Cui Wei, Wylie Diana, Aslam Samena, Dinnyes Andres, King Tim, Wilmut Ian, Clark A John
Department of Gene Expression and Development, Roslin Institute, Roslin, Midlothian EH25 9PS, United Kingdom.
Biol Reprod. 2003 Jul;69(1):15-21. doi: 10.1095/biolreprod.102.013250. Epub 2003 Feb 19.
Telomere shortening and lack of telomerase activity have been implicated in cellular senescence in human fibroblasts. Expression of the human telomerase catalytic reverse transcriptase subunit (hTERT) in these cells reconstitutes telomerase activity and immortalizes the cells without tumor transformation. In this report, we show that sheep fibroblasts are similar to human cells. They do not have detectable telomerase activity and undergo only a finite numbers of cell divisions before replicative senescence. Telomere lengths in sheep fibroblasts are similar to those reported for human cells and shorten at a rate of 50-200 base pairs (bp) each cell division. Expression of the human telomerase catalytic subunit restored the telomerase activity in the sheep cells and extended their proliferative life span. None of the telomerase positive sheep fibroblasts exhibited a transformed phenotype after 200 days of continuous culture, and the higher hTERT expressing cells maintained their telomere lengths and normal cell characteristics for more than 500 days in culture. In cloning experiments using one of these cell lines as a nuclear donor, the reconstructed karyoplasts were reprogrammed and developed to the blastocyst stage at a similar frequency to that observed with the parental, telomerase negative cell line. After embryo transfer the blastocysts exhibited a relatively high frequency of implantation, early fetal development, and organogenesis. No fetuses survived beyond 40 days of development, however, showing that although these cells could be substantially reprogrammed, they were not fully competent for nuclear transfer.
端粒缩短和端粒酶活性缺失与人类成纤维细胞的细胞衰老有关。在这些细胞中表达人类端粒酶催化逆转录酶亚基（hTERT）可重建端粒酶活性，并使细胞永生化而不发生肿瘤转化。在本报告中，我们表明绵羊成纤维细胞与人类细胞相似。它们没有可检测到的端粒酶活性，在复制性衰老前仅经历有限次数的细胞分裂。绵羊成纤维细胞中的端粒长度与报道的人类细胞相似，每次细胞分裂以50 - 200个碱基对（bp）的速率缩短。人类端粒酶催化亚基的表达恢复了绵羊细胞中的端粒酶活性，并延长了它们的增殖寿命。在连续培养200天后，没有一个端粒酶阳性的绵羊成纤维细胞表现出转化表型，并且高表达hTERT的细胞在培养中500多天保持其端粒长度和正常细胞特征。在使用这些细胞系之一作为核供体的克隆实验中，重建的核质体被重编程并发育到囊胚阶段，频率与亲代端粒酶阴性细胞系观察到的相似。胚胎移植后，囊胚表现出相对较高的着床、早期胎儿发育和器官发生频率。然而，没有胎儿在发育40天后存活，这表明尽管这些细胞可以被大量重编程，但它们不完全适合核移植。
