1 ABERRANT DNA METHYLATION IN PORCINE IN VITRO-, PARTHENOGENETIC-, AND NUCLEAR TRANSFER-PRODUCED BLASTOCYSTS
1 ABERRANT DNA METHYLATION IN PORCINE IN VITRO-, PARTHENOGENETIC-, AND NUCLEAR TRANSFER-PRODUCED BLASTOCYSTS
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1 猪体外、单性生殖和核移植产生的囊胚中的异常 DNA 甲基化
DOI：
10.1071/rdv18n2ab1
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发表时间：
2006
期刊：
影响因子：
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通讯作者：
R. Prather
中科院分区：
文献类型：
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作者：
A. Bonk;M. Samuel;L. Lai;Y. Hao;Rongfeng Li;Zhonghua Liu;C. Murphy;E. Antoniou;R. Prather
体外、孤雌生殖和核移植衍生胚胎的异常 DNA 甲基化与早期胚胎的低发育能力有关。基因组的去甲基化在受精后立即发生，并持续到囊胚阶段。基因组的再甲基化或重编程发生在植入前后并维持在体细胞组织中。本研究的目的是分析猪配子和囊胚中的 DNA 甲基化。通过差异 DNA 甲基化杂交分析配子和囊胚表观基因组中 Bstu I 位点 (CGCG) 的甲基化状态。从屠宰场收集的卵巢中吸出生殖囊卵母细胞，从新鲜精液中分离精子，并从体内、体外、核移植和孤雌生殖来源衍生和收集囊胚。根据 Bstu I 位点的存在从猪 CpG 岛文库中选择基因组克隆。这些克隆的插入片段经过 PCR 扩增并点在载玻片上。 DNA 用 Mse I 消化，连接至接头，并用 Bstu I 消化。具有甲基化 Bstu I 位点的片段保持完整，而具有未甲基化 Bstu I 位点的片段被切割。通过 PCR 扩增完整片段并用氨基烯丙基-dUTP 标记。肝脏DNA作为参考，用Cy5标记；其他样品均用 Cy3 标记。使用 Axon Genepix 4000B 扫描仪（Axon Instruments, Inc., Union City, CA, USA）扫描载玻片。使用 GenePix Pro 4.0 软件对微阵列图像进行初步分析。通过使用 Genespring 7.0 方差分析 (P < 0.05) 进行的额外分析，确定了 221 个克隆在配子或囊胚的至少一种生物学条件上存在显着差异。对 46 个克隆进行了测序，BLAST 分析鉴定出 18 个独特的克隆、16 个没有相似性的克隆以及 12 个与多个基因具有相似性的克隆。在几个克隆中鉴定出核糖体（RPS20、RPL18）和原卟啉原氧化酶（PPOX）基因。还鉴定了免疫系统的组成部分（CCR、TLR）、转录因子（ATF2）和胚胎特异性基因（WNT8B）。根据标准相关相似性度量为被识别为显着不同的点创建条件树。条件树显示，在生囊卵母细胞、孤雌生殖囊胚、核移植囊胚、体外产生的囊胚和精子中，甲基化谱最为相似。体内产生的囊胚与其他样本分开分组。这些结果与之前的研究一致，之前的研究表明，配子在受精后到囊胚阶段经历去甲基化，此时基因组被重新甲基化。此外，这些结果表明，体内产生的囊胚发育过程中发生的重编程事件不太可能发生在体外、核移植和孤雌生殖产生的囊胚中。这项工作由 NIH (RR13438) 和 21 世纪食品基金资助。
Aberrant DNA methylation of in vitro-, parthenogenetic-, and nuclear transfer-derived embryos has been implicated in the low developmental competence of early embryos. Demethylation of the genome occurs immediately after fertilization and continues through the blastocyst stage. Remethylation or reprogramming of the genome occurs around the time of implantation and is maintained in somatic tissues. The aim of this study was to analyze DNA methylation in porcine gametes and blastocysts. Differential DNA methylation hybridization was conducted to analyze the methylation status of the Bstu I site (CGCG) in the gamete and blastocyst epigenomes. Germinal vesicle oocytes were aspirated from ovaries collected at an abattoir, sperm was isolated from a fresh ejaculate, and blastocysts were derived and collected from in vivo, in vitro, nuclear transfer, and parthenogenetic sources. Genomic clones were selected from a porcine CpG Island library based on the presence of a Bstu I site. The inserts from these clones were PCR amplified and spotted on glass slides. DNA was digested with Mse I, ligated to linkers, and digested with Bstu I. Fragments with methylated Bstu I sites remained intact whereas fragments with unmethylated Bstu I sites were cut. Intact fragments were amplified by PCR and labeled with amino allyl-dUTP. Liver DNA served as the reference and was labeled with Cy5; the other samples were labeled with Cy3. An Axon Genepix 4000B scanner (Axon Instruments, Inc., Union City, CA, USA) was used to scan the slides. Initial analysis of the microarray image was performed with GenePix Pro 4.0 software. Additional analysis, performed by using Genespring 7.0 ANOVA (P < 0.05), identified 221 clones as being significantly different in at least one of the biological conditions of the gametes or the blastocysts. Forty-six clones were sequenced and BLAST analysis identified 18 clones that were unique, 16 clones that had no similarity, and 12 clones that had similarity to multiple genes. Ribosomal (RPS20, RPL18) and protoporphyrinogen oxidase (PPOX) genes were identified in several clones. Components of the immune system (CCRs, TLRs), a transcription factor (ATF2), and an embryo-specific gene (WNT8B) were also identified. A condition tree was created according to the standard correlation similarity measure for the spots identified as significantly different. The condition tree shows that the methylation profiles are most similar in the germinal vesicle oocyte, parthenogenetic blastocyst, nuclear transfer blastocyst, in vitro-produced blastocyst, and sperm. In vivo-produced blastocysts grouped separately from the other samples. These results are consistent with previous studies that have shown that gametes undergo demethylation after fertilization on through the blastocyst stage when the genome is remethylated. Additionally, these results suggest that the reprogramming events that occur during the development of the in vivo-produced blastocysts are less likely to occur in in vitro-, nuclear transfer-, and parthenogenetic-produced blastocysts. This work was funded by a grant from the NIH (RR13438) and Food for the 21st Century.