Assessment of telomerase activity and telomere length in cloned bovine embryos, fetuses and offspring derived by somatic cell nuclear transfer
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Successful cloning requires the reprogramming of the donor nuclei from pluripotent or differentiated cells to an undifferentiated state to permit the temporal and spatial re-expression of genes and events involved in embryo and fetal development. Nuclear Reprogramming requires the removal and reversal of epigenetic modifications imposed on the chromatin during cellular differentiation. Telomeres are long repeating DNA sequences (TTAGGG)n and associated protein at the ends of chromosomes. They are critical structures that function in the stability, segregation and replication of the chromosome during mitosis. Since most somatic cells do not contain the enzyme telomerase, telomeric sequences are lost with every in vivo and in vitro cell division. Telomerase is a multi-subunit, reverse transcriptase, which synthesizes telomeric repeats in cells of the germline and other immortal cells and tumors. To determine whether or not telomerase activity is reprogrammed and telomere loss is repaired after nuclear transfer of cultured bovine somatic cells, we assessed the telomerase activity and telomere length status of a number of bovine cell lines, reconstructed embryos and cloned fetal and cloned newborn calf cells/tissues. In this report, telomerase activity was assayed in bovine cell lines using the telomeric repeat amplification protocol (TRAP). Bovine embryonic stem (ES)-like cells exhibited a considerably higher (p < 0.05) level of telomerase activity in relation to fibroblast cells. Fetal fibroblasts displayed a relative telomerase activity significantly higher (p < 0.05) than adult fibroblasts. Telomerase activity of both the bovine embryonic stem (ES)-like cells and bovine fetal fibroblasts significantly (p < 0.05) decreased from early to late cell passage and by day 5 and 10 of serum starvation conditions. To examine the effect of in vitro cell division on telomere length, the mean terminal restriction fragment (TRF) length was determined for early and late passage cells. Mean TRF length decreased with passage number in fetal fibroblasts, and surprisingly in telomerase-positive, bovine ES-like cells. Reprogramming of telomerase activity was evident as early as the blastocyst stage in cloned bovine embryos whose nuclear material was derived from bovine cells with non-detectable/low telomerase activity. This expression of telomerase activity in reconstructed embryos is delayed compared to in vitro fertilization-derived bovine embryos that express telomerase activity at the morulae stage. Although telomerase activity is reprogrammed as early as the blastocyst stage of post cloning embryonic development, telomere length may not be fully rebuilt in cloned bovine fetuses and offspring. These results demonstrate that cloned offspring inherit genomic modifications acquired during the donor nuclei's in vivo and in vitro period prior to nuclear transfer. These results suggest that incomplete nuclear reprogramming allows the production of cloned offspring with non-repaired genetic alterations. (Funded by NSERC, the Barbara Graham Trust and the Texas Co-ordinating Board of Higher Education, Advanced Technology Program.).
