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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 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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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
Bibliography
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 (cpmb unit 2.1A) and mammalian cell tissue culture (cpmb appendix 3F)

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

Alternate Protocol: 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 CaCl₂ (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% 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 A.1C.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.