Therapeutic cloning by somatic cell nuclear transfer (SCNT) holds great promise as a vehicle for patient-specific, stem cell-based treatment of a variety of diseases or debilitations. However, this approach will require the use of donor somatic cells and manipulations in vitro of enucleated oocytes and reconstituted embryos from which embryonic stem (ES) cells can be derived. This is the first work to examine the potential introduction de novo of mutations as a result of the cloning process. To address this, we utilized the Big Blue^® transgenic mouse system from Stratagene to determine the spontaneous mutation frequency and spectrum in germ cells, fetuses generated by natural mating or assisted reproductive technologies, three different somatic donor cell types typically used for cloning, and in cloned fetuses produced from each of these donor cell types. Results from this study show that the spontaneous mutation frequency in female germ cells is similar to that found in male germ cells at fetal and neonatal stages, and that these frequencies are significantly lower than those in developmentally matched somatic cells. This result suggests that SCNT embryos produced from somatic cells might carry more mutations than those produced by natural reproduction. However, our study found that the frequency of mutations in fetuses generated by assisted reproductive technologies is similar to the frequency found in fetuses conceived naturally, indicating neither maintenance nor manipulation of embryos in vitro introduce additional mutations in surviving embryos. Finally, results of our study of cloned fetuses showed that embryos generated by SCNT maintain a spontaneous mutation frequency comparable to embryos produced by natural conception, irrespective of the donor cell typed used, due to a "bottleneck effect" that limits propagation of acquired mutations during either natural reproduction, assisted reproduction or cloning. Importantly, our results show that because cloned fetuses maintain a relatively low frequency of point mutations (at least at a single test locus), comparable to that found in fetuses produced by natural reproduction, epigenetic mechanisms responsible for maintaining genetic integrity appear to be reprogrammed during the cloning process. Collectively, these results suggest that the genetic integrity of stem cells derived from therapeutically cloned blastocysts will be similar to that of stem cells derived from blastocysts produced by natural conception or assisted reproductive technologies. This study, conducted in a model transgenic system, represents an important step in the examination of the safety of therapeutic cloning before application to human patients is considered.