Cloning Mice

Part 1: Preparing Micropipettes

• For efficient enucleation and injection with a piezo-driven micromanipulation device (Fig. 2), the wall of the enucleation and injection pipettes must be thin (less than one-fourth the size of the radius). The tips of the pipettes must be blunt. Using tapered tips often results in low oocyte-survival rates after enucleation or injection.

View larger version:

• In this window
• In a new window

Figure 2.

Impact unit of the piezo-driven micromanipulator system (right) attached to an injection pipette holder on an inverted microscope.

• 1. Break the tips of the pipettes vertically using a microforge to create a blunt end (see Fig. 1A in Protocol: Pronuclear Transplantation in the Mouse Embryo [McGrath et al. 2017]).

  • Blunt-ended pipettes are able to break the zona pellucida and oolemma but cause minimal damage to the oocyte. The inner diameter of the enucleation pipettes is between 7 and 8 μm.

  • It is possible to thin the wall of the capillary with hydrofluoric acid, which may slightly improve the oocyte survival rate, but it is not necessary.

• 2. Adjust the inner diameter of the injection micropipettes according to the size and hardness of the donor cells.

  • Inner diameters of 4–5 μm for cumulus cells and primordial germ cells, 3–4 μm for immature Sertoli cells, and 7–9 μm for fibroblast cells are recommended.

• 3. Prepare holding pipettes the same way as for other mouse embryo manipulations (see Protocol: Making Holding Pipettes [Nagy et al. 2006a]).

Part 2: Setting Up the Enucleation and Injection Micropipettes

• This step has a significant effect on the efficiency of nuclear transfer. If the setup is performed correctly and carefully, 100–150 oocytes can be injected before the injection pipette needs to be changed.

• There are two reagents that can be used to stabilize piezo-driven microcapillaries. Mercury is the traditional choice, although it is toxic and requires specialized handling and disposal. A newer alternative is perfluoro compounds, such as Fluorinert, also called FC-77. Fluorinert is chemically stable, nonflammable, and considered nontoxic, although it can cause eye, skin, and respiratory irritation. Both options are described here; perform either Step 4 or Step 5.

• 4. Use mercury when setting up the micropipettes.

  • i. Load a small quantity of mercury (1–2 mm long when in the micropipette) into the enucleation/injection micropipette from the proximal end using a small, flexible plastic capillary tube attached to a 1-mL syringe.

  • ii. Attach the enucleation/injection micropipette that is connected to the oil-filled injector to the instrument holder of the piezo impact drive. After expelling the air from the tip of the enucleation/injection micropipette, wash the inside of the tip several times with oil while it is in the micromanipulation chamber (Fig. 3A).

  • iii. Wash the enucleation/injection micropipette in PVP medium until the mercury inside the pipette can be moved smoothly and without any time lag (Fig. 3B).

    • Take care to completely remove any oil adhering to the inside of the pipette.

  • iv. Place a small volume of oil between the PVP and the CZB-HEPES medium to prevent the mercury from contaminating the micromanipulation medium (Fig. 3C).

  • v. Proceed to Step 6.

• 5. Use Fluorinert when setting up the micropipettes.

  • i. Load Fluorinert to backfill the entire enucleation/injection micropipette from the proximal end using a small, flexible plastic capillary tube.

  • ii. Attach the enucleation/injection micropipette that is connected to the oil-filled injector to the instrument holder of the piezo impact drive.

  • iii. Expel the air from the tip of the enucleation/injection micropipette, and then wash the inside of the tip several times in injection medium while it is in the micromanipulation chamber. Maintain a single medium–Fluorinert interface close to the tip of the needle to ensure smooth operation.

  • iv. Proceed to Step 6.

• 6. Use the top of a 50-mm plastic Petri dish as the micromanipulation chamber.

  • If Nomarski (differential interference contrast) optics are used, replace the center of the chamber with a glass coverslip.

• 7. Place 2- to 4-μL drops of the following media on the dish and cover with silicone or mineral oil: (1) PVP drops for pipette cleaning, (2) drops of CZB-HEPES containing cytochalasin B for enucleation of oocytes, and (3) CZB-HEPES drops for donor nucleus injection.

View larger version:

• In this window
• In a new window

Figure 3.

