Microinjection

After U2 snRNA and pre-mRNA have been transcribed and healthy oocytes have been separated and kept at 18°C, the next crucial step is to carry out three sequential microinjections (Fig. 1). The microinjection protocols, including the preparatory steps of making the needles, are described next.

3.3.1. Preparation of Needles for Microinjection

To carry out successful microinjections, good needles are essential. The needles are made using a micropipet puller. We use 7-in. Drummond tubes with the following program to create a fine tip: heat = 310, pull = 65, velocity = 50, time = 150. The needles can be made in a large batch and stored. The tip of the needle is snapped off to create a blunt end. We suggest trying different pulling conditions to find the best one for each laboratory.

3.3.2. Microinjection Protocols

3.3.2.1. Day 1: Cytoplasmic Injection of an Antisense U2 DNA Oligonucleotide (Fig. 6A)

1. Press "empty" on the injector to near completion.

2. Fill the needle with mineral oil using a syringe.

3. Assemble the needle onto the injector (Fig. 5).

4. Set the volume to 50.6 nL and set speed to "fast speed" on a dip switch.

5. Pipet 2 ||L of an antisense U2 DNA oligonucleotide (3 |g/|lL) on a piece of stretched-out parafilm (for a control experiment, pipet 2 |L of water instead) (see Note 8).

6. Adjust the microscope so that both the sample on the parafilm and the needle tip are in the field of view.

7. Lower the needle so that the tip is placed inside of the drop of the sample. Make sure the tip is not touching the parafilm.

8. Press "fill" to fill the needle with the sample (see Note 9).

9. Place a batch of 20 oocytes to be injected on a meshed Petri dish filled with 1X MBS buffer (see Note 10).

10. Place the needle above the buffer, press "inject," and observe that a small drop of sample exits (see Note 11).

11. Using a pair of forceps, position the oocytes with their midlines (between animal hemisphere and vegetal hemisphere) directly toward the tip of the needle (Fig. 6A) (see Note 12).

12. Inject the sample into the cytoplasm at the midline, pause 1 s after the injection, and take out the needle slowly after the pause (see Note 13).

13. Inject the sample into the rest of the oocytes, repeating step 10 after every five oocytes (see Note 14).

14. Incubate overnight (see Note 15).

Fig. 5. The assembly of the needle onto the injector is shown.

3.3.2.2. Day 2: Cytoplasmic Injection of Rescuing U2 snRNA (Fig. 6A)

15. Follow steps 1 to 4.

16. Pipet 2 ||L of rescuing U2 (50 ng/|L, wild-type or 5FU-substituted U2) on a piece of stretched-out parafilm (for a control, pipet 2 |L of water instead).

3.3.2.3. Day 3: Nuclear Injection of Radiolabeled pre-mRNA (Fig. 6B) (see Note 17)

18. Follow steps 1 to 3.

19. Set the volume to 13.8 nL and set speed to "fast speed" on a dip switch.

20. Pipet 1 |L of adenovirus pre-mRNA (500,000 cpm/|L) on a piece of stretched-out parafilm.

21. Follow steps 6 to 10.

22. Using a pair of forceps, position the oocytes with the center of the animal hemisphere toward the tip of the needle.

23. Aim the tip at the center of the animal hemisphere (Fig. 6B) and carefully lower the needle so that the tip has entered the outer membrane and the nucleus but has not gone completely through the nucleus.

Fig. 6. The relative positions of the glass needle and the oocytes in (A) cytoplasmic injection and (B) nuclear injection are shown.

24. Press "inject," pause 2 s, and take out the needle slowly after the pause.

25. Inject the rest of the oocytes, repeating step 10 after every five oocytes.

26. Incubate for 1 h in 1X MBS buffer.

27. Proceed with nuclear isolation.

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