Nuclear Transfer into Mouse Oocytes

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 …

Discussion

Good luck!

Materials

Material Name	Type	Company	Catalogue Number	Comment
Cytochalastin B		Sigma		100x stock = 500 ug/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	P-8136	cold-soluble
HCZB with 11% w/v PVP				Start with 20 ml HCZB medium in a 50 mL conical tube. Place 22g PVP on top of liquid. Close tube and store undisturbed at 4˚ C for 72 – 96 hrs, at which time the PVP will have entered solution. Filter sterilize through an 8 mm filter and store at 4˚ C.
PVP		ICN Biochemicals		MW 360000

Please check for additional buffers compositions in the Protocol part.