ES cell microinjection into blastocysts. | © dkfz.de

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 10-15 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
• Transfer of the embryos via transfer capillaries in the uteri of the foster female
• Mating of the born chimeras
• Analysis of the born F1-pubs for the presence of the targeting construct