47 SUCCESSFULLY PRODUCING CLONED MICE FROM SOMATIC CELLS OF AGED MICE VIA ESTABLISHED ntES CELL LINES

Recent nuclear transfer techniques have enabled us to produce cloned animals from somatic cell nuclei in a variety of animal species and are to date the only way to obtain offspring from infertile animals. Despite very aged mice often showing an infertile phenotype, the decreasing rate of cloning success with increased age makes it almost impossible to produce cloned mice or offspring from these animals. Other studies, however, have demonstrated that ES cell lines have been established from cloned blastocysts through somatic cell nuclear transfer (ntES cells), irrespective of sex, strains, or organs. These cells are subsequently capable of differentiating into all three germ layers in vitro, or even into spermatozoa and oocytes in chimeric mice. Thus, ntES cells have received considerable attention recently in regenerative medicine. Importantly, the success rate of establishing ntES cell lines from cloned blastocysts is ten times higher than that of producing cloned mice, which may allow us to establish ntES cell lines even from such 'unclonable' aged mice. ntES cells also have the potential to be a good donor source for nuclear transfer as they have the same DNA as their donor somatic cells and can indefinitely proliferate in their undifferentiated states. In this study, we attempted to establish ntES cell lines from aged mice and analyze their normality. We then tried to produce cloned mice via nuclear transfer using established ntES cell lines. We obtained donor cells from tail-tip fibroblast cells of BDF1 and BCF1 male and female mice that were over two years old. Following nuclear transfer, we transferred a proportion of the cleaved cloned embryos to pseudopregnant ICR female mice. The remaining embryos were cultured for 72 h, and cloned embryos that developed into morulae or blastocysts were plated on feeder cells. We then examined all established ntES cell lines for normality by Oct4 and Nanog expression using immunofluorescence staining and pluripotency by chimeric mice formation, for which ntES cells were injected into fertilized ICR embryos. Finally, we attempted to produce cloned mice from the nuclei of these ntES cell lines. In each experiment, 25, 37, 73, 63, and 75 cloned embryos from aged mice tail-tip cells were used in attempts to produce cloned mice, and 20, 20, 27, 35, and 40 cloned embryos were used to derive ntES cell lines, respectively. No cloned mice were obtained by direct nuclear transfer of the aged mice tail-tip cells; however, we were successful in establishing ntES cell lines from all experiments, with an establishment rate between 10 and 25%. All established ntES cell lines expressed Oct4 and Nanog and contributed to somatic cells in chimeric mice. Some chimeric mice produced offspring derived from ntES cells after mating. We were also able to produce cloned mice even from a 2-year-old and a 9-month-old BCF1 male mouse by nuclear transfer using ntES cells as donor nuclei. These results clearly show that normal ntES cell lines can be established from infertile, aged mice and this technique can now be used to produce offspring, irrespective of donor conditions.