54 SUCCESS OF MOUSE CLONING FROM AN OUTBRED STRAIN BY TRICHOSTATIN A TREATMENT AFTER SOMATIC NUCLEAR TRANSFER

Abstract

Although the somatic cloning technique has been used for numerous applications and basic research of reprogramming in various species, the extremely low success rates have plagued this technique for a decade. Further, in mice, the clonable strains have been limited mainly to the hybrid F1 strains such as B6D2F1. Recently, we have reported a new efficient cloning technique using trichostatin A (TSA) where reconstructed oocytes are activated by 5 mM strontium with 5 nM TSA for 6 h, followed by 3 h of culture in KSOM medium containing the same concentration of TSA. After the TSA treatment, cloned embryos were cultured in KSOM medium without TSA. This TSA treatment leads to a 2–5-fold increase in success rates for mouse cloning of B6D2F1 cumulus cells. In this study, to further test the validity of this TSA cloning technique, we tried to clone the adult ICR mouse, an outbred strain, which has never been directly cloned before. Only when TSA was used did we obtain both male and female cloned mice from cumulus and fibroblast cells of adult ICR mice with 4–5% success rates, which is comparable to 6–7% of B6D2F1. Thus, the TSA cloning technique now allows us to successfully clone outbred mice, demonstrating that this technique not only improves the success rates of cloning from the hybrid strains but also enables mouse cloning from normally unclonable strains. Further, our results provide insight into the mechanism underlying why only limited strains can be cloned using the current standard cloning technique.