Selection of Transfected Mammalian Cells

Richard Mortensen1, Jonathan D. Chestnut2, James P. Hoeffler2, Robert E. Kingston3

1 Brigham and Women's Hospital, Boston, Massachusetts, 2 Invitrogen Corporation, Carlsbad, California, 3 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.6
DOI:  10.1002/0471142301.ns0406s00
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 (the efficiency can vary depending on the cell type). Therefore, a dominant, selectable marker is used to permit isolation of stable transfectants. In the first part of this unit, the procedure for determining selection conditions and the resulting stable transfection is presented and the most commonly used selectable markers are discussed. The second protocol includes conditions for thirteen markers commonly used for selection of mammalian cells. A third protocols describes selection of transfected cells from the total population soon after transfection with plasmids that express both the gene of interest and a selection tag. Optimization of transfection conditions can be facilitated by a simple staining assay detailed in a support protocol.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Stable Transfer of Genes into Mammalian Cells
  • Support Protocol 1: Picking Stable Colonies Using Cloning Cylinders
  • Basic Protocol 2: Selectable Markers for Mammalian Cells
  • Basic Protocol 3: Rapid Selection of Transfected Mammalian Cells
  • Support Protocol 2: Optimization of Contransfection Conditions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Stable Transfer of Genes into Mammalian Cells

  Materials
  • Complete medium
  • Selective medium (see protocol 3)
  • Additional reagents and equipment for picking stable colonies (see protocol 2), mammalian cell culture and counting cells (CPMB APPENDIX ) and transfection (see CPMB UNITS and appendix 1A in this manual)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be followed accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Picking Stable Colonies Using Cloning Cylinders

  • Cloning cylinders
  • 0.05% trypsin/0.06 mM EDTA in PBS ( appendix 2A), 37°C

Basic Protocol 2: Selectable Markers for Mammalian Cells

  Materials
  • Gene of interest
  • Cell line for transfection and appropriate complete medium (CPMB APPENDIX )
  • Capture‐Tec system (Invitrogen) consisting of pHook‐1, pHook‐2, or pHook‐3 plasmid (three different kits are sold) and Capture‐Tec magnetic beads
  • 3 mM EDTA in PBS
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Cell scraper
  • 60‐mm tissue culture plates
  • Magnetic stand (e.g., Invitrogen; other models may be used) or strong magnet
  • End‐over‐end rotating mixer
  • Additional reagents and equipment for mammalian cell culture and counting cells, transfection and subcloning genes (see CPMB APPENDIX 3F, CPMB UNITS and CPMB UNIT , respectively and appendix 1A of this manual)
NOTE: All reagents and equipment coming into contact with live cells must be sterile and proper sterile technique should be followed accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 3: Rapid Selection of Transfected Mammalian Cells

  Materials
  • Magnetically selected and unselected (supernatant) control cells (see protocol 4) transfected with:
    • pHook‐1 and control plasmid pcDNA3.1/His/lacZ (if using pHook‐1)
    • pHook‐2/lacZ (if using pHook‐2)
    • pHook‐3/lacZ (if using pHook‐3)
    • Magnetically selected and unselected (supernatant) mock transfected cells
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Xgal staining solution (see recipe)
  • Tissue culture dishes
  • Inverted microscope
NOTE: All reagents and equipment coming into contact with live cells must be sterile and proper sterile technique should be followed 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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