Developmental potential of mouse embryos reconstructed from metaphase embryonic stem cell nuclei.
Mice have recently been successfully cloned from embryonic stem (ES) cells. However, these fast dividing cells provide a heterogeneous population of donor nuclei, in terms of cell cycle stage. Here we used metaphases as a source of donor nuclei because they offer the advantage of being both unambiguously recognizable and synchronous with the recipient metaphase II oocyte. We showed that metaphases from ES cells can provide a significantly higher development rate to the morula or blastocyst stage (56--70%) than interphasic nuclei (up to 28%) following injection into a recipient oocyte. Selective detachment of mitotic cells after a demecolcin treatment greatly facilitates and accelerates the reconstruction of embryos by providing a nearly pure population of cells in metaphase and did not markedly affect the developmental rate. Most of the blastocysts obtained by this procedure were normal in terms of both morphology and ratio of inner cell mass and total cell number. After transfer into pseudopregnant recipients at the one- or two-cell stage, the ability of metaphase to be fully reprogrammed was demonstrated by the birth of two pups (1.5% of activated oocytes). Although the implantation rate was quite high (up to 32.9% of activated oocytes), the postimplantation development was characterized by a high and rapid mortality. Our data provide a clear situation to explore the long-lasting effects that can be induced by early reprogramming events.
Zhou
Q
Q
Unité de Biologie du Développement et Biotechnologie, Institut National de la Recherche Agronomique, 78352 Jouy en Josas, France.
Jouneau
A
A
Brochard
V
V
Adenot
P
P
Renard
J P
JP
D000818:Animals; D001755:Blastocyst / Q000502:physiology; D002453:Cell Cycle; D048429:Cell Size; D019976:Cloning, Organism; D004247:DNA / Q000032:analysis; D010064:Embryo Implantation; D004624:Embryo Transfer; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology*; D005314:Embryonic and Fetal Development; D005434:Flow Cytometry; D007399:Interphase; D008677:Metaphase; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D009028:Morula / Q000502:physiology; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000648:ultrastructure; D013234:Stem Cells / Q000648:ultrastructure*
D004247:DNA