ES cell microinjection

ES cell microinjection into blastocysts.

ES cell microinjection into blastocysts.

With this technique one could generate a specific genetic modification in mice.

Embryo isolation

  • Superovulation of donor females with i.p. injection of Gonadotropin and 47 hrs. later with Chorionic Gonadotropin
  • Mating of the treated females with fertile males
  • Identification of the mated females by plug check
  • Euthanasia of the donor females by cervical dislocation and preparation of the embryos under the microscope
  • Washing of the embryos through several droplets of medium and incubation in the incubator

Blastocyst injection

  • Thawing and culturing of the ES cell clone on a layer of inactivated mouse embryonic fibroblasts (feeder)
  • Harvesting and separation of ES cells and fibroblasts
  • Isolation of blastocysts from superovulated donor females
  • Transfer of 10 embryos in a droplet of medium covered with silicon oil in the injection chamber
  • Microinjection of 5-15 genetically modified ES cells into the blastocyst cavity
  • Incubation of the injected embryos in a CO2 incubator for 1-2 hrs

Transfer of injected blastocysts

  • Mating of the foster females with vasectomized males to induce pseudopregnancy
  • Transfer of the embryos via transfer capillaries into the uteri of the foster female
  • Mating of the born chimeras
  • Analysis of the born F1-pups for the presence of the targeting construct

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