体细胞核移植（somatic cell nuclear transplantation，SCNT）技术是将高度分化的体细胞的细胞核转移到去核的卵母细胞中，使重构胚胎发育成完整动物体的技术，是哺乳动物诱导细胞重编程最早的例子。核移植后的重构胚胎可以将一个高度分化的供体细胞核赋予全能性，经过无数次的分裂和分化最终形成完整的多细胞生物，在该过程中，染色质重编程起决定性作用。有研究发现，在重构胚胎激活的最初几个小时内，染色质的核小体已经发生了大规模的改变。因此对胚胎激活后最初几小时内染色质动态变化的研究可以从全基因组的水平深入解析重编程的机制。
孤雌激活（Parthenogenetic activation，PA）技术可以通过人工的方法，在无需精子进入卵母细胞的情况下，将高度分化的卵母细胞转变为胚胎。在PA胚胎中仅含有全部从卵母细胞中继承的基因组，是研究母体基因组表观遗传效应的理想模型。因此对PA胚胎中表观遗传重塑的研究，可以为胚胎重编程过程母体遗传信息的重塑机制提供理论参考。
ATAC-seq（Assay for Transposase-Accessible Chromatin sequencing）是近年来兴起的一种进行表观遗传学相关研究的关键技术，可以在全基因组水平上检测细胞染色质可及性。然而，目前没有适用于猪卵母细胞及胚胎ATAC-seq文库构建的方法。不仅由于卵母细胞和胚胎中存在数量相当庞大的线粒体DNA，严重影响了ATAC-seq文库质量，且与体细胞相比，卵母细胞和胚胎样本数量极少，不易获得。因此，为了建立适合猪卵母细胞及胚胎ATAC-seq文库构建的方法，并探究SCNT胚胎重编程过程染色质可及性的动态变化。该研究首先以猪胎儿成纤维细胞（pig embryonic fibroblasts，PEFs）和猪GV（Germinal Vesicle）期卵母细胞为研究模型，尝试构建用于猪卵母细胞和胚胎的微量细胞ATAC-seq，将其命名为“Pmini-ATAC-seq（pig mini ATAC-seq）”，并通过qPCR、Agilent 2100 Bioanalyzer生物分析仪和高通量测序等手段对文库质量进行检测。之后，通过孤雌激活和徒手克隆等技术收集激活10 h后的胚胎进行Pmini-ATAC-seq文库构建，库检合格后的样品使用Illumina Hiseq X ten平台进行第二代高通量测序，并通过生物信息学的方法将测序结果与PEFs的ATAC-seq数据进行联合分析。旨在研究孤雌激活和克隆胚胎重编程过程最初几个小时内染色质可及性的动态变化规律，并揭示其在调控体细胞重编程中的重要意义。
本研究分为三部分，主要研究内容和结果如下：
试验一 Pmini-ATAC-seq文库构建方法的建立
本研究中，以PEFs为细胞模型，首先主要从细胞裂解液、收集细胞的方法以及扩增循环数等方面进行优化，尝试进行PEFs常量细胞和微量细胞ATAC-seq文库的构建。之后，重点研究细胞裂解时间对ATAC-seq文库质量的影响，并在此基础上利用核质分离的原理对卵母细胞进行线粒体DNA的去除。结果发现，裂解时间为5 min的细胞最终文库浓度非常低，仅为12.93 nmol / L和8.47 nmol / L，该浓度无法满足高通量测序的要求；对于裂解时间分别为15 min、30 min和45 min的PEFs进行测序和分析后，发现随着裂解时间的增加，测序结果中来自细胞核基因组DNA的比例有所增加，但比对到参考基因组的比例却有所降低；此外，裂解时间分别为30 min和45 min的文库，插入片段大小大于15 min的组。这些结果表明，PEFs裂解时间会影响ATAC-seq文库的质量，并且在15 min的裂解时间下可以获得更高质量的ATAC-seq文库。基于细胞裂解的最优时间，本研究最终创建了一种新的高效去除卵母细胞或胚胎线粒体DNA污染的方法，并验证了该方法去除线粒体DNA的效率。凝胶电泳结果显示，采用1.2 μm的滤膜过滤后，可以将卵母细胞中线粒体部分过滤去除；Agilent 2100 Bioanalyzer对文库片段大小检测后发现，经过线粒体去除后的样品第一个峰出现在200 bp；高通量测序的结果表明，去除线粒体前，非核比例为78.61%；去除线粒体后，非核比例降低至27.51%。以上结果表明，该研究可以将卵母细胞ATAC-seq文库中的线粒体DNA有效去除，并成功构建Pmini-ATAC-seq文库的方法，为进一步利用猪卵母细胞及胚胎进行表观遗传相关研究奠定了基础。
试验二 Pmini-ATAC-seq解析猪孤雌激活初期胚胎染色质开放位点
