A reliable, noninvasive technique for spindle imaging and enucleation of mammalian oocytes

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Abstract
Factors affecting the efficiency of animal cloning remain to be elucidated1,2. Enucleation of recipient oocytes is a critical step in cloning procedures and typically is performed by aspirating a portion of the cytoplasm underlying the first polar body. Enucleation is evaluated using epifluorescence after Hoechst staining for DNA3,4,5,6, which may disrupt functions of the cytoplast, especially mitochondria7. Mitochondrial DNA in Dolly and other cloned sheep has been shown to derive exclusively from recipient oocytes8. Not only might evaluation of the aspirated karyoplast portion5 inadequately reflect the state of the cytoplast, it is also time consuming. Here we report a reliable, noninvasive technique for spindle imaging and enucleation of oocytes using a new microscope, the Pol-Scope9. The efficiency of enucleation was 100%, and only 5.5% of the oocytes' mitochondria entered the karyoplast upon Pol-Scope-directed removal of the spindle. Moreover, Pol-Scope imaging of spindles and micromanipulation did not compromise the developmental competence of reconstituted oocytes and cytoplasts.