Misregulation of apoptosis in cloned pig embryos

描述（由申请人提供）：本研究的目的是探讨克隆猪胚胎的细胞凋亡模式，以及与体外受精胚胎相比，核移植（NT）胚胎细胞凋亡增加的可能原因。猪胚胎克隆因其在生物医学领域的巨大应用潜力而成为生物医学研究领域的一个重要新课题。由于猪与人在生理上的异常相似，人们正在生产转基因猪，作为人类的组织和器官捐献者，并作为人类疾病的模型。然而，由于克隆效率低（在猪中约为1%），其原因知之甚少，因此实际应用很困难。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基因表达异常，被认为是发育失败和胚胎缺陷的主要原因之一。有人提出，表观遗传因素的不当重编程可能会影响发育的后期，从而导致植入失败，这占克隆失败的很大比例。在胚胎着床前发育过程中，错误的细胞凋亡导致内细胞团（ICM）和滋养外胚层（TE）细胞的不平衡，从而对胚胎着床产生负面影响。胎盘植入不足是克隆胎儿丢失的主要原因。为了了解NT胚胎中细胞凋亡频率的增加，我们提出以下具体目标：分析1a)供体细胞线粒体是否分布不均并导致特定细胞的凋亡，从而导致发育过程中细胞凋亡增加；1b)无线粒体转染的供细胞核是否表现出较低的凋亡发生率；2a)错靶向层粘连蛋白是否导致细胞凋亡增加；2b)核有丝分裂器（NuMA）的错误调节是否在发育过程中凋亡中起重要作用。我们的研究将使用带有荧光线粒体识别信号（Aim 1）的供体细胞来追踪整个发育过程中的供体细胞线粒体。发生凋亡的细胞将通过TUNEL试验进行鉴定。将采用分子和成像方法，包括Western免疫印迹和免疫荧光显微镜对固定卵母细胞、卵裂期胚胎和发育到囊胚期进行观察。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囊胚阶段联系起来。这些实验将为克隆猪胚胎着床前发育过程中线粒体、层蛋白B、A/C和NuMA的分布可能参与的不平衡凋亡模式提供新的信息。未来的研究目标是通过控制胚胎的异常凋亡来提高克隆效率和克隆胚胎的正常发育。R03小额资助项目研究的试点数据将用于通过R01机制申请资助。

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.