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Related Experiment Videos

Calcium phosphate transfection.

Robert E Kingston1, Claudia A Chen, Hiroto Okayama

  • 1Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.

Current Protocols in Cell Biology
|January 30, 2008
PubMed
Summary
This summary is machine-generated.

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Two calcium phosphate-based methods efficiently transfect eukaryotic cells for transient and stable expression. These protocols introduce plasmid DNA via precipitate, with glycerol or DMSO enhancing efficiency in some cases.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biotechnology

Background:

  • Eukaryotic cell transfection is crucial for genetic studies and biotechnology.
  • Calcium phosphate precipitation is a common method for introducing nucleic acids into cells.
  • Optimizing transfection efficiency for both transient and stable expression remains a key challenge.

Purpose of the Study:

  • To present two distinct calcium phosphate-based methods for eukaryotic cell transfection.
  • To evaluate the efficacy of these methods for both transient and stable transfection.
  • To identify factors influencing transfection efficiency, such as chemical additives.

Main Methods:

  • Two calcium phosphate precipitation techniques were employed for cell transfection.
  • Method 1: Direct layering of a HEPES-buffered calcium phosphate precipitate onto monolayer cell cultures.

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  • Method 2: A BES-buffered system allowing gradual precipitate formation and deposition onto cells, enhancing efficiency.

    • Main Results:

    • Both methods successfully introduced plasmid DNA into eukaryotic cells.
    • The BES-buffered method demonstrated high efficiency for stable transformation with circular plasmid DNA.
    • Similar transfection efficiencies were observed for linear plasmid or genomic DNA, and for transient expression, using both protocols.

    Conclusions:

    • Calcium phosphate-based transfection offers versatile options for eukaryotic cell genetic modification.
    • The choice of buffer system (HEPES vs. BES) impacts efficiency, particularly for stable transformation.
    • Both methods provide reliable means for transient and stable transfection, adaptable with additives like glycerol or DMSO.