Cloned Mice: DISCUSSION(2)
Given the relatively low rate of IE, oocytes whose chromatin would have only been incompletely compartmentalized in PBs were also used in the creation of cytoplasts for nuclear transfer experiments. Thus, the relative developmental competence of both oocyte types was not considered. In vitro development of embryos cloned by transfer of ES cell nuclei into activated/demecolcine-treated cyto-plasts was generally inferior to that following transfer into unactivated MII cytoplasts, which were then activated. The latter approach has been successfully exemplified as an effective means of cloning mice, cattle, pigs, goats, and most recently, cats. However, enucleation of active spindles and emerging PBs by aspiration has also yielded viable cytoplasts capable of supporting development to term, as exemplified by the use of mouse oocytes at TI and goat and bovine oocytes at TII. canadian neighbor pharmacy online
Delayed activation of MII cytoplasts has been suggested to be important for the cloning of mice and other species by allowing an increased opportunity for the removal and/ or recruitment of developmentally restrictive and potentiating chromatin-associated factors, respectively. Our results suggest that this requirement may not be absolute. However, using activated/demecolcine-treated cytoplasts, the efficiency of initiating development and the production of live young were impaired. Although oocytes induced to self-enucleate could benefit by the acquisition of factors released from the spindle during meiotic resumption, the preinitiation of development limits the time over which nuclear remodeling can occur. For this reason, activated cytoplasts may have a greater requirement for previous nuclear readiness for remodeling after nuclear transfer. This may be achieved by previous synchronization of nuclear donor cells in G0/G1 by serum deprivation or confluence. Although ES cells used in our study were cultured under serum-reduced conditions for 1 day before nuclear transfer, with smaller cells selected as nuclear donors, their cell-cycle status was unknown.