Misregulation of apoptosis in cloned pig embryos

描述（由申请人提供）：本研究的目的是探索克隆猪胚胎中细胞凋亡的模式以及与体外受精胚胎相比，核移植（NT）胚胎中细胞凋亡增加的可能原因。由于猪胚胎克隆技术在生物医学领域的巨大应用潜力，猪胚胎克隆技术已成为生物医学研究中的一个重要的新课题。转基因猪的生产是为了作为人类的组织和器官捐赠者，并作为人类疾病的模型，因为猪与人类在生理上非常相似。然而，由于克隆效率低，实际应用困难。1%的猪），其原因知之甚少。移植后供体细胞核的不完全或异常重塑，导致整个发育过程中的印记失败和异常基因表达，被认为是发育失败和胚胎缺陷的主要原因之一。有人提出，表观遗传因素造成的不当重编程可能会影响发育的后期阶段，导致植入失败，这在克隆失败的原因中占很大比例。在着床前发育过程中，错误的细胞凋亡导致内细胞团（ICM）和滋养外胚层（TE）细胞失衡，对着床产生负面影响。胎盘形成不足是克隆胎儿丢失的主要原因。为了了解NT胚胎中凋亡频率的增加，我们提出了以下具体目标：分析1a）供体细胞线粒体是否分布不均匀并导致特定细胞的凋亡，从而导致发育过程中凋亡的增加; 1b）不含线粒体的供体细胞核是否表现出较低的凋亡发生率; 2a）核纤层蛋白的错误定位是否导致凋亡细胞的增加;以及2b）核有丝分裂器（NuMA）的失调是否在发育期间的细胞凋亡中起作用。我们的研究将采用具有荧光线粒体识别信号（Aim 1）的供体细胞来在整个发育过程中追踪供体细胞线粒体。将使用TUNEL试验鉴定经历凋亡的细胞。将采用分子和成像方法，包括固定卵母细胞、卵裂期胚胎和囊胚期发育的Western免疫印迹和免疫荧光显微镜检查。我们将线粒体染色模式和TUNEL染色与细胞凋亡和胚胎发育到胚泡阶段相关联。这些实验将提供新的信息不平衡的凋亡模式在克隆猪胚胎植入前发育过程中，线粒体，核纤层蛋白B，A/C，和NuMA的分布可能发挥作用。未来的研究目标是操纵胚胎以防止异常凋亡，以提高克隆效率和克隆胚胎的正常发育。R 03小额赠款计划研究的试点数据将用于通过R 01机制申请资金。

DESCRIPTION (provided by applicant): The purpose of this research is to explore the patterns of apoptosis in cloned pig embryos and possible causes for increased apoptosis in nuclear transfer (NT) embryos as compared to in vitro fertilized embryos. Cloning of pig embryos has become an important new topic in biomedical research because of the high potential for biomedical applications. Genetically modified pigs are being produced to serve as tissue and organ donors for humans and as a model for human disease because of the exceptional physiological similarity of pigs with humans. However, practical applications are difficult because of the low cloning efficiency (ca. 1% in pigs) for which causes are only little understood. Incomplete or abnormal remodeling of the donor cell nucleus following transfer, resulting in imprinting failures and abnormal gene expression throughout development, are thought to be among the primary reasons for developmental failures and embryo defects. It has been proposed that improper reprogramming by epigenetic factors may affect later stages of development that can result in implantation failures, which account for a large percentage of the causes for cloning failures. Misguided apoptosis during preimplantation development results in an imbalance of inner cell mass (ICM) and trophectoderm (TE) cells that negatively affects implantation. Insufficient placentation is the primary cause for cloned fetal loss. To understand the increased frequency of apoptosis in NT embryos we propose the following specific aims: to analyze 1a) whether donor cell mitochondria are distributed unequally and contribute to apoptosis in specific cells resulting in increased apoptosis during development; 1b) whether donor cell nuclei transferred without mitochondria exhibit lower incidences of apoptosis; 2a) whether mistargeting of lamins produces increased cells undergoing apoptosis; and 2b) whether misregulation of the nuclear mitotic apparatus (NuMA) plays a rolls in apoptosis during development. Our studies will employ donor cells with a fluorescent mitochondria recognition signal (Aim 1) to trace donor cell mitochondria throughout development. Cells undergoing apoptosis will be identified with the TUNEL assay. Molecular and imaging methods including Western immunoblotting and immunofluorescence microscopy of fixed oocytes, cleavage stage embryos, and development to the blastocyst stages will be employed. We will correlate mitochondria staining patterns and TUNEL staining with apoptosis and with embryo development to the blastocyst stages. These experiments will provide new information on imbalanced apoptosis patterns in cloned pig embryos during preimplantation development in which the distribution of mitochondria, lamins B, A/C, and NuMA might play a role. Future research is aimed at manipulating embryos to prevent abnormal apoptosis to increase cloning efficiency and normal development of cloned embryos. Pilot data from this R03 small grants program research will be used to apply for funding through the R01 mechanism.