基于的Pmini-ATAC-seq技术，本研究分别收集50枚GV期猪卵母细胞和50枚孤雌激活后10 h的胚胎进行Pmini-ATAC-seq建库，在库检合格后对样品进行高通量测序分析，主要对卵母细胞和胚胎染色质可及性位点的生物学功能，以及GV和PA差异开放位点的重要作用进行研究。结果表明，与GV相比，PA胚胎中富集到的peaks数量非常少，仅有11152个；且胚胎染色质开放位点在基因组的覆盖比例非常低，仅有0.0772%。虽然GV和PA染色质开放位点在基因功能区的占比规律类似，但根据GO富集分析结果说明，两者染色质开放位点功能不同。GV胚胎开放位点主要涉及染色质结构、卵母细胞成熟的过程。PA胚胎开放位点主要涉及组蛋白修饰和转录活性的调控过程。GV与PA差异开放位点相关基因KEGG富集分析结果并没有筛选出显著富集的生物学通路，但结果发现这些差异位点可以参与细胞多能性调控的生物学过程。以上结果表明，GV期卵母细胞与PA胚胎染色质可及性具有很大的不同。为进一步进行胚胎重编程过程染色质可及性的研究提供理论基础。
试验三 猪SCNT胚胎中染色质开放位点的变化
本研究通过徒手克隆（Hand-made Cloning）的技术构建了50枚SCNT胚胎，在激活后10 h收集胚胎进行Pmini-ATAC-seq文库构建。在库检合格后对样品进行ATAC-seq高通量测序分析，同时，将该研究的测序结果与其他研究中PEFs的ATAC-seq测序结果进行联合分析，比较SCNT与PEFs染色质开放模式的异同，检测PEFs各分类基因在胚胎重编程过程中可及性的变化规律，并比较PEFs与SCNT重构胚胎中存在差异的染色质可及位点及其重要的细胞生物学功能。结果表明，与PEFs相比，SCNT胚胎和PA胚胎中富集到的peaks数量很少，仅有3710个；将这些信号峰映射到参考基因组时发现，SCNT胚胎染色质开放位点在基因组的覆盖比例非常低，仅有0.0267%。此外，在胚胎重编程过程中，SCNT与PA胚胎基因组中PEFs高表达的基因和X染色体基因可及性消失。GO富集分析表明，SCNT胚胎中可及性染色质相关的分子功能主要涉及转录调节活性，生物学过程主要涉及组蛋白乙酰化正调控。SCNT和PEFs之间差异染色质开放位点主要与转录活性和组蛋白修饰有关。这些结果表明，在胚胎重编程的早期阶段染色质结构紧密，但仍检测到一些主要分布在基因间区的染色质开放位点，且重编程过程中染色质可及性的动态变化主要与转录活性和组蛋白修饰有关。该研究为胚胎重编程早期阶段染色质可及性的动力学和重要性提供了新的见解。
综上所述，本研究以猪卵母细胞和胚胎为研究对象，成功建立了适用于猪卵母细胞和胚胎微量细胞ATAC-seq建库分析的新方法，称之为“Pmini-ATAC-seq”。同时，该研究利用Pmini-ATAC-seq检测了胚胎重编程过程初期染色质可及性位点的变化，发现在胚胎重编程的早期阶段染色质结构紧密，但仍存在一些主要与转录活性和组蛋白修饰有关的染色质开放位点。该研究为胚胎重编程早期阶段染色质可及性动态变化的解析提供新的见解，也为推动表观遗传技术在家畜胚胎研究中的应用奠定一定的基础。

Somatic cell nuclear transplantation (SCNT) can transform a highly differentiated donor nucleus into a pluripotent nucleus. After numerous divisions and differentiation steps, the reconstructed embryo finally forms a complete multicellular organism. Radical changes in gene expression patterns in cells are critically important for SCNT. Chromatin nucleosome positioning has been reported to undergo large-scale dynamic reorganization in the first few hours after the activation of the SCNT embryo, suggesting that chromatin has already been reprogrammed at the early stage of embryo activation. Therefore, studies examining the dynamic changes of chromatin in the first few hours after embryo activation could provide insight into the mechanisms and significance of genome-wide reprogramming.
