Selection of Transfected Mammalian Cells

Richard M. Mortensen1, Robert E. Kingston2

1 University of Michigan, Ann Arbor, Michigan, 2 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 9.5
DOI:  10.1002/0471142727.mb0905s86
Online Posting Date:  April, 2009
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Abstract

To determine the function of a gene in vitro, expression in heterologous cells is often employed. This can be done by transient expression, but often requires a more permanent expression of the gene and the creation of a cell line. This process can involve decisions as to the nature of construct used for expression, and invariably uses some strategy to select the transfected cells. Typically, these strategies use one of a number of genes that confer resistance to an added drug that will kill untransfected cells but not the transfected cells (positive selection). Alternatively, sometimes the strategy uses a gene that will confer sensitivity to a compound and kills the transfected cells (negative selection). This chapter discusses some of the strategies and genes used in creating cell line for in vitro study of gene function. Curr. Protoc. Mol. Biol. 86:9.5.1‐9.5.13. © 2009 by John Wiley & Sons, Inc.

Keywords: stable integration; selection; mammalian cell; stable transfection; selection marker; Cre‐lox; Flp‐FRT; expression

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Stable Transfer of Genes into Mammalian Cells
  • Basic Protocol 2: Selectable Markers for Mammalian Cells
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Stable Transfer of Genes into Mammalian Cells

  Materials
  • Complete medium ( appendix 3F)
  • Selective medium (see protocol 2)
  • Cloning cylinders (see recipe; also see Fig. 16.23.2)
  • Additional reagents and equipment for mammalian cell culture and counting cells ( appendix 3F) and transfection (units 9.1, 9.3, & 9.4)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

Videos

Literature Cited

Literature Cited
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