Mitochondria distribution in cloned pig embryos

描述(申请人提供)：本研究的目的是探索线粒体分布作为克隆猪胚胎发育潜力的标记。近年来，哺乳动物胚胎的克隆因其在生物医学和农业应用方面的巨大潜力而受到人们的关注。克隆猪作为组织和器官捐赠者是人们非常感兴趣的，因为它与人类具有特殊的生理兼容性。然而，实际应用还不可行，因为克隆效率较低(猪的克隆效率约为0.2%)，其原因尚不清楚。一个原因可能是线粒体分布不对称，可能导致ATP生成能力降低，无法支持正常的细胞功能。我们认为，线粒体聚集和微管组织的特定模式将使我们能够预测克隆胚胎的发育潜力，并提高克隆效率。我们的具体目标是分析：1a)在形态正常的卵裂期胚胎队列中单个卵裂球之间的线粒体分布是否存在差异；1b)细胞内pH的变化是否与线粒体组织的破坏和体外发育减少有关；2a)微管组织是否在克隆胚胎核移植后对线粒体分布起作用；以及2b)核移植后中心体分离不均匀是否对线粒体分布起作用。扫描激光共聚焦显微镜将观察线粒体在固定卵母细胞和活卵母细胞、卵裂期胚胎和发育到囊胚期的分布。MitoTracker Green FM和MitoTracker-X-Rosamine将用于对线粒体进行染色。用抗微管蛋白和抗中心体抗体进行双重和三重免疫荧光染色将确定线粒体和微管组织之间的关系。我们将通过特征线粒体荧光模式将存活率与囊胚阶段相关联。这些实验将提供核克隆后线粒体分布和微管组织的新信息，并使未来的研究旨在选择最有可能存活的胚胎，并增加克隆动物的活产。R03小额助学金项目研究的试点数据将用于通过RO1机制申请资金。

DESCRIPTION (provided by applicant): The purpose of this research is to explore mitochondrial distribution as markers for developmental potential of cloned pig embryos. Cloning of mammalian embryos has become attractive in recent years because of the high potential for biomedical and agricultural applications. Cloning of pigs as tissue and organ donors is of high interest because of the exceptional physiological compatibility with humans. However, practical applications are not yet feasible because of the low cloning efficiency (ca. 0.2% in pigs) for which causes are only little understood. One reason may be an asymmetrical mitochondria distribution that can result in reduced ATP generating capacity and an inability to support normal cell functions. We propose that specific patterns of mitochondria aggregation and microtubule organization will allow us to predict developmental potential of cloned embryos and increase cloning efficiency. Our specific aims are to analyze: 1a) whether differences in mitochondrial distribution occur among individual blastomeres in cohorts of morphologically normal cleavage stage embryos; 1b) whether changes in intracellular pH are associated with disruption of mitochondrial organization and reduced development in vitro; 2a) whether microtubule organization plays a role in mitochondrial distribution after nuclear transfer in cloned embryos; and 2b) whether unequal centrosome separation after nuclear transfer plays a role in mitochondrial distribution. The distribution of mitochondria will be examined by scanning laser confocal microscopy in fixed and live oocytes, in cleavage stage embryos, and development to the blastocyst stages. MitoTracker Green FM and Mitotracker-X-Rosamine will be used to stain mitochondria. Double and triple immunofluorescence staining with anti-tubulin and anti-centrosome antibodies will determine the relationship between mitochondria and microtubule organization. We will correlate survival to the blastocyst stages with characteristic mitochondria fluorescence patterns. These experiments will provide new information on mitochondria distribution and microtubule organization after nuclear cloning and allow future research aimed at selecting embryos that are most likely to survive and increase live birth of cloned animals. Pilot data from this RO3 small grants program research will be used to apply for funding through the RO1 mechanism.