Summary
This movie and the protocol are intended to help learning nuclear transfer.
Abstract
Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).Protocol
Preparations:
- A detailed description of superovulation and microdrop embryo culture can be found elsewhere 2.
- Prepare a petri dish with microdrops of embryo culture medium (e.g. KSOM, Chemicon). Cover with mineral oil (mineral oil batches should be tested for compatibility with embryo culture). Equilibrate at 37°C in air plus 5% CO2. (In Thermo-Electron 3110 water jacketed incubator or equivalent)
- Prepare a dish for the isolation of oocytes: Place drops of hepes buffered CZB & hyaluronidase (0.1% w/v, Sigma) in one half of the dish and HCZB in the other half. Cover with mineral oil. Keep the dish warm on a heated stage of a dissection microscope.
- Prepare the microscope: Place a holding pipette of an outer diameter of about 50µm (slightly smaller than the diameter of the oocyte), and an opening of about 20µm in one pipette holder. Holding pipettes are easy to make with the necessary equipment, a needle puller (Sutter Instruments) and a microforge (Narishige), but they may also be purchased from Humagen.
Load 3-5 µl mercury into the tail of a micropipette (inner diameter 8µm). Avoid mercury spills. Prepare a dish with several small drops each of 11% w/v PVP (polyvinylpyrrolidone) in HCZB and 5µg/ml cytochalasin B in HCZB. Use the lid of a Petri dish rather than the dish itself as the lid has a short rim that will not interfere with the movement of the holding and the enucleation pipettes. Align the holding pipette and the enucleation pipette in an HCZB drop. Aspirate a little HZCB into the tip of the holding pipette. - Preparation of donor cells depends largely on the cell type and the experiment the investigator intends to perform. In general, donor cells should be treated with a proteolytic enzyme such as trypsin and kept on ice to reduce stickiness until transfer.
- Humanely euthanize mice 14-15 hours after the administration of hCG. Harvest oviducts and dissect them in the warm HCZB-hyaluronidase drops. After approx. 5 min, oocytes will be mostly free from cumulus cells. They should then be washed several times in HCZB, then washed further in the pre-equilibrated KSOM drops, and kept in the incubator until enucleation.
Enucleation
Manipulations are done with hydraulic micromanipulators, (Narishige) on an inverted microscope, such as the Nikon TE200. A piezo micromanipulator (Primetech) is used for drilling the zona pellucida. Flat-tipped enucleation and transfer pipettes can be purchased from Humagen. Clean and lubricate the enucleation pipette in the PVP drops by expelling microscopic mercury droplets and by aspirating and expelling PVP. To prevent stickiness of the needle, the same procedure should be done between each group of oocytes that are either enucleated or transferred.
Place a small group of oocytes into the HCZB-cytochalasin B drop. The size of the group should be adapted to the level of experience of the investigator. Oocytes should not be kept on the stage for longer than 20-30 min. Move the instruments to the HCZB-cytochalasin B drops. The holding pipette and the enucleation pipette should be brought into alignment with the equatorial plane of the oocyte. This will be possible if the holding pipette has an outer diameter that is slightly smaller than the oocyte itself. Rotate an oocyte until the differently refractive metaphase spindle can be seen. If a beginner cannot see the spindle, the metaphase plate can be identified using the DNA dye Hoechst and UV illumination, however, that is not compatible with efficient embryonic development. Position the metaphase spindle at 3 o’clock and hold it well with the holding pipette. Apply piezo pulses to penetrate the zona pellucida. Touch the metaphase plate with the enucleation pipette. Once the resistance of the metaphase spindle can be ‘felt’, aspirate the spindle and withdraw the needle. The oocyte cytoplasm is fluid, the metaphase spindle however moves as a clump when touched with the pipette. Try to reduce the amount of cytoplasm that is removed with the spindle. After a group of oocytes has been enucleated, wash them through embryo culture medium and place them into the incubator until transfer. Some enucleated oocytes should not be transferred but should be activated and serve as a control for enucleation. These oocytes will fragment within several hours, indicating successful enucleation.
Nuclear transfer
Place cells into PVP solution (11% PVP in HCZB). If many clones arrest at the 1-cell stage, the PVP concentration should be lowered to 5% or less. Mix cells well with PVP. Pick up cells with a pipette with an inner diameter slightly smaller than the diameter of the cell. The membrane of the cell should break upon aspiration. In some cases, repetitive expelling and aspiration as well as the application of gentle piezo pulses can be used to break the donor cell. A new drop of donor cells should be made with each group of oocytes being transferred, as donor cells become sticky after some time. Inject the broken donor cell shortly after breaking the membrane. For the transfer, focus on the equatorial plane of the oocyte itself rather than the equatorial plane of the zona pellucida, and position the transfer and the holding pipette in the same plane. Hold the oocyte including a part of its cytoplasm firmly. Penetrate the zona pellucida using piezo pulses. Bring the donor nucleus to the tip of the pipette, then push the pipette tip almost to the opposite side of the enucleated oocyte, close to the holding pipette, making a deep furrow in the oocyte. Aspirate a very small amount of oocyte cytoplasm into the pipette, apply a single weak piezo pulse to break the oocyte membrane, eject the donor nucleus, withdraw the needle rapidly while aspirating at the cytoplasmic membrane at the right end of the furrow. By simultaneously withdrawing the needle and aspirating, it should be possible to close the hole left behind by NT. This 'hole removal technique will reduce the number of oocytes that lyse during NT. Wash the reconstructed oocytes in KSOM and return them to the incubator for 1-3 hours.
