Cloned Mice: INTRODUCTION

INTRODUCTION

During nuclear transfer in mammals, the meiotic spindle and associated cytoplasm is removed by micromanipulation. The extent to which this procedure perturbs essential cellular processes remains unknown, although the birth of cloned offspring from several species shows that it is still compatible with subsequent development. As an alternative to mechanical enucleation, methods of inducing enucleation might provide superior cytoplasts for nuclear transfer by minimizing the extent to which cytoplasmic integrity is compromised. During polar body (PB) emission in meiotically maturing or activated oocytes, enucleation can be induced by treatment with reagents that interfere with chromosome segregation and spindle function. These include treatment with etoposide (ETO), which inhibits topoisomerase II-mediated DNA cutting and repair mechanisms, and cycloheximide (CHX), which inhibits protein synthesis. However, to date, only limited development has been observed using cytoplasts derived from mouse oocytes whose enucleation was induced by these reagents. Reconstitution of mouse 2-cell blastomeres with oocytes enucleated during first PB emission using the combination of ETO-CHX results in development only as far as the 4-cell stage. In contrast, reconstitution of 2-cell blastomeres with metaphase (M) II cytoplasts enucleated by treatment with ETO alone before activation and for 4 h afterward yielded blastocysts at a low frequency. Recently, the promise of producing developmentally viable cytoplasts following induced enucleation (IE) was renewed by a brief report of mice cloned from cumulus cells using oocytes enucleated by the timed administration of the microtubule depolymerizing agent, demecolcine. natural asthma treatment

In the present study, our aim was to evaluate the utility of demecolcine to induce the enucleation of activated mouse oocytes. In addition, the developmental competence of resulting cytoplasts was examined in nuclear transfer experiments using embryonic stem (ES) cells as nuclear donors. We report that the timed administration of demecol-cine yields a proportion of oocytes whose IE is complete, although reversible compartmentalization of chromatin is also evident in a significant proportion. Cytoplasts prepared by the mechanical removal of PBs from oocytes whose chromatin has undergone IE or compartmentalization support ES cell nuclear transfer development to term, albeit at lower rates than traditional MII cytoplasts that are mechanically enucleated.

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