Stable Transfer of Genes into Mammalian Cells

Robert E. Kingston1

1 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.17A
DOI:  10.1002/0471142735.im1017as12
Online Posting Date:  May, 2001
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Abstract

Analysis of gene function frequently requires the formation of mammalian cell lines that contain the studied gene in a stably integrated form. Approximately one in 104 cells in a transfection will stably integrate DNA, so a dominant selectable marker is used to permit isolation of stable transfectants. Appropriate selection conditions for the parental cell line are first determined, and the gene to be studied is then transfected into that cell line with a gene that expresses a selectable marker. The cells are allowed to grow under selection for approximately 10 doublings before individual colonies are picked and expanded into cell lines. This unit includes conditions for six markers commonly used for selection of mammalian cells. An alternative, specialized procedure for transferring immunoglobulin chains into lymphoid cells is presented in Unit .

     
 
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Table of Contents

  • Selectable Markers and Selective Medium for Mammalian Cells
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1:

  Materials
  • Exponentially growing eukaryotic cells
  • Complete medium (appropriate for cell line)
  • Selective medium (see )
  • Plasmid DNA (CsCl purified; units 10.3 & 10.13)
  • 10‐cm tissue culture dishes
  • Cloning cylinders
  • Additional reagents and equipment for transfection (units 10.13, 10.15 & 10.16)
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Figures

Videos

Literature Cited

Literature Cited
   Perucho, M., Hanahan, D., and Wigler, M. 1980. Genetic and physical linkage of exogenous sequences in transformed cells. Cell 22:309‐317.
   Robins, D.M., Ripley, S., Henderson, A.S., and Axel, R. 1981. Transforming DNA integrates into the host chromosome. Cell 23:29‐39.
   Wigler, M., Silverstein, S., Lee, L‐S., Pellicer, A., Cheng, Y‐C., and Axel, R. 1977. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell 11:223‐232.
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