Calcium Phosphate Transfection

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

1 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, 2 National Institute of Mental Health, Bethesda, Maryland, 3 Osaka University, Osaka, Japan
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Appendix 1C
DOI:  10.1002/0471142301.nsa01cs01
Online Posting Date:  May, 2001
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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

  • Basic Protocol 1: Transfection Using Calcium Phosphate–DNA Precipitate Formed in HEPES
  • Support Protocol 1: Glycerol/DMSO Shock of Mammalian Cells
  • Alternate Protocol 1: High‐Efficiency Transfection Using Calcium Phosphate–DNA Precipitate Formed in BES
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: 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 CaCl 2 (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 (CPMB UNIT ) and mammalian cell tissue culture (CPMB APPENDIX )
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% CO 2 incubator unless otherwise specified.

Support Protocol 1: Glycerol/DMSO Shock of Mammalian Cells

  • 10% (v/v) glycerol solution or DMSO in complete medium, sterile
  • PBS ( appendix 2A), sterile

Alternate Protocol 1: High‐Efficiency Transfection Using Calcium Phosphate–DNA Precipitate Formed in BES

  Materials
  • Exponentially growing mammalian cells (see Critical Parameters)
  • Complete medium: Supplemented DMEM/10% FBS ( appendix 2A)
  • CsCl‐purified plasmid DNA
  • TE buffer, pH 7.4 ( appendix 2A)
  • 2.5 M CaCl 2 (see recipe)
  • 2× BES‐buffered solution (BBS; see recipe)
  • PBS ( appendix 2A)
  • Selection medium (see unit 4.6; optional)
  • 10‐cm tissue culture dishes
  • 35°C, 3% CO 2 humidified incubator
  • 35° to 37°C, 5% CO 2 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

Videos

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.
  Provides the basis for BES‐mediated transfection.
   Ishiura et al., 1982. See above.
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