DNA microinjection | © dkfz.de

With this technique most DNA-sequences can be incorporated into the mouse genome. The integration of the exogenous DNA is non-specific.

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
• Separation of the cumulus cells and the zygotes by short treatment with hyaluronidase
• Washing of the embryos through several droplets of medium and incubation in the incubator

DNA injection

• Transfer of 20-30 embryos in a droplet of medium covered with silicon oil in the injection chamber
• Fixation of an embryo at the holding pipette by negative pressure
• Injection of 1-2-pl DNA in one of the pronuclei of the 1-cell embryo (zygote)
• Incubation of the injected embryos in a CO2 incubator for 1-2 hrs. or overnight

Transfer of the injected embryos

• Mating of the foster females with vasectomized males
• Transfer of the 1 or 2-cell embryos via transfer capillaries into the infundibulum of the oviduct of a foster female
• Analysis of the born pubs for transgenesis