Cloned Mice Derived from Embryonic Stem Cell Karyoplasts and Activated Cytoplasts Prepared by Induced Enucleation

Abstract

Our objective was to induce enucleation (IE) of activated mouse oocytes to yield cytoplasts capable of supporting development following nuclear transfer. Fluorescence microscopy for microtubules, microfilaments, and DNA was used to evaluate meiotic resumption after ethanol activation and the effect of subsequent transient treatments with 0.4 micro g/ml of demecolcine. Using oocytes from B6D2F1 (C57BL/6 x DBA/2) donors, the success of IE of chromatin into polar bodies (PBs) was dependent on the duration of demecolcine treatment and the time that such treatment was initiated after activation. Similarly, variations in demecolcine treatment altered the proportions of oocytes exhibiting a reversible compartmentalization of chromatin into PBs. Treatment for 15 min begun immediately after activation yielded an optimized IE rate of 21% (n = 80) when oocytes were evaluated after overnight recovery in culture. With this protocol, 30-50% of oocytes were routinely scored as compartmentalized when assessed 90 min postactivation. No oocytes could be scored as such following overnight recovery, with 66% of treated oocytes cleaving to the 2-cell stage (n = 80). Activated cytoplasts were prepared by mechanical removal of PBs from oocytes whose chromatin had undergone IE or compartmentalization. These cytoplasts were compared with mechanically enucleated, metaphase (M) II cytoplasts whose activation was delayed in nuclear transfer experiments using HM-1 embryonic stem cells. Using oocytes from either B6D2F1 or B6CBAF1 (C57BL/6 x CBA) donors, the in vitro development of cloned embryos using activated cytoplasts was consistently inferior to that observed using MII cytoplasts. Live offspring were derived from both oocyte strains using the latter, whereas a single living mouse was cloned from activated B6CBAF1 cytoplasts.

Effect of the enucleation procedure on the reprogramming potential and developmental capacity of mouse cloned embryos treated with valproic acid

“…The observed efficiencies of ESC derivation from our control embryos are similar to those previously reported by Gong et al (2008) in the hybrid B6CBAF1 strain, which was 15.3% for parthenogenetic blastocysts. Owing to the infrequent use of hybrid B6CBAF1 animals in SCNT experiments (Gasparrini et al 2003, Maalouf et al 2009, Costa-Borges et al 2010, no ntESC lines have been derived from this genetic background until now, but the efficiencies obtained in ntESC derivation in this study are similar to those reported for other hybrid strains (Wakayama et al 2005).…”

“…The discrepancy of results in the mouse species can be probably attributed to strain-specific differences, as not all HDACis are equally effective in all genetic backgrounds (Kishigami et al 2007, Van Thuan et al 2009). With regard to the IE procedure, although Gasparrini et al (2003) previously reported the production of a cloned mouse using cytoplasts prepared by IE, we were unable to obtain live offspring from the IE group. It should be mentioned, however, that they used ESCs as karyoplast donors, which are known to be easier to reprogram than the cumulus cells used in our study (Hochedlinger & Jaenisch 2006).…”

“…The enucleation efficiencies of IE are generally low (20% in the mouse; Ibáñez et al 2003), but aspiration of the PB2 with a micropipette can increase the efficiency up to 90%, as it avoids the reintegration of the chromosomes into the cytoplasm in some oocytes that cannot extrude the PB2 completely (Costa-Borges et al 2011). The cytoplasts prepared according to this method proved to be competent to support embryonic development to term after reconstruction with embryonic cell nuclei, but the embryonic developmental rates were lower than those when ME cytoplasts were used (Gasparrini et al 2003). Recently, we demonstrated that if NT of the somatic cells is performed prior to the activation and the antimitotic treatment, blastocyst rates can be increased to values similar to those obtained with ME cytoplasts (Costa-Borges et al 2011).…”

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

“…To synchronize the fused tetraploid embryos into metaphase, we used the microtubule-destabilizing agent DC, which was previously used for induced enucleation in nuclear transfer experiments [11,[29][30]. Regardless of the polyploidizing nature of DC [31], no reports regarding its effects on full-term embryo development were available.…”

“…The cleavage time of the tetraploid embryos is strongly correlated with diploid embryo cleavage time (Supplementary information, Table S1). Synchronized tetraploid embryos with two distinct nuclei (obtained from blastomeres) were generated in media containing demecolcine (DC), a colchicine-related drug that depolymerizes microtubules and limits spindle formation during metaphase (Supplementary information, Table S2) [11]. This process appeared to be reversible, since the tetraploid embryos could regain mitotic activity and continue through repeated cell cycles upon release from DC exposure (Supplementary information, Table S3).…”