Parthenogenetic Activation (PA) technology can transform highly differentiated oocytes into embryos through artificial methods without sperm. The PA embryo only contains the genome inherited from the oocyte, which is an ideal model for studying the epigenetic effects of the maternal genome. Therefore, studies examining epigenetic remodeling in PA embryos can provide a theoretical reference for maternal genome remodeling mechanism during embryo reprogramming.
ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) has emerged as one of the most powerful approaches to detect cell chromatin accessibility at the whole genome level. However, no methods have been reported for pig oocyte or embryo ATAC-seq library construction. This mainly due to the large amount of mtDNA (mitochondrial DNA), which severely reduced the quality of the ATAC-seq library. Therefore, in order to find an effective ATAC-seq library construction method for pig oocytes and embryos, and to study the dynamic changes of chromatin accessibility during SCNT embryo reprogramming. We firstly used pig fetal fibroblasts (PEFs) and GV oocytes as models to try to construct ATAC-seq method for pig oocytes and embryos, wich was named as "Pmini-ATAC-seq (pig mini ATAC-seq)". And the library quality was tested by qPCR, Agilent 2100 Bioanalyzer and high-throughput sequencing methods. Then, Pmini-ATAC-seq library preparation was constructed by collecting embryos after activation for 10 h via PA and SCNT. And ATAC-seq sequencing were performed on qualified libraries through Illumina Hiseq X ten platform. Along with pig embryonic fibroblast (PEFs) ATAC-seq, the results were analyzed by bioinformatics methods. The goal of this study was to characterize dynamic changes in chromatin accessibility and the importance of genome-wide chromatin accessibility in the first few hours of PA and SCNT embryo programming. 
The studies were divided into three parts and the specific research content and results are as follows:
Experiment 1. Establishment of pig mini ATAC-seq (Pmini-ATAC-seq) on Porcine Oocytes and Embryos
In this study, we firstly used PEFs as a model to optimize conditions such as cell lysates, cell collection methods, and the number of amplification cycles during ATAC-seq library construction. And we tried to build ATAC-seq library respectively using normal and micro amount of PEFs. Then, we tested the effects of lysis time on the quality of ATAC-seq libraries and tried to remove mitochondria from oocytes and embryos. The results showed that cells with a lysis time of 5 min had very low final library concentrations, only 12.93 nmol/L and 8.47 nmol/L, which were too low to meet the requirements for high-throughput sequencing. After PEFs with lysis times of 15 min, 30 min, and 45 min were sequenced and analyzed, we found that with an increased lysis time, the nuclear DNA ratio increased to a certain extent, but the mapped ratio decreased, and the duplication ratio increased. Moreover, the insert fragment sizes of the libraries with lysis times of 30 min and 45 min were larger than the 15 min group. These results indicate that the PEFs lysis time affects the ATAC-seq library quality, and a higher quality ATAC-seq library can be obtained with a lysis time of 15 minutes. Based on the results of cell lysis time, we further explored methods to remove mtDNA. The agarose gel electrophoresis results showed the mitochondrial in oocytes. had been removed partly. Agilent 2100 Bioanalyzer showed the main peak of the sample was 200bp after mitochondrial clearance. The Illumina Hiseq sequencing results showed that Before mitochondrial clearance, the proportion of non-nuclear DNA was as high as 78.61%, whereas after mitochondrial clearance, the proportion of non-nuclear DNA was reduced to 27.51%. These results indicated that we successfully reduced the amount of mtDNA in oocytes and embryos and established an effective Pmini-ATAC-seq method. This study provided possible for further epigenetic research on porcine oocytes and embryos.
