66 SUCCESSFUL ESTABLISHMENT OF PLURIPOTENT Ntes CELL LINES FROM AGED MICE

Nuclear transfer technique has enabled us to produce cloned animals from somatic cell nuclei in various animal species to date. Moreover, it has been demonstrated that ES cell lines have been established from cloned blastocysts by somatic cell nuclear transfer (ntES cell), irrespective of sex, strains, or organs. These cells are capable of differentiating into all 3 germ layers in vitro, or even into spermatozoa and oocytes in chimeric mice. So ntES cells have gotten a lot of attention recently in the field of regenerative medicine. However, it is unclear whether ntES cells can be established from aged individuals because, in general, the cloning success rate was higher when young donor cells were used, such as fetus cells rather than adult. To answer this question, we tried to establish ntES cell lines from aged mice and then examined their pluripotency. The donor cells were obtained from tail-tip fibroblast cells of 11-month-old to 15-month-old male and female mice. After nuclear transfer, we succeeded in establishing 8 ntES cell lines from 3 aged BDF1 males and 6 ntES cell lines from 2 aged BCF1 females. The normality of these ntES cell lines was examined after passages 5 times. Karyotypes were analyzed using SKY-Fish painting, and pluripotency was examined by chimeric mice formation, in which ntES cells were injected into fertilized ICR blastocysts. As a result, most of the ntES cell lines examined had normal karyotypes, and all of the ntES cell lines tested could contribute to somatic cells of chimeric mice. Now we are examining whether these ntES cells have germ line transmission ability in chimeric mice by natural mating.