 3A; optional)
  • 10-cm tissue culture dishes
  • 35°C, 3% CO2 humidified incubator
  • 35° to 37°C, 5% CO2 humidified incubator
  • Fyrite gas analyzer (optional; Fisher Scientific or Curtin Matheson)

NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.

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Figures

  • Figure 20.3.1
    Formation of calcium phosphate precipitate.

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Literature Cited

Literature Cited
    Chen, C. and Okayama, H. 1987. High efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell. Biol. 7:2745-2752.
    Chen, C. and Okayama, H. 1988. Calcium phosphate–mediated gene transfer: A highly efficient system for stably transforming cells with plasmid DNA. BioTechniques 6:632-638.
    Graham, F.L. and van der Eb, A.J. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456.
    Ishiura, M., Hirose, S., Uchida, T., Hamada, Y., Suzuki, Y., and Okada, Y. 1982. Phage particle–mediated gene transfer to cultured mammalian cells. Mol. Cell. Biol. 2:607-616.
    Wigler, M., Pellicer, A., Silverstein, S., and Axel, R. 1978. Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor. Cell 14:725.
 Key References
    Chen and Okayama, 1987. See above.
    Ishiura et al., 1982. See above.

Provides the basis for BES-mediated transfection.

     
 
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