Grafting Epiblast Fragments to 65D Embryos

1. Place two drops of PB1 (+10% FCS) medium approx 1 cm apart on the coverslip, and set up the manipulation chamber as Subheading 3.3.2., steps 1-4.

2. Set up the manipulation apparatus, and position the pipets as described in Subheading 3.3.2., steps 5-9.

3. Position and hold the embryo as in Subheading 3.3.2., steps 14-16, except that the embryos is held on the side opposite to the site of grafting (Fig. 8).

4. Pick up the cell clumps into the grafting pipet with a small amount of medium. Draw the cell clumps about 20 | m into the grafting pipet.

5. Insert the grafting pipette by a sharp jabbing action through all tissue layers into the pro-amniotic cavity at the site of transplantation. If necessary, a beveled pipet may be used.

6. Apply a positive pressure to the de Fonbrune syringe to expel the graft from the pipet as the pipet is slowly withdrawn from the embryo (Note 16). A coordinated movement of the pipet and the expulsion of the graft is critical to the precise positioning of the graft in the epiblast (Note 17).

7. Return the embryo to the culture medium (Note 18) (Subheading 1.).

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