21 Transgenie Animals
325
DNA
Fig. 1. Microinjection of DN A into the pronucleus. 1, polar body; 2, zona pellucida; 3, male
pronucleus ; 4, rest of sperm; 5, female pronucleus.
One drop of culture medium is placed in the middle of a glass slide, which
is then overlaid with a drop of paraffin-oil. Oocytes aretransferred into the
drop of medium on the slide using a paraffin-filled glass pipette. Microinjection can only be performed if two pronuclei can be distinguished clearly.
If the two pronuclei are not visible, oocytes are returned into medium and
are examined again later. Ifboth pronuclei can be identified, microinjection
can be performed under 400-fold magnification. Using a micromanipulator, a needle is inserted into the male pronucleus which is slightly larger than
the female pronucleus and 1 pl/oocyte (1 fg/oocyte) of DNA is injected. If
injection is successful, the pronucleus will swell visibly. After injection, oocytes are incubated in M16 medium until oviduct transfer is performed.
Oviduct transfer
Successful transfer of embryos is dependent on synchronization between
embryonie development and timing of pseudopregnancy. The pseudopregnant rat is anesthetized with the Ketavet/Rompun mixture, the mause with
A vertin, as described above.
After opening the body wall by a midline incision in the lower back, the
ovaries and the oviducts are surgically exposed. Hernarrhage from ruptured
325
DNA
Fig. 1. Microinjection of DN A into the pronucleus. 1, polar body; 2, zona pellucida; 3, male
pronucleus ; 4, rest of sperm; 5, female pronucleus.
One drop of culture medium is placed in the middle of a glass slide, which
is then overlaid with a drop of paraffin-oil. Oocytes aretransferred into the
drop of medium on the slide using a paraffin-filled glass pipette. Microinjection can only be performed if two pronuclei can be distinguished clearly.
If the two pronuclei are not visible, oocytes are returned into medium and
are examined again later. Ifboth pronuclei can be identified, microinjection
can be performed under 400-fold magnification. Using a micromanipulator, a needle is inserted into the male pronucleus which is slightly larger than
the female pronucleus and 1 pl/oocyte (1 fg/oocyte) of DNA is injected. If
injection is successful, the pronucleus will swell visibly. After injection, oocytes are incubated in M16 medium until oviduct transfer is performed.
Oviduct transfer
Successful transfer of embryos is dependent on synchronization between
embryonie development and timing of pseudopregnancy. The pseudopregnant rat is anesthetized with the Ketavet/Rompun mixture, the mause with
A vertin, as described above.
After opening the body wall by a midline incision in the lower back, the
ovaries and the oviducts are surgically exposed. Hernarrhage from ruptured