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Mitochondria distribution in cloned pig embryos

克隆猪胚胎中的线粒体分布

基本信息

批准号：
6832809
负责人：
HEIDE SCHATTEN
金额：
$ 7.25万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-12-08 至 2006-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6832809
关键词：
acidity /alkalinity cell component structure /function cell morphology centromere confocal scanning microscopy embryo /fetus embryogenic cleavage immunocytochemistry immunofluorescence technique intracellular microtubules mitochondria nuclear transfer swine

项目摘要

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.

描述（由申请人提供）：本研究的目的是探索作为克隆猪胚胎发育潜力标记的线粒体分布。由于在生物医学和农业应用方面的巨大潜力，哺乳动物胚胎的克隆近年来变得具有吸引力。克隆猪作为组织和器官供体是高度感兴趣的，因为它与人类具有特殊的生理相容性。然而，由于克隆效率低（在猪中约为0.2%），其原因尚不清楚，因此实际应用尚不可行。其中一个原因可能是线粒体分布不对称，导致ATP生成能力降低，无法支持正常细胞功能。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，提高克隆效率。我们的具体目的是分析：1a)在形态正常的卵裂期胚胎群中，线粒体分布是否在单个卵裂球中发生差异；1b)细胞内pH的变化是否与线粒体组织破坏和体外发育减少有关；2a)克隆胚胎核移植后微管组织是否在线粒体分布中起作用；2b)核转移后中心体分离不均是否对线粒体分布有影响。线粒体的分布将通过扫描激光共聚焦显微镜在固定和活卵母细胞、卵裂期胚胎和发育到囊胚期进行检查。将使用MitoTracker Green FM和MitoTracker - x - rosamine染色线粒体。抗微管蛋白和抗中心体抗体的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。我们将把生存与囊胚阶段与线粒体荧光模式的特征联系起来。这些实验将提供细胞核克隆后线粒体分布和微管组织的新信息，并允许未来的研究旨在选择最有可能存活的胚胎和增加克隆动物的活产率。该RO3小额资助计划研究的试点数据将用于通过RO1机制申请资助。