Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle

Abstract

Nuclear reprogramming requires the removal of epigenetic modifications imposed on the chromatin during cellular differentiation and division. The mammalian oocyte can reverse these alterations to a state of totipotency, allowing the production of viable cloned offspring from somatic cell nuclei. To determine whether nuclear reprogramming is complete in cloned animals, we assessed the telomerase activity and telomere length status in cloned embryos, fetuses, and newborn offspring derived from somatic cell nuclear transfer. In this report, we show that telomerase activity was significantly (P < 0.05) diminished in bovine fibroblast donor cells compared with embryonic stem-like cells, and surprisingly was 16-fold higher in fetal fibroblasts compared with adult fibroblasts (P < 0.05). Cell passaging and culture periods under serum starvation conditions significantly decreased telomerase activity by approximately 30 -50% compared with nontreated early passage cells (P < 0.05). Telomere shortening was observed during in vitro culture of bovine fetal fibroblasts and in very late passages of embryonic stem-like cells. Reprogramming of telomerase activity was apparent by the blastocyst stage of postcloning embryonic development, and telomere lengths were longer (15-23 kb) in cloned fetuses and offspring than the relatively short mean terminal restriction fragment lengths (14 -18 kb) observed in adult donor cells. Overall, telomere lengths of cloned fetuses and newborn calves (Ϸ20 kb) were not significantly different from those of age-matched control animals (P > 0.05). These results demonstrate that cloned embryos inherit genomic modifications acquired during the donor nuclei's in vivo and in vitro period but are subsequently reversed during development of the cloned animal.

Mammalian nuclear transfer

“…For example, by applying restriction landmark genome scanning (RLGS) in two seemingly healthy cloned mice, it was shown that methylation patterns at several sites in each clone differed from those in the controls (Ohgane et al, 2001). It should be emphasized, however, that reprogramming that occurs postzygotically such as X-chromosome re-activation and subsequent inactivation (Eggan et al, 2000) and telomere length adjustment (Lanza et al, 2000a;Tian et al, 2000;Wakayama et al, 2000a;Betts et al, 2001) are faithfully accomplished in apparently normal cloned embryos. Nevertheless, X inactivation errors and chromosomal aberrations have been seen in visibly abnormal cloned embryos (Xue et al, 2002;Nolen et al, 2005).…”

Reprogramming of genome function through epigenetic inheritance

“…Another measure of reprogramming of somatic nuclei is the potential to restore the telomere size and to overcome senescence [65][66][67][68] . Although there is some evidence that telomere length is restored in somatic nuclei after transplantation into oocytes, most DNA mutations in the somatic nucleus cannot be repaired.…”

Nuclear Cloning, Epigenetic Reprogramming, and Cellular Differentiation

“…Indeed, results obtained in our laboratory and by others indicate that the fraction of abnormally expressed genes in cloned newborns is substantially higher in the placenta as compared to somatic tissues (Humpherys et al 2002;Fulka et al 2004). In contrast to epigenetic reprogramming that occurs prezygotically, it appears that postzygotic reprogramming such as X-chromosome inactivation (Eggan et al 2000) and telomere length adjustment (Lanza et al 2000;Tian et al 2000;Wakayama et al 2000;Betts et al 2001) are faithfully accomplished after nuclear transfer and, therefore, would not be expected to impair survival of cloned animals.…”