Oocyte activation
Prepare one half of a dish with droplets of calcium free MCZB and the other half with droplets of calcium free MCZB plus 10mM Sr2+ and 5µg/ml cytochalasin B to inhibit polar body extrusion. Equilibrate for 30 min. under 5% CO2 in air. Wash NT embryos well in calcium-free MCZB and then place them in small groups in different drops of calcium-free MCZB. Return the dish to the incubator and culture for 5-6 hours. To control for the artificial activation protocol and for embryo culture conditions, non-enucleated oocytes should be used. They will develop as parthenogenetic embryos. Following activation, wash the embryos through KSOM and place embryos in the incubator for long-term culture. Pronuclei should be visible at this time point, indicating successful transfer.
Embryo Culture Media Cookbook
Master Salts
Begin with 980 mL ultrapure H2O in sterile 1 L bottle
Add dry components:
NaCl 4760 mg (81mM) Sigma S-5886
KCl 360 mg (5mM) Sigma P-5405
MgSO4 • 7H2O 290 mg (1.18mM) Sigma M-2773
KH2PO4 160 mg (1.17mM) Sigma P-5655
EDTA 2NA 40 mg (0.1mM) Sigma E-6635
Glucose (D) 1000 mg (5.5mM) Sigma G-6152
Add liquid Components
Na-lactate (lactic acid) 5.3 mL Sigma 44263
Pen’Strep 10 mL Gibco 15140-122
For MCZB Stock Salts:
Filter sterilize 500 mL master salts (good for 3-4 months; store at 4° C)
For HCZB Stock Salts:
Start with 500 mL master salts
Add 50 mg PVA (cold-soluble; Sigma P-8136)
Stir for 30-60 min and sterile filter (good for 3 months; store at 4° C)
Ca++ Free MCZB (for oocytes activation in 5% CO2)
Start with 99 mL MCZB stock salts
Add dry components:
NaHCO3 211 mg Sigma S-5761
Na-pyruvate (pyruvic acid) 3 mg Sigma P-4562
L-Glutamine 15mg Sigma G-8540
Bovine serum albumin (BSA) 500 mg Sigma A-3311
Swirl until all components are dissolved; sterile filter.
HCZB (for micromanipulation in ambient atmosphere)
Start with 99 mL HCZB stock salts
Add dry components:
Hepes-Na 520 mg Sigma H-3784
NaHCO3 42 mg Sigma S-5761
Add liquid components:
128 mM CaCl2 (18.8g/l) 1 mL Sigma C-7902
Adjust pH to 7.5 with 1 N HCl
Swirl until dissolved; sterile filter.
Subscription Required. Please recommend JoVE to your librarian.
Discussion
Good luck!
Subscription Required. Please recommend JoVE to your librarian.
Disclosures
The authors have nothing to disclose.
Acknowledgments
DE would like to thank Dr. Hide Akutsu for sharing his NT tricks and Dr. Stephen Sullivan and Garrett Birkhoff for critical reading of the protocol.
Materials
| Name | Company | Catalog Number | Comments |
| Cytochalastin B | Sigma-Aldrich | 100x stock = 500 µg/ml Cytochalasin B in DMSO. Store at -20 ˚C. | |
| Strontium Chloride | 10x stock = 100 mM SrCl2 in H2O. Store at room temperature. | ||
| MCZB Stock Salts | Filter sterilize 500 ml master salts (good for 3-4 months; store at 4 ˚C) | ||
| HCZB Stock Salts | Start with 500 mL master salts. Add 50 mg PVA. Stir for 30-60 min and sterile filter (good for 3 months; store at 4 ˚C). | ||
| PVA | Sigma-Aldrich | P-8136 | cold-soluble |
| HCZB with 11% w/v PVP | Start with 20 ml HCZB medium in a 50 ml conical tube. Place 22 g PVP on top of liquid. Close tube and store undisturbed at 4 ˚C for 72 – 96 hr, at which time the PVP will have entered solution. Filter sterilize through an 8 mm filter and store at 4 ˚C. | ||
| PVP | ICN Biomedicals | MW 360,000 | |
| Please check for additional buffers compositions in the Protocol part. | |||
References
- Eggan, K., Jaenisch, R. Mammalian and Avian Transgenesis- New Approaches. Pease, S., Lois, C. , Springer. (2006).
- Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. Manipulating the mouse embryo. Press, C. S. H. L. , (2003).
- Wakayama, T., Perry, A. C., Zuccotti, M., Johnson, K. R., Yanagimachi, R. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature. 394, 369-374 (1998).
