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Antibiotic selection of gene targeted mESCs

This protocol describes the use of selectable markers during gene targeting.

Category
Protocol
Record History
Added on May 14, 2010 at 2:09 PM by Lindner, Jill
Modified on June 25, 2012 at 12:30 PM by Lindner, Jill
Shared with (contributions)
VCSCBi: Training / Protocols Master

Reagents, supplies, and equipment:

  • cES media + antibiotic(s) (warm to 37ºC)
  • Geneticin (G-418) at 200 µg/ml for vector containing the neo cassette (used for positive/single selection only)
  • Hygromycin B at 200 µg/ml for hygro cassette
  • Puromycin at 1.5µg/ml for pu-Δtk cassette
  • Gancyclovir at 2 µM for vector containing the HSVtk cassette

    Note: double selection (both positive and negative selection): Select with G-418 or hygromycin B (at 200 µg/ml) + gancyclovir at 2 µM for vector containing both  neo or hygro and the HSVtk cassette.

Procedure: Single selection

  1. Begin selection 24 or 48 hr after electroporation by feeding each 150 mm dish with 25 ml cES + G-418 (or hygromycin B or puromycin).
  2. Feed daily until all non-targeted colonies have died and surviving colonies may be picked, usually 6-8 days.

Procedure: Double selection

  1. Begin selection 24 hr after electroporation by feeding each 150 mm dish with 25 ml cES + G-418 (or hygromycin B). Feed with single selection media for 2 days.
  2. Begin second selection 72 hr after electroporation by feeding all but one 150 mm dish with cES + G-418 (or hygromycin B) + ganciclovir. Leave one dish with single selection media to use as a control for double selection efficiency. There should be considerable difference in the number of surviving colonies between the single and double selection.
  3. Feed daily until all non-targeted colonies have died and surviving colonies may be picked, usually 7-10 days.

    Note: Vectors containing DT (diphtheria toxin) as a negative selector, no additional component is added to the media.

Written by Susan Hipkens
Revised: 9/16/05