Expression data from somatic cell nuclear transfer bovine embryo. Expression data from somatic cell nuclear transfer bovine embryo

Since the first cloned animal Dolly Sheep was successfully created by using somatic cell nuclear transfer(SCNT) technique. It has become an irreplaceable tool to understand nuclear reprogramming and totipotency and holds huge potentials for regenerative medicine. However, extremely poor development rate of SCNT embryos indicates it is still questionable. The nature of reprogramming oocyte factors and their mechanism of action remain largely unknown.It is evident that the major barrier that hinders the developing iSCNT embryo mainly appears at the time of embryonic genome activation (EGA), which primarily occurs at the eight-cell stage in mammalian. The interspecies somatic cell nuclear transfer (iSCNT) is desired model for nuclear reprogramming research and a powerful tool for discovering the master genome activation genes. In this study, a valuable transcriptome recourse of iSCNT embryos was established, which derived from more than 2000 clone embryos of four different inter-family donor cells. Based on weighted gene co-expression network (WGCNA) approach, we provide an extensive transcriptome analysis of differentially expressed genes(DEG) for iSCNT embryos. The total gene expression patterns of different iSCNT embryos were discussed. 26 cell-specific modules with were identified, and those module significance and GO enriched categories were analyzed. The regulatory pathways of reprogramming barriers were further enriched. As master genome trigger genes, the transcripts related to TFIID subunit, RNA polymerase and Mediator were incomplete activated in iSCNT embryos. This indicated that pioneer factors, present in the cytoplasm of the oocyte, were failed to bind the sequence target on the heterology nuclear genome. This genomic incompatibility between the nuclear donor cell and the cytoplast may be as a major contributing factor causes the developmental failure of iSCNT cloned embryos. This study demonstrates that the iSCNT embryos undergoes only partial or incomplete reprogramming at eight-cell stage in mammalian. Our results offered convincing evidence that the abnormal expression of key master pathways may be caused the embryo developmental block of cloning embryo. This work will contribute to a better understanding of the molecular interaction between nuclear–cytoplasmic interaction and provides insight into the molecular determinants of nuclear reprogramming, human embryonic stem cell (hESC)-based therapies and rescuing highly endangered species. Overall design: In this study, the Affymetrix gene chip bovine genome array was used for gene array analysis. The Affymetrix Gene Chip Bovine Genome array contains 24,027 probe sets corresponding to approximately 23,000 transcripts including assemblies from ~19,000 UniGene clusters. The arrays images were first quantified using Gene Chip Operating Software (GCOS, Affymetrix). Samples are divided into two groups, one is embryos, another is the somatic cells. In embryos group, we collected two biological replicates of bovine oocytes, contains 1150 oocytes (BMII, ID-1) and 1000 occytes (BMII, ID-2) respectively; Two biological replicates of bovine-bovine intraspecies cloned embryos (BBNT), replicates contain 309(ID-3) and 552(ID-4) BBNT embryos; 527(ID-5) Przewalski's gazelle –Bovine interspecies nuclear transfer 8- to 16-cell stage embryos (PBNT) , 521(ID-6) Yak-Bovine interspecies nuclear transfer 8- to 16-cell stage embryos ,515(ID-7) Tibetan–bovine interspecies nuclear transfer 8- to 16-cell stage embryos (TBNT). In somatic cell group, we collected two biological replicates Luxi cattle somatic cells (LC, ID- 8,9), Mongolia cattle somatic cells(MC, ID-10,11) , Yak somatic cells(YC, ID-13,14) and Tibetan somatic cells(TC, ID-15,16) respectively. In addition, we also collected Holstein somatic cells (HC, ID-12) and three replicates Przewalski’s gazelle somatic cells(PC, ID-17,18,19)

自首例利用体细胞核移植（somatic cell nuclear transfer, SCNT）技术成功克隆出绵羊多利以来，该技术已成为解析细胞核重编程与细胞全能性不可或缺的工具，在再生医学领域拥有巨大应用潜力。然而体细胞核移植胚胎的发育成功率极低，表明该技术仍存在诸多待解问题。卵母细胞因子介导重编程的本质及其作用机制目前仍未完全阐明。目前已知，阻碍种间体细胞核移植（interspecies somatic cell nuclear transfer, iSCNT）胚胎发育的主要障碍出现于胚胎基因组激活（embryonic genome activation, EGA）时期，该过程在哺乳动物中主要发生于8细胞期。种间体细胞核移植是细胞核重编程研究的理想模型，也是挖掘核心基因组激活基因的有力工具。本研究构建了一套珍贵的iSCNT胚胎转录组资源，该资源源自4种不同科间供体细胞的逾2000枚克隆胚胎。本研究通过加权基因共表达网络（weighted gene co-expression network, WGCNA）分析方法，对iSCNT胚胎的差异表达基因（differentially expressed genes, DEG）开展了全面的转录组学分析，并探讨了不同iSCNT胚胎的整体基因表达模式。研究共鉴定出26个细胞特异性模块，并对这些模块的显著性及GO富集类别进行了分析，进一步富集了重编程障碍相关的调控通路。作为核心基因组激活因子，与TFIID亚基、RNA聚合酶及中介体（Mediator）相关的转录本在iSCNT胚胎中未被完全激活。这表明存在于卵母细胞胞质中的先驱因子未能结合异源细胞核基因组上的靶序列。供体细胞核与胞质之间的这种基因组不相容性，可能是导致iSCNT克隆胚胎发育失败的主要诱因之一。本研究证实，哺乳动物iSCNT胚胎在8细胞期仅发生部分或不完全的重编程。本研究结果有力证明，关键核心通路的异常表达可能是导致克隆胚胎发育阻滞的原因。本研究将有助于我们更好地理解核质互作的分子机制，并为阐明细胞核重编程的分子决定因素、基于人胚胎干细胞（human embryonic stem cell, hESC）的治疗策略以及拯救极度濒危物种提供新的见解。实验设计：本研究采用Affymetrix牛基因组基因芯片进行基因芯片分析。该芯片包含24027个探针组，对应约23000条转录本，涵盖约19000个UniGene簇的组装序列。芯片图像首先通过Gene Chip Operating Software（GCOS, Affymetrix）进行定量分析。实验样本分为两组：胚胎组与体细胞组。胚胎组收集了2个生物学重复的牛卵母细胞：分别为1150枚卵母细胞（BMII，ID-1）与1000枚卵母细胞（BMII，ID-2）；2个生物学重复的牛-牛种内克隆胚胎（BBNT），分别包含309枚（ID-3）与552枚（ID-4）BBNT胚胎；527枚（ID-5）普氏原羚-牛种间核移植8-16细胞期胚胎（PBNT）、521枚（ID-6）牦牛-牛种间核移植8-16细胞期胚胎、515枚（ID-7）藏牛-牛种间核移植8-16细胞期胚胎（TBNT）。体细胞组收集了2个生物学重复的鲁西黄牛体细胞（LC，ID-8、9）、蒙古牛体细胞（MC，ID-10、11）、牦牛体细胞（YC，ID-13、14）与藏牛体细胞（TC，ID-15、16）；此外还收集了荷斯坦奶牛体细胞（HC，ID-12）以及3个生物学重复的普氏原羚体细胞（PC，ID-17、18、19）