Experiment 2 Pmini-ATAC-seq analyzed the changes of chromatin open sites in the early stage of porcine parthenogenetic activation
Based on Pmini-ATAC-seq we performed, 50 GV porcine oocytes and 50 PA embryos 10 h after activation were colledted for Pmini-ATAC-seq library construction. After qualified the libraies, the samples were subjected to high-throughput sequencing. Then, we studied the biological functions of oocytes and embryo chromatin accessible sites, and explored important role of GV and PA differential accessible sites. The results showed that compared with GV, fewer peaks were enriched in PA embryos, which were 11152; and lower coverage ratio of accessible sites in the genome, which was 0.0772%. The distribution of open sites among the gene functional area was similar between GV and PA, but chromatin accessible sites in GV and PA had different functions according to the results of GO enrichment analysis. The accessible sites in GV embryos mainly involved the chromatin structure and oocyte maturation, while in PA embryos were mainly involved in the regulation of histone modification and transcriptional activity. No significant enrichment of KEGG pathways were found between GV and PA differential accessible sites, but we found that these differential sites related to cell pluripotency regulation process. These results indicated that the chromatin accessibility of GV oocytes and PA embryos was significantly different and provided a theoretical basis for further research on chromatin accessibility during embryo reprogramming.
Experiment 3. Identification of chromatin accessibility at the early stage of pig SCNT embryo by ATAC-seq.
We collected 50 pig SCNT embryos 10 h after activation by HMC (Hand-made Cloning). Then we conducted ATAC-seq sequencing on qualified libraries, and mainly analyzed the accessible chromatin landscape during an early stage of embryo reprogramming, the distribution of chromatin accessibility sites in gene functional areas, and the molecular function of accessible chromatin sites. Along with PEFs ATAC-seq data, we detected the chromatin accessibility of genes with different expression patterns in PEFs, compared the different accessibility sites during embryo reprogramming and their biological function. The results showed that compared with PEFs, fewer peaks were enriched in SCNT embryos, which were 3710. And SCNT had much lower coverage ratios of peaks in the genome, which were 0.0267%. The X chromosome also became inaccessible during embryo reprogramming. GO enrichment analysis revealed that the molecular functions related to accessible chromatin in SCNT primarily included transcriptional regulatory activity and Histone acetylation. The differentially accessible chromatin sites between SCNT and PEFs were primarily related to transcriptional activity and histone modification. These results indicated that despite the tight chromatin structure during the early stage of embryo reprogramming, some accessible chromatin sites, which were primarily distributed in the intergenic region, were still detected. Dynamic changes in chromatin accessibility during reprogramming were primarily related to transcriptional activity and histone modification. Generally, this study provided new insight into the dynamics and importance of chromatin accessibility during the early stages of embryo reprogramming.
In summary, we used pig oocytes and embryos as the modle, successfully established a new ATAC-seq method for pig oocytes and embryonic trace cells, which were called "Pmini-ATAC-seq". Then, we detected the dynamic changes of chromatin accessibility sites in the early stage of embryo reprogramming using Pmini-ATAC-seq. And found that despite the tight chromatin structure during the early stage of embryo reprogramming, some accessible chromatin sites, which were associated with transcriptional activity and histone modification, were still detected. This research provides new insights into the importance of dynamic changes in chromatin accessibility in the early stages of embryo reprogramming, and also makes a great contribution to promoting the application of epigenetic technology in livestock embryo researches.