Production of cloned rabbits by somatic nuclear transfer.

TWO TECHNICAL FACTORS appear to be critical to the success of rabbit cloning: timing of activation of the recipient oocyte (Collas and Robl, 1990) and synchronization of the recipient uterus physiology with the development state of the reconstructed embryo (Chesne et al., 2002). In the rabbit cloning protocol (Chesne et al., 2002), each donor cell is electrofused with an enucleated and non-activated metaphase II oocyte. The nucleus of the donor cell is then transferred into the cytoplasm of the recipient oocyte. The resulting nuclear transfer (NT) embryos are left for 1 h to ensure nuclear envelope breakdown and premature chromosome condensation (PCC) because of the activity of maturation/metaphase promoting factor (MPF), which remains high in the cytoplasm of non-activated oocyte, (Campbell et al., 1996). The NT embryos are then activated through a second set of electrostimulation, and incubated in the presence of both cycloheximide (a protein synthesis inhibitor) and 6-dimethylaminopurine (a kinase inhibitor), to reduce the MPF activity and permit entrance into S phase. Because the rabbit zygote enters S phase very early after fertilization (Oprescu and Thibault, 1965; Szollosi, 1966), the exposure period to these two drugs has been reduced to 1 h. Special care should also be taken in handling oocytes. Rabbit oocytes can be activated easily. In vitro, spontaneous activation occurs 20–22 h post ovulation. Variations in temperature can also accelerate activation. To date, producing live cloned rabbit has only been possible using freshly collected cells (Chesne et al., 2002). Because of the interests of using the rabbit as an experimental model for several human pathologies (e.g., atherosclerosis, AIDS, cystic fibrosis, . . .), we are actively working to produce live animals from somatic donor cells cultured for a number of passages compatible with the realization of targeted genetic modification.