Setting up an enucleation pipette. The procedure is the same for injection pipettes (see text for details). Note that the PVP drop and pipette are under oil. (A) Air is expelled from the tip of the enucleation/injection micropipette; the tip is washed several times with oil and then with PVP medium (B) until the mercury is moved smoothly inside the pipette. (C) A small volume of oil is placed between the PVP and the CZB-HEPES medium to prevent the mercury from contaminating the micromanipulation medium.

Part 3: Collecting and Enucleating Oocytes

• 8. Inject mature female mice intraperitoneally with 7.5 IU of PMSG. Let the mice rest for 48 h. Inject them with 7.5 IU of hCG.

• 9. Collect mature oocytes that are surrounded by cumulus cells from the ampullae of the oviducts 15–17 h after the hCG injection.

  • Oocytes retrieved from the oviducts sooner than recommended (e.g., 13 h) after hCG treatment generally show better developmental ability but are less tolerant to intracytoplasmic injection.

• 10. Place the collected oocytes in CZB medium containing 0.1% hyaluronidase, and then place them in a 37°C 5% CO₂ humidified incubator until the cumulus cells disperse (2–3 min).

• 11. Wash the oocytes, and place them in a microdrop culture dish in the incubator.

• 12. Place a group of 10–20 oocytes into a drop of CZB-HEPES medium containing cytochalasin B in a micromanipulation chamber.

• 13. Hold the oocytes with the metaphase plate between the 2 o’clock and 4 o’clock positions.

• 14. Place the tip of the enucleation pipette onto the surface of the zona pellucida.

• 15. Use a few piezo pulses (controller settings: speed 3–6, intensity 1–4) to advance the pipette while applying very slight negative pressure.

  • To avoid damaging the oolemma, apply slight positive pressure when the perivitelline space narrows under the impacted area just before the entire zona is pierced.

• 16. Remove the chromosomes by suction, along with a small volume of cytoplasm.

  • Warming the microscope stage is recommended because the spindle microtubules will remain well polymerized and form a small clear area that can be easily distinguished from the surrounding opaque cytoplasm.

  • Metaphase II chromosomes are visible without the use of fluorescent dye under Nomarski or Hoffman optics. Oocytes with a clear cytoplasm (such as B6D2F1 oocytes; see above) can be more easily enucleated than those with granulated masses (such as B6 and B6CBAF1 oocytes).

• 17. After a group of oocytes has been enucleated, wash the oocytes several times in drops of fresh CZB medium.

• 18. Leave the oocytes in the incubator for 30 min to 2 h before donor nucleus injection or electrofusion.

  • Oocytes are very sensitive and can easily lyse just after enucleation. However, their viability is usually restored after they have been incubated as described.

Part 4: Preparing Nucleus Donor Cells

• 19. Collect cumulus cells from freshly ovulated donor oocytes by treatment with 0.1% bovine testicular hyaluronidase (Sigma-Aldrich H3884) in CZB medium, as described above in Part 3: Collecting and Enucleating Oocytes (and Protocol: Collecting Zygotes and Removing Cumulus Cells with Hyaluronidase [Nagy et al. 2006b]).

Part 5: Injecting Donor Nuclei

• In the original trials of intracytoplasmic injection of mouse oocytes, the micromanipulation temperature was maintained at 17°C–18°C to enhance the survival rate. However, this is not necessary, and the operation can be performed at room temperature without any problems. A heated stage may cause adverse effects such as oocyte lysis.

• 20. Gently mix the donor cumulus cells into the PVP drops in the micromanipulation chamber.

  • Use cumulus cells from an F₁ hybrid for initial mouse cloning attempts.

• 21. Place a group of 10–20 enucleated oocytes in a drop of CZB-HEPES in a micromanipulation chamber.

• 22. Remove the nuclei from the donor cells by gently aspirating them in and out of the injection pipette. Then, draw up the donor nuclei in a line within the injection pipette (Fig. 4A,B).

  • When the operator becomes sufficiently skilled, several (three to 10) nuclei can be aspirated into the injection pipette and injected at one time. This operation saves time handling the oocytes in vitro.

• 23. After advancing the injection pipette through the zona pellucida with a few piezo pulses, as described in Step 15, insert the pipette deep into the ooplasm while pushing forward a donor nucleus to the tip of the pipette (Fig. 4C,D).

