Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation
Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation
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DOI：
10.1530/rep.1.01206
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发表时间：
2007-01-01
期刊：
影响因子：
3.8
通讯作者：
Zakhartchenko, Valeri
中科院分区：
文献类型：
--
作者：
Yang, Feikun;Hao, Ru;Zakhartchenko, Valeri
供体核的表观遗传状态对体细胞核移植（SCNT）胚胎的发育潜力有重要影响。在这项研究中，我们将培养的兔卵丘细胞（RCC）和胎儿成纤维细胞（RFF）从遗传标记的兔（Alicia/Basilea）转移到中期11卵母细胞，并分析组蛋白H3-赖氨酸9-赖氨酸14乙酰化（acH 3 K9/14）在供体细胞和克隆胚胎的水平。我们还评估了供体细胞和克隆胚胎的组蛋白乙酰化状态与其发育潜力之间的相关性。为了测试组蛋白乙酰化状态的改变是否会影响克隆胚胎的发育，我们用丁酸钠（NaBu）（一种组蛋白去乙酰化酶抑制剂）处理供体细胞。此外，我们试图通过嵌合互补的克隆胚胎与卵裂球在体内受精或孤雌生殖胚胎，以提高克隆效率。acH 3 K9/14在RCC中的表达高于RFF（P < 0.05）。虽然供体细胞的类型并不影响囊胚发育，但在移植到受体体内后，RCC克隆胚胎诱导了更高的初始妊娠率（40 vs 20%）。然而，几乎所有的怀孕与任何类型的克隆胚胎丢失的中期妊娠，只有一个完全发育，活的RCC衍生兔获得。NaBu处理组acH 3 K9/14水平显著高于对照组（49% vs 33%，P < 0.05），核移植胚胎发育至囊胚的比例显著高于对照组（33% vs 49%，P < 0.05）。acH 3 K9/14在任一组克隆胚胎中的分布与体内受精胚胎中的分布不相似，这表明该表观遗传标记的重编程在克隆兔胚胎中是异常的，并且不能通过用NaBu处理供体细胞来纠正。从NaBu处理的RFF克隆的胚胎与来自体内衍生胚胎的囊胚的聚集改善了胚泡的发育，但没有获得克隆后代。只有在克隆胚胎与孤雌生殖卵裂球聚集后，才能从这种供体细胞类型产生两只活的克隆兔。我们的研究表明，供体细胞和克隆胚胎中组蛋白乙酰化水平与其发育潜力相关，可能是一个有用的表观遗传标记，以预测兔SCNT的效率。
The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase 11 oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres from in vivo fertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P < 0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF, P < 0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that in in vivo fertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres from in vivo derived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.