Developmental Potential of Mouse Embryos Reconstructed from Metaphase Embryonic Stem Cell Nuclei1

Abstract

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.

Somatic Nucleus Reprogramming Is Significantly Improved by m-Carboxycinnamic Acid Bishydroxamide, a Histone Deacetylase Inhibitor

“…1-2 h after nuclear transfer, the reconstructed embryos were activated by 10 mM SrCl2 in calcium-free CZB medium in the presence of 5 g/ml cytochalasin B for 6 h and cultured in CZB medium at 37°C, 5% CO 2 for 4 days. The one-step micromanipulation method was also used in the R1 ES nuclear transfer process, and the method to prepare the R1 donor cell was performed as previously described (21).…”

Cell cycle analysis and interspecies nuclear transfer of cat cells treated with chemical inhibitors

“…In previous studies, donor cells at the G2/M phase could also be reprogrammed by the MII oocytes, and the reconstructed embryos were able to develop to the blastocyst stage (Korfiatis et al, 2001;Zhou et al, 2001). In the present study, we examined the effect of different inhibitors on the cell-cycle synchronisation of fi- Hungarica 62, 2014 broblast cells from the cat ovary and the development of iSCNT embryos after reconstruction.…”

“…Use of donor cells in the G0/G1 phase provided better results in the development of a cloned embryo when a metaphase II (MII) oocyte with a high level of maturation-promoting factor (MPF) was used as recipient ooplasm (Campbell et al, 1996). On the other hand, some studies showed that cloned embryos also had developmental capacity after the transfer of somatic cells in the G2/M phase into enucleated MII oocytes (Korfiatis et al, 2001;Zhou et al, 2001). Roscovitine, a cyclin-dependent kinase inhibitor, is commonly used to arrest cells in the G0/G1 phase (Gibbons et al, 2002), while demecolcine, a microtubule inhibitor, can induce cells to enter the G2/M phase (Li et al, 2005a).…”

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

“…Mitotic donor cells were selected under DIC optics and injection pipettes with inner diameter 10 µm for ES cells and 12-13 µm for fibroblasts, blastomeres were used to transfer the donor cell chromosomes. Spindle chromosome complex removal from the recipient embryo and nuclear transfer of donor nuclei was done in one step using the hole removal method [43]. The entire manipulation process was completed within a 5-30 min window after transfer of embryos into the chamber.…”