ABSTRACT
Since 2002, our INRA laboratory (Biologie du Développement et de la Reproduction) has developed a method to produce live somatic clones in rabbit, one of the mammalian species considered as difficult to clone. This chapter presents the technical protocol used nowadays to achieve the goal to obtain cloned embryos able to develop to term using fresh somatic cumulus cells.
Subject(s)
Embryo Culture Techniques , Nuclear Transfer Techniques , Oocyte Retrieval/methods , Animals , Blastocyst/cytology , Blastocyst/physiology , Cloning, Organism/methods , Female , Micromanipulation/instrumentation , Micromanipulation/methods , Nuclear Transfer Techniques/instrumentation , Ovarian Follicle/cytology , RabbitsABSTRACT
We have developed a method to produce live somatic clones in the rabbit, one of the mammalian species considered up to now as difficult to clone. To do so, we have modified current cloning protocols proven successful in other species by taking into account both the rapid kinetics of the cell cycle of rabbit embryos and the narrow window of time for their implantation after transfer into foster recipients. Although our method still has a low level of efficiency, it has produced several clones now proven to be fertile. Our work indicates that cloning can probably be carried out successfully in any mammalian species by taking into account physiological features of their oocytes and embryos. Our results will contribute to extending the use of rabbit models for biomedical research.
Subject(s)
Cell Differentiation , Cloning, Organism/methods , Nuclear Transfer Techniques , Animals , Blastocyst/cytology , Blastocyst/physiology , Cells, Cultured , Embryonic and Fetal Development , Female , Oocytes/cytology , Oocytes/physiology , RabbitsABSTRACT
When in vitro-matured oocytes were enucleated, aged and kept at 10°C before reconstitution, the in vitro development of nuclear transfer embryos to the blastocyst stage did not differ from that obtained with in vitro fertilization. This suggests that these recipient cytoplasts constitute a suitable environment for the development of the nuclear transplant. The aim of the present study was to investigate, at the biochemical level, the result of the preparation of recipient oocytes, including enucleation, ageing and cooling. For this purpose the phosphorylation profiles of four groups of in vitro-matured bovine oocytes (aged oocytes, aged-cooled oocytes, enucleated-aged oocytes and enucleated-aged-cooled oocytes (recipient cytoplasts)) were analyzed. These recipient cytoplasts exhibited a phosphorylation profile similar to that of activated oocytes. Maturation promoting factor (MPF) activity, which was high in young metaphase II oocytes, in aged oocytes, in enucleated-aged oocytes and in aged-cooled oocytes, dropped to the basal level in enucleated-aged-cooled oocytes (recipient cytoplasts), while mitogen-activated protein kinase (MAPK) activity remained elevated. The combination of enucleation, ageing and cooling following oocyte in vitro maturation resulted in an interphase-like stage cytoplasm having a phosphorylation profile and low MPF activity similar to activated oocytes, but exhibiting high MAPK activity.