• 24. After leaving the pipette inside the oocyte for a few seconds, apply an empirically determined single piezo pulse of minimal intensity. The plasma membrane will be punctured at the pipette tip, as evidenced by a rapid relaxation of the membrane. Expel the nucleus into the ooplasm with a minimal amount of medium (Fig. 4D–F).

  • For successful transfer of nuclei from more solid cells, such as fibroblasts, it is necessary to remove the entire plasma membrane before injection. If the rate of nuclear formation is poor (<50%) among surviving oocytes, nuclear transfer by electrofusion is recommended (Ogura et al. 2000b). For transfer of nuclei from soft cells, such as cumulus cells (Wakayama et al. 1998), ES cells (Wakayama et al. 1999), immature Sertoli cells (Ogura et al. 2000a), or primordial germ cells (Lee et al. 2002), partial damage to the plasma membrane is usually sufficient for the nuclei to intermingle with the ooplasm.

• 25. Gently withdraw the pipette (relatively rapidly at first and then slowly).

• 26. Leave the injected oocytes for 5–10 min at room temperature. Then, wash and culture the injected oocytes in CZB medium until they are activated with strontium.

View larger version:

• In this window
• In a new window

Figure 4.

Injection of a cumulus cell nucleus into an enucleated oocyte. (A) Dispersed cumulus cells; (B) cumulus cell nuclei (arrowheads) in injection pipette; (C) piercing the zona pellucida with the injection pipette; (D) pushing the injection pipette deep into the enucleated oocyte but not breaking the oolemma. (E) Application of the piezo pulse breaks the oolemma, causing a visual relaxation of the membrane (cf. the lengths of the double arrows in D and E). (F) Injection of the donor cumulus cell nucleus (arrowhead) into the ooplasm.

Part 6: Activating Embryos and Culturing

• Reconstructed embryos require a signal to activate development. This is induced by culturing the reconstructed embryos in medium containing strontium.

• 27. Approximately 1 h after nuclear transfer, activate the oocytes in Ca²⁺-free CZB medium containing 2.5–10 mm strontium chloride (CZB-Sr) and 5 µg/mL cytochalasin B at 37°C in 5% CO₂.

  • The optimal concentration of strontium chloride and time of exposure to the activation medium may vary between laboratories. Contamination of the CZB-Sr medium for activation by Ca²⁺ carryover from the CZB medium decreases the effect of Sr²⁺. Therefore, the reconstructed embryos should be washed free of CZB before being activated in CZB-Sr.

  • The presence of 1% DMSO in the CZB-Sr activation medium can enhance the reconstructed embryos to develop into blastocysts (Wakayama and Yanagimachi 2001).

• 28. Approximately 1 h later, place the activated oocytes in CZB medium containing 5 µg/mL cytochalasin B. Culture for 5 h.

  • Cytochalasin B is added to the activation medium to prevent extrusion of the donor chromosomes as a polar body. The effects of cytochalasin B and cytochalasin D on cloning experiments do not appear to differ.

• 29. Wash the reconstructed embryos several times in fresh CZB medium and then culture until the embryos are transferred.

  • It is important to wash away the cytochalasin thoroughly and quickly because concentrations of cytochalasin <5 μg/mL often cause deformation of the oocyte surface.

  • Treatment with TSA, a histone deacetylase inhibitor, for 10 h following oocyte activation can enhance the reconstructed embryos to develop into term offspring (Kishigami et al. 2006).

• 30. Roughly assess the success of nuclear transfer by examining the pseudopronuclei of the reconstructed embryos. The embryos should have two or three well-developed pseudopronuclei when they are retrieved from the cytochalasin-containing medium. If none or only one nucleus has formed, the oocytes were activated following nuclear transfer because of inappropriate in vitro handling. Such embryos rarely develop to term (Wakayama and Yanagimachi 2001).

Part 7: Transferring Cloned Embryos

• 31. Transfer the cloned embryos into the oviducts or uteri, as in other experiments (see Protocol: Oviduct Transfer [Nagy et al. 2006c] and Protocol: Uterine Transfer [Nagy et al. 2006d], respectively).

  • Special care must be exercised (see Discussion) because cloned embryos are much more sensitive to changes in physical and physiological conditions than are embryos produced by conventional in vitro techniques, such as in vitro fertilization.