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7.14A: Putting Foreign DNA into Cells

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    9318
  • [ "article:topic", "authorname:boundless" ]

    The methods used to get DNA into cells are varied (e.g., transformation, transduction, transfection, and electroporation).

    LEARNING OBJECTIVES

    Describe the methods of introducing foreign DNA into cells

    KEY TAKEAWAYS

    Key Points

    • When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation.
    • In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed transfection.
    • Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present).

    Key Terms

    • transformation: The alteration of a bacterial cell caused by the transfer of DNA from another, especially if pathogenic.
    • microorganisms: A microorganism or microbe is a microscopic organism that comprises either a single cell (unicellular), cell clusters, or multicellular relatively complex organisms.

    The DNA mixture, previously manipulated in vitro, is moved back into a living cell, referred to as the host organism. The methods used to get DNA into cells are varied, and the name applied to this step in the molecular cloning process will often depend upon the experimental method that is chosen (e.g., transformation, transduction, transfection, electroporation).

    When microorganisms are able to take up and replicate DNA from their local environment, the process is termed transformation, and cells that are in a physiological state such that they can take up DNA, are said to be competent. In mammalian cell culture, the analogous process of introducing DNA into cells is commonly termed transfection. Both transformation and transfection usually require preparation of the cells through a special growth regime and chemical treatment process that will vary with the specific species and cell types that are used.

    Electroporation uses high voltage electrical pulses to translocate DNA across the cell membrane (and cell wall, if present). In contrast, transduction involves the packaging of DNA into virus-derived particles, and using these virus-like particles to introduce the encapsulated DNA into the cell through a process resembling viral infection. Although electroporation and transduction are highly specialized methods, they may be the most efficient methods to move DNA into cells.

    image

    Electroporation: Diagram of the major components of an electroporator with cuvette loaded.

    Whichever method is used, the introduction of recombinant DNA into the chosen host organism is usually a low efficiency process; that is, only a small fraction of the cells will actually take up DNA. Experimental scientists deal with this issue through a step of artificial genetic selection, in which cells that have not taken up DNA are selectively killed, and only those cells that can actively replicate DNA containing the selectable marker gene encoded by the vector are able to survive.

    When bacterial cells are used as host organisms, the selectable marker is usually a gene that confers resistance to an antibiotic that would otherwise kill the cells, typically ampicillin. Cells harboring the vector will survive when exposed to the antibiotic, while those that have failed to take up vector sequences will die. When mammalian cells (e.g., human or mouse cells) are used, a similar strategy is used, except that the marker gene (in this case typically encoded as part of the kanMX cassette) confers resistance to the antibiotic Geneticin.