Murray John Isaac, Bao Zhirong, Boyle Thomas J, Waterston Robert H
Department of Genome Sciences, University of Washington Box 357730, Seattle, Washington 98195, USA.
Nat Protoc. 2006;1(3):1468-76. doi: 10.1038/nprot.2006.222.
Lineage analysis of Caenorhabditis elegans is a powerful tool for characterizing developmental phenotypes and embryonic gene-expression patterns. We present a detailed protocol for the lineaging of embryos by computational analysis of 4D images of embryos that ubiquitously express histone-GFP (green fluorescent protein) fusion proteins through the 350 cell stage followed by manual editing. We describe how to optimize imaging settings for this purpose, the use of the lineage-extraction software, StarryNite, and the lineage-editing software, AceTree. In addition, we describe a useful polymer bead mounting technique for C. elegans embryos that has several advantages compared with the standard agar pad mounting technique. The protocol requires about 1 h of user time spread over 2 days to generate the raw lineage, and an additional 2 or 4 h to edit the lineage to the 194- or 350-cell stage, respectively.
秀丽隐杆线虫的谱系分析是表征发育表型和胚胎基因表达模式的有力工具。我们提供了一个详细的方案，用于通过对胚胎的4D图像进行计算分析来绘制胚胎谱系，这些胚胎在350细胞阶段之前普遍表达组蛋白-GFP（绿色荧光蛋白）融合蛋白，之后进行手动编辑。我们描述了如何为此目的优化成像设置、使用谱系提取软件StarryNite和谱系编辑软件AceTree。此外，我们描述了一种用于秀丽隐杆线虫胚胎的有用的聚合物珠固定技术，与标准的琼脂垫固定技术相比，它有几个优点。该方案需要用户在2天内分散约1小时来生成原始谱系，分别再额外花费2或4小时将谱系编辑到194细胞或350细胞阶段。
