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Selection of Transfected Mammalian Cells

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

1Brigham and Women's Hospital, Boston, Massachusetts
2Invitrogen Corporation, Carlsbad, California
3Massachusetts 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

  • Unit Introduction
  • 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 Basic Protocol 2)
  • Additional reagents and equipment for picking stable colonies (see Support Protocol 1), mammalian cell culture and counting cells (cpmb appendix 3F) and transfection (see cpmb units 9.1-9.4 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% CO2 incubator unless otherwise specified.


Support Protocol 1: Picking Stable Colonies Using Cloning Cylinders

 Additional Materials (also see Basic Protocol 1)
  • Cloning cylinders
  • 0.05% trypsin/0.06 mM EDTA in PBS (appendix 2A), 37°C

Basic Protocol 3: Rapid Selection of Transfected Mammalian Cells

 Materials
  • Gene of interest
  • Cell line for transfection and appropriate complete medium (cpmb appendix 3F)
  • 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 3F, cpmb units 9.1-9.4 and cpmb unit 3.16, 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% CO2 incubator unless otherwise specified.


Support Protocol 2: Optimization of Contransfection Conditions

 Materials
  • Magnetically selected and unselected (supernatant) control cells (see Basic Protocol 3) 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% CO2 incubator unless otherwise specified.


Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 4.6.1
    Generalized construct for expression of genes in mammalian cells. Each integrated construct must contain a promoter region with a transcriptional start site and a coding sequence. Features that increase expression include translational start sites according to Kozak's rules, intervening sequences with splice donor and acceptor sites and polyadenylation signals. Abbreviation: IVS; intervening sequence.

    View Image
  • Figure 4.6.2
    Using the Cre-lox system to remove a selectable marker after integration at a unique site. Transient expression of Cre recombinase causes the excision of sequences between the lox sites.

    View Image
  • Figure 4.6.3
    Using the Cre-lox system to integrate an expression construct at a specific reproducible site. The target site is a previous integrated defective form of a selectable marker (lacking a promoter and ATG start codon). Transfection with a plasmid containing the gene of interest leads to integration at the lox site and adds a promoter and translational start site, restoring expression of the selectable marker.

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  • Figure 4.6.4
    Placement of cloning cylinder around CHO colony.

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  • Figure 4.6.5
    Plasmid maps of pHook-1, pHook-2 and pHook-3. pHook-1 expresses a fusion protein consisting of the signal peptide from Ig V-J2-C region fused with the anti-phOx sFv and the transmembrane domain from the PDGF receptor. pHook-2 and pHook-3 express the same fusion protein but also contain a separate expression cassette consisting of the CMV promoter, a multiple cloning site and a polyadenylation signal. Definitions: EM-7, synthetic bacterial promoter; HA, hemagglutinin epitope tag; myc-1, myc epitope tag (see text for additional definitions).

    View Image
  • Figure 4.6.6
    Magnetic cell-selection protocol. (A) Cells are transfected with one of the pHook vectors and returned to culture for 2 to 24 hr. (B) The cell population, including transfected cells displaying the sFv hook (dark circles) and untransfected cells (open circles), is then harvested and incubated with antigen-coated magnetic beads (small dots) for 30 min. (C) Selection of transfected cells is achieved by exposing the cell/bead mixture to a magnet and discarding cells that are not retained by the magnet.

    View Image

Literature Cited

Literature Cited
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