Stark Danny A, Kasemeier-Kulesa Jennifer C, Kulesa Paul M
Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
CSH Protoc. 2008 Mar 1;2008:pdb.prot4975. doi: 10.1101/pdb.prot4975.
INTRODUCTIONTracing cell movements in a living embryo or embryo slice culture remains a challenging problem due to difficulties in cell accessibility and in accurate delivery of fluorescent labels into an individual cell or subgroup of cells. Here, we describe a photoactivation cell-labeling technique in avian embryos that allows for selective marking of individual cells or groups of cells at precise times and spatial locations normally not accessible using previous techniques. The current protocol is also less invasive than previously described methods. We provide details of fluorescent protein delivery into cells of interest utilizing microinjection and in ovo electroporation, as well as the optical parameters needed for photoactivation and imaging of both single- and dual-color photoactivatable fluorescent proteins (PAFPs). We present applications to label single cells and small subgroups of cells throughout the head and trunk of the developing vertebrate embryo, using the avian neural crest as our model cell population.
引言
在活胚胎或胚胎切片培养物中追踪细胞运动仍然是一个具有挑战性的问题，这是因为细胞难以接近，并且难以将荧光标记精确地递送至单个细胞或细胞亚群中。在此，我们描述了一种在鸡胚中进行光激活细胞标记的技术，该技术能够在精确的时间和空间位置对单个细胞或细胞群进行选择性标记，而这些位置通常是以前的技术无法到达的。当前的方案相比先前描述的方法侵入性也更小。我们提供了利用显微注射和卵内电穿孔将荧光蛋白递送至感兴趣细胞的详细信息，以及单双色光激活荧光蛋白（PAFP）光激活和成像所需的光学参数。我们展示了使用鸡胚神经嵴作为模型细胞群体，在发育中的脊椎动物胚胎的头部和躯干中标记单个细胞和小细胞亚群的应用。
