• Culture dishes containing microdrops of M16 or KSOM, overlaid with mineral oil

• Micromanipulator assembly, consisting of an inverted fixed-stage microscope (or upright fixed-stage microscope with image-erected optics), micromanipulators fitted with a holding pipette and a microinjection pipette, and a micromanipulation chamber or glass depression slide

1 Set up microdrops of M2 or FHM in the microinjection chamber, under oil.

2 Secure an embryo to the holding pipette by applying negative pressure to the zona pellucida.

3 Push the fine needle through the zona pellucida and into the perivitelline space of the embryo, and out again through the zona pellucida (Figure 1A).

4 Release the embryo from the holding pipette whilst it remains impaled on the needle

5 Press the needle repeatedly against the wall of the holding pipette to produce a slit through the zona pellucida, along 10-20% of the length of the embryo (Figure 1C): be careful not to press too hard against the holding pipette, else the needle will break.b

6 Reorientate the embryo by micromanipulation with the holding pipette, so that the holding pipette is opposite to the slit.

7 Replace the needle with a microinjection pipette, and inject 5-10 mES cells through the slit in the zona pellucida and into the perivitelline space of the embryo (Figure 1D).

8 Pool and culture manipulated and injected embryos in microdrops of M16 or KSOM.

9 After 1-2 d of culture, transfer those embryos having reached the compacted morula or blastocyst stages to recipient females (see Section 2).

a Alternatively, mES cells may be injected into tetraploid blastocysts, which are obtained by electrofusion at the two-cell stage (Protocol 2) followed by culture for 3 d in microdrops of M16 or KSOM. Typically, 5-10 mES cells are injected per blastocyst.

bBy extending the slit to 50% of the length of the embryo, this manipulation may conveniently be used to remove entirely the zonae pellucidae from small numbers of embryos (see Protocol 3).

(D) -^

Figure 1 Diagram illustrating the technique for cutting the zona pelluclda of a tetraplold morula, and microinjection of mES cells. Stages (A) to (D) of the procedure are explained in Protocol 5.

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