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 microscope equipped with Nikon Narashige hydraulic micromanipulators (Nikon Ltd., Kingston Upon Thames, U.K.). Although selected oocytes were prestained with 5 |xg/ml of Hoechst 33342 in hCZB for 5 min, only pipettes were normally exposed to ultraviolet light to confirm removal of spindle-associated chromatin contained in aspirated PBs. After aspiration of PBs, oocytes were collected and kept in CZB medium in the incubator before piezo-mediated injection of ES cell nuclei as described for nuclear transfer using MII cytoplasts. my canadian pharmacy online
Nuclear Transfer Using MII Cytoplasts (Control)
As a positive control to evaluate the developmental competence of activated cytoplasts, nuclear transfer was also performed on mechanically enucleated MII cytoplasts according to a slightly modified version of the method of Wakayama et al.. This micromanipulation and nuclear transfer was performed on a Nikon Eclipse TE300 microscope separate from that used to remove PBs from demecolcine-treated oocytes and equipped with Eppendorf TransferMan NK micromanipulators (Eppendorf UK Ltd., Cambridge, U.K.) and a Piezo Micromanipulator Controller PMM150 (Prime Tech Ltd., Ibaraki, Japan). Before enucleation, oocytes were treated for 3-5 min with 5 ^g/ml of cytochalasin B (CB) in hCZB (before removal of the MII spindle and associated PB was performed using a 6- to 8-^m pipette in the same medium). Enucleated oocytes were transferred into hCZB medium before nuclear injection. Embryonic stem cells were prepared for nuclear injection by mixing an equal volume of cells suspended in hCZB with 10% polyvinyl-pyrrolidone (PVPK 90, 360 kDa; ICN, Aurora, OH). Cells with a comparatively smaller diameter (<10 |xm) were selected for injection, and their membranes were ruptured using an injection pipette with a inner diameter of 5 |xm to free nuclei. The same pipette was used to aspirate four to six nuclei in a row that were subsequently injected one at a time into the enucleated oocytes. Nuclear reconstructed cytoplasts derived from MII oocytes were cultured in CZB for 1-3 h before activation. Activation was by treatment for 5-6 h in calcium-free CZB medium containing 10 mM SrCl2 and 5 |xg/ml of CB, the latter to inhibit PB extrusion.