ES cell culture / Gene Targeting

ES cells in culture.

ES cells in culture.
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Via homologous recombination in embryonic stem cells (ES cells), it is possible to generate a specific genetic modification at a specific location in the genome.

  • Day 1: Preparation of ES cell medium with LIF (leukaemia inhibitory factor) and thawing of feeder (inactivated mouse embryonic fibroblasts)
  • Day 2: Thawing and plating of ES cells on feeder plates
  • Day 4: Sub-confluent ES cell colonies are washed twice with PBS and treated with trypsin to obtain a single cell suspension. The ES cells are either replated with a 1 to 4 dilution or 5x10E6 cells are electroporated with 20-50 µg targeting construct
  • Day 5: Start of the selection (e.g. G418)
  • Day 14: Isolation of ES cell clones
  • Later: Freezing and DNA isolation of part of the ES cell clones. Detection of positive clones with PCR or Southern Blotting.

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