DNA microinjection

DNA microinjection

DNA microinjection into 1-cell embryo (zygote)
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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 to induce pseudopregnancy
  • Transfer of the 1 or 2-cell embryos via transfer capillaries into the oviduct of a foster female
  • Analysis of the newborn pups for transgenesis

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