Archive for the ‘Cell’ Category - Part 5

Cloned Mice: DISCUSSION(1)

We observed that transient treatment with the microtubule-destabilizing agent demecolcine during meiotic resumption resulted in the irreversible enucleation and reversible compartmentalization of chromatin in activated mouse oocytes. This treatment was effective in different mouse strains, with its efficacy being dependent on both the timing and the duration of its administration. Activated cytoplasts produced following mechanical […]

Cloned Mice: RESULTS(5)

Nuclear Transfer Using B6CBAF1 Cytoplasts In five replicate experiments using B6CBAF1 oocytes, activated and MII cytoplasts were compared in nuclear transfer experiments with HM-1 ES cell nuclei. Again, in each experiment, a sample of oocytes was also partheno-genetically activated with ethanol and cultured to the blastocyst stage. As with B6D2F1, high rates of parthenoge-netic development […]

Cloned Mice: RESULTS(4)

Substantial differences were also observed between cy-toplasts in their rate of development after nuclear reconstruction (Table 4). Whereas high proportions of MII cy-toplasts cleaved and formed morulae/blastocysts, these stages of development were only reached by 14% and 2% of activated cytoplasts, respectively. Of oocytes parthenogenetically activated with ethanol, 85% and 75% cleaved and reached the […]

Cloned Mice: RESULTS(3)

To assess whether IE by the timed administration of demecolcine was reversible and whether oocytes scored as compartmentalized eventually became enucleated if allowed to develop further, oocytes were treated for 15 min with demecolcine beginning immediately after activation and fixed at 90 min pa or after overnight culture. In four replicate experiments, no significant difference […]

Cloned Mice: RESULTS(2)

Intermediate phenotypes could also be found, which emphasized the need to assess the effect of demecolcine by fluorescence microscopy. For the third category of PB phenotypes, Hoechst-stained chromatin in the oocyte at the site of the budding PB was apparent at both low and high magnification, indicating that enucleation had not been successful (Fig. 2, […]

Cloned Mice: RESULTS(1)

Meiotic Resumption Following Ethanol Activation of B6D2F1 Oocytes The timing of meiotic progression after ethanol activation was characterized in oocytes recovered from B6D2F1 mice (Table 1 and Fig. 1). At 15 min pa, the majority of oocytes sampled (85%, n = 41) had reached anaphase (A) II. At 30 min pa, oocytes were roughly equal […]

Cloned Mice: MATERIALS AND METHODS(4)

Parthenogenetic Activation As a positive control for oocyte quality and the method of activation used with the IE protocol, a sample of oocytes in each nuclear transfer experiment were activated with 7% ethanol in hCZB for 7 min, washed in hCZB, and cultured in the presence of 5 |xg/ml of CB to inhibit extrusion of […]

Cloned Mice: MATERIALS AND METHODS(3)

Nuclear Transfer with Activated Cytoplasts Demecolcine-treated oocytes were assessed 1.5 h pa at a magnification of 50X using a stereomicroscope. Those oocytes exhibiting either a long flat PB or two closely apposed PBs were selected for aspiration of PBs using a 12-|xm pipette. Aspirations were in hCZB at room temperature on a Nikon Eclipse TE300 […]

Cloned Mice: MATERIALS AND METHODS(2)

Immunocytochemistry Oocytes were fixed and immunostained for microtubules and microfilaments using a modification of the method described by Messinger and Albertini. Briefly, oocytes were fixed and extracted for 30 min at 37°C in a microtubule-stabilizing buffer (0.1 M Pipes [pH 6.9], 5 mM MgCl26H2O, and 2.5 mM EGTA) containing 2% formaldehyde, 0.5% Triton X-100, 50% […]

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 […]

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