Reproductive Biotechnology Centre, Post Box 299003, Dubai, United Arab Emirates.
Reproductive Biotechnology Centre, Post Box 299003, Dubai, United Arab Emirates.
Theriogenology. 2022 Jan 1;177:151-156. doi: 10.1016/j.theriogenology.2021.10.012. Epub 2021 Oct 20.
There are controversial reports on the restoration of eroded telomere length in offspring produced by somatic cell nuclear transfer (SCNT) in different animal species. To the best of our knowledge, no earlier studies report the telomere length in naturally produced or cloned animals in any of the camelid species. Therefore, the present study was conducted to estimate the telomere length in dromedary camels produced by SCNT, the donor cells, and their age-matched naturally produced counterparts by Terminal Restriction Fragment (TRF) length analysis and real-time Q PCR T/S ratio methods. Genomic DNA was extracted from venous blood collected from 6 cloned animals and their age-matched counterparts. Using the southern blot technique, digested DNA was blotted onto a positively charged nylon membrane, and its hybridization was carried out using telomere (TTAGGG)n specific, DIG-labeled hybridization probe (Roche Diagnostics, Germany) at 42 °C for 4 h. Stringent washes were carried out at the same temperature, followed by a chemiluminescence reaction. The signals were captured using the Azure Biosystems C600 gel documentation system. A TeloTool program from MATLAB software with a built-in probe intensity correction algorithm was used for TRF analysis. The experiment was replicated three times, and the data, presented as mean ± SEM, were analyzed using a two-sample t-test (MINITAB statistical software, Minitab ltd, CV3 2 TE, UK). No difference was found in the mean telomere length of cloned camels when compared to their naturally produced age-matched counterparts. However, the telomere length was more (P < 0.05) than that of the somatic cells used for producing the SCNT embryos. A moderate positive Pearson correlation coefficient (r = 0.6446) was observed between the telomere lengths estimated by TRF and Q PCR T/S ratio method. In conclusion, this is the first study wherein we are reporting telomere length in naturally produced and cloned dromedary camels produced by somatic cell nuclear transfer. We found that telomere lengths in cloned camels were similar to their age-matched naturally produced counterparts, suggesting that the camel cytoplast reprograms the somatic cell nucleus and restores the telomere length to its totipotency stage.
关于不同动物物种的体细胞核移植(SCNT)产生的后代中,端粒长度的恢复存在争议报道。据我们所知,在任何骆驼科动物中,以前没有研究报告过自然产生或克隆动物的端粒长度。因此,本研究通过末端限制性片段(TRF)长度分析和实时 Q-PCR T/S 比方法,评估 SCNT 产生的单峰驼、供体细胞及其年龄匹配的自然产生对应体的端粒长度。从 6 只克隆动物及其年龄匹配的对照动物采集静脉血提取基因组 DNA。使用Southern blot 技术,将消化的 DNA印迹到带正电荷的尼龙膜上,并在 42°C 下用端粒(TTAGGG)n 特异性、DIG 标记的杂交探针(罗氏诊断,德国)进行杂交 4 小时。在相同温度下进行严格洗涤,然后进行化学发光反应。使用 Azure Biosystems C600 凝胶文件系统捕获信号。使用 MATLAB 软件中的 TeloTool 程序进行 TRF 分析,该程序带有内置的探针强度校正算法。该实验重复了三次,数据以平均值±SEM 表示,使用双样本 t 检验(MINITAB 统计软件,Minitab ltd,CV3 2TE,英国)进行分析。与自然产生的年龄匹配的对照相比,克隆骆驼的平均端粒长度没有差异。然而,端粒长度比用于产生 SCNT 胚胎的体细胞更长(P<0.05)。通过 TRF 和 Q-PCR T/S 比方法估计的端粒长度之间观察到中度正的 Pearson 相关系数(r=0.6446)。总之,这是第一项研究报告体细胞核移植产生的自然产生和克隆单峰驼的端粒长度。我们发现克隆骆驼的端粒长度与其年龄匹配的自然产生对照相似,这表明骆驼胞质体重新编程了体细胞核,并将端粒长度恢复到其全能性阶段。