Cloned Mice: MATERIALS AND METHODS(1)
The present study was conducted following approval by the Roslin Institute Animal Ethics Committee and within a project license issued under the Animal (Scientific Procedures) Act 1986. Unless otherwise stated, all reagents were purchased from Sigma (Poole, U.K.).
In Vivo Oocyte Collection and Embryo Culture
Female B6D2F1 or B6CBAF1 mice (age, 8-10 wk) were superovulated by injection of 5 IU of eCG and, 48 h later, 5 IU of hCG. Oocytes were recovered as cumulus-oocyte complexes (COCs) either 13-14 or 1618 h post-hCG, depending on whether enucleation was to be by mechanical aspiration of MII oocytes or by induction following activation, respectively. Whereas embryo culture was in CZB medium at 5% CO2 in air and 37°C, handling of COCs, mature and activated oocytes, and embryos was in ambient atmosphere in Hepes-buffered CZB (hCZB), in which bicarbonate buffer was replaced with 20 mM Hepes. Before enucleation or activation, oocytes were denuded of cumulus by treatment with 300 IU/ml of hyaluronidase in hCZB followed by several washes in hCZB alone. canadian family pharmacy com
Unless otherwise noted, MII oocytes with a first PB were activated for IE by exposure to 7% ethanol in hCZB for 7 min. This was followed by culture in CZB containing 0.4 |xg/ml of demecolcine beginning at different times postactivation (pa) and for different durations as defined by each experiment. After demecolcine treatment, oocytes were normally incubated in CZB until 90 min pa, at which point they were either assessed for the success of IE or selected for use in nuclear transfer (see below). In experiments to optimize the efficiency of IE, the position of Hoechst 33342-stained chromatin was evaluated relative to immunostained spindle microtubules and cortical microfilaments in fixed oocytes at high magnification (1000X).