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Nat. Prot.:线虫胚胎后发育荧光活体显微成像方法

来源:中科院生物物理研究所 2013-01-14 15:58

中国科学院生物物理研究所欧光朔研究组在2012年12月期的Nature Protocols上发表题为Live imaging of cellular dynamics during Caenorhabditis elegans postembryonic development的文章,介绍他们发展的研究线虫胚胎后发育的荧光活体显微成像方法。

胚胎后发育是生命体一个重要的发育时期。例如,线虫的959个体细胞中有400多个是在胚胎后时期产生的。观察线虫胚胎时期发育的显微成像技术相对成熟,而研究线虫胚胎后发育的活体荧光显微成像方法缺乏。

该文章系统介绍了观察活体线虫胚胎后发育时期细胞动态的方法,并对可能的技术难点进行了讨论。欧光朔研究组将这项成像技术与线虫遗传学的结合,发现了迁移细胞的分子标识(Ou & Vale, Journal of Cell Biology, 2009)、 一种新的细胞不对称分裂方式(Ou et al., Science, 2010) 、自体吞噬基因在凋亡细胞降解中的作用(Li et al., Journal of Cell Biology, 2012)等。

该项工作得到科技部、国家自然科学基金委和“青年千人计划”的资助。(生物谷Bioon.com)

Live imaging of cellular dynamics duringCaenorhabditis elegans postembryonic development

Yongping Chai Wei Li Guoxin Feng Yihong Yang Xiangming Wang Guangshuo Ou

Postembryonic development is an important process of organismal maturation after embryonic growth. Despite key progress in recent years in understanding embryonic development via fluorescence time-lapse microscopy, comparatively less live-cell imaging of postembryonic development has been done. Here we describe a protocol to image larval development in the nematode Caenorhabditis elegans. Our protocol describes the construction of fluorescent transgenic C. elegans, immobilization of worm larvae and time-lapse microscopy analysis. To improve the throughput of imaging, we developed a C. elegans triple-fluorescence imaging approach with a worm-optimized blue fluorescent protein (TagBFP), green fluorescent protein (GFP) and mCherry. This protocol has been previously applied to time-lapse imaging analysis of Q neuroblast asymmetric division, migration and apoptosis, and we show here that it can also be used to image neuritogenesis in the L1 larvae. Other applications are also possible. The protocol can be completed within 3 h and may provide insights into understanding postembryonic development.

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