Live Embryo Imaging to Follow Cell Cycle and Chromosomes Stability After Nuclear Transfer
Live Embryo Imaging to Follow Cell Cycle and Chromosomes Stability After Nuclear Transfer
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DOI：
10.1007/978-1-4939-1594-1_11
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发表时间：
2015-01-01
期刊：
影响因子：
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通讯作者：
Boiani, Michele
中科院分区：
文献类型：
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作者：
Balbach, Sebastian T.;Boiani, Michele
核移植（NT）到小鼠卵母细胞产生一个转录和功能异质性群体的克隆胚胎。大多数NT胚胎的研究仅考虑处于预定义关键阶段的胚胎（例如，桑椹胚或胚泡），也就是说，在大量的重编程已经发生之后。这些回顾性方法在阐明重编程机制和预测发育成功方面的作用有限。使用活胚胎电影摄影术观察克隆胚胎发育有可能揭示否则无法检测到的胚胎特征。然而，活细胞电影摄影所需的曝光是高度毒性的克隆embryos.在这里，我们描述了一个协议相结合的明场和荧光活细胞成像组蛋白H2 b-GFP表达小鼠胚胎，记录细胞分裂到囊胚阶段。该方案可以适用于观察其他报告基因，如Oct 4-GFP或Nanog-GFP，使我们能够定量分析克隆胚胎的卵裂动力学。
Nuclear transfer (NT) into mouse oocytes yields a transcriptionally and functionally heterogeneous population of cloned embryos. Most studies of NT embryos consider only embryos at predefined key stages (e.g., morula or blastocyst), that is, after the bulk of reprogramming has taken place. These retrospective approaches are of limited use to elucidate mechanisms of reprogramming and to predict developmental success. Observing cloned embryo development using live embryo cinematography has the potential to reveal otherwise undetectable embryo features. However, light exposure necessary for live cell cinematography is highly toxic to cloned embryos.Here we describe a protocol for combined bright-field and fluorescence live-cell imaging of histone H2b-GFP expressing mouse embryos, to record cell divisions up to the blastocyst stage. This protocol, which can be adapted to observe other reporters such as Oct4-GFP or Nanog-GFP, allowed us to quantitatively analyze cleavage kinetics of cloned embryos.