国家自然科学基金资助项目(30270647),北京市自然科学基金项目(5012007),农业生物技术国家重点实验室开放课题和国家高技术"863"计划项目(2002AA206311)资助.
This work was supported by grants from The National Natural Sceinces Foundation of China (30270647), Beijing Municipal Natural Sciences Foundation, China (5012007) and State 863 High Technology R&D Project of China (2002AA206311).
用 RT-PCR 引物分别扩增成年昆明 (KM) 小鼠睾丸、脾脏、肾脏、肝脏和胸腺组织的总 RNA 发现,端粒酶催化亚基基因 tert 在这些组织中都有转录,目标产物正确组装到 PMD 18-T 载体后测序,结果与已知 cDNA 序列一致 . PMSG/hCG 超数排卵方法获得 KM 小鼠成熟卵母细胞和 CZB 溶液体外培养的胚胎 (KM ♀× KM ♂ ) ,用酸性 Tyrode's 溶液消化透明带后,采用巢式 RT-PCR ,同时分析 tert 基因和持家基因 hprt 的转录发现,对于单个样品来说 , 全部卵母细胞 (15 h-post hCG , 10/10) 都存在 hprt 转录本,其中,只有 40% (4/10) 还同时存在 tert 转录本 . 原核形成初期 (20 h-post hCG , 6/6) 和原核晚期 (30 h post-hCG , 8/8) 的受精卵,以及发育至 2-C 早期的胚胎 (35 h-post hCG , 7/7) 都不转录 tert 基因,只有 hprt mRNA 存在; 2-C 晚期 (50 h-post hCG) 时,两个基因同时转录 (4/8) 和一个基因单独转录 (4/8) 的胚胎各占 50% ;从 4-C 阶段 (65 h-post hCG , 4/4) 开始,包括 8-C 阶段 (75 h-post hCG , 4/4) ,桑椹胚阶段 (93 h-post hCG , 4/4) ,直至囊胚阶段 (118 h-post hCG , 4/4) ,所有的胚胎都同时转录 tert 和 hprt 基因,而且转录水平明显升高 . 以 20 枚胚胎量为模板进行 RT-PCR 发现,原核早期,原核晚期的胚胎中仍然没有 tert 基因转录,只有 hprt mRNA ,但是,在 2-C 早期胚胎中同时检测到了 hprt 和 tert 两种 mRNA. 结果表明,持家基因 hprt 在成熟卵母细胞受精前后,以及胚胎早期发育过程中均存在转录本 . 40% 卵母细胞中存在的 tert mRNA 在受精后很快降解,检测不到;胚胎基因组在 2-C 早期开始转录 tert mRNA ,转录水平逐渐上升 . 结果暗示,小鼠胚胎的基因组 DNA 在 2-C 早期开始启动,功能基因 tert 也在此时开始转录,可能与胚胎发育初期的染色体保护有关 .
Telomerase is a ribonucleoprotein complex that elongates telomeres, allowing to stabilize the ends of chromosomes during cell proliferation. Here nested PCR method was used to describe the transcriptional patterns of mTERT (mouse telomerase reverse transcriptase) gene, encoding the catalytic subunit of mouse telomerase expressed both in murine adult tissues and preimplantation embryos cultured in CZB medium from zygote to blastocyst. The analysis revealed that the mTERT mRNA expression in adult tissues, such as testis, spleen, kidney, liver, and thymus. For single-copy zona-free oocyte or embryo samples, the tert transcript in 60% (6/10) of matured oocytes (15 h-post hCG) had been not found, while the transcripts of housekeeping gene hprt located in all matured oocytes (10/10). No tert mRNA was present in single-copy embryo samples at early (20 h-post hCG, 6/6), late (30 h post-hCG, 8/8) pronuclear stage, or early 2-C stages (35 h-post hCG, 7/7), although the hprt mRNA transcriptions were found during this period. Interestingly, the embryos at early 2-C stage expressed of tert and hprt gene together in multi-copy (20 embryos/sample) embryo samples. Half of single-copy embryos (4/8) shown expression of two genes simultaneous at late 2-C stage (50 h-post hCG), while the other half of them (4/8) only transcript either tert (2/4) or hprt gene (2/4). Expression of tert and hprt genes were detected simultaneous in all single-copy embryos developed at 4-C (65 h-post hCG, 4/4), 8-C (75 h-post hCG, 4/4), morulae (93 h-post hCG, 4/4) and blastocyst (118 h-post hCG, 4/4) stages and the level of transcription increased. These results suggested that tert gene was began to express in embryos at early 2-C stage during the process of embryonic genome activation and the level of transcription was increased during embryogenesis.
王 宇,姚 慧,安晓荣,陈永福,苟克勉.端粒酶催化亚基基因在小鼠 胚胎发育早期的转录活性[J].生物化学与生物物理进展,2005,32(3):282-287
复制生物化学与生物物理进展 ® 2025 版权所有 ICP:京ICP备05023138号-1 京公网安备 11010502031771号