Huss David, Choi Harry M T, Readhead Carol, Fraser Scott E, Pierce Niles A, Lansford Rusty
Keck School of Medicine, University of Southern California, Children's Hospital Los Angeles, California 90027;
Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, California 91125;
Cold Spring Harb Protoc. 2015 Mar 2;2015(3):259-68. doi: 10.1101/pdb.prot083832.
Multiplexed fluorescent hybridization chain reaction (HCR) and advanced imaging techniques can be used to evaluate combinatorial gene expression patterns in whole mouse embryos with unprecedented spatial resolution. Using HCR, DNA probes complementary to mRNA targets trigger chain reactions in which metastable fluorophore-labeled DNA HCR hairpins self-assemble into tethered fluorescent amplification polymers. Each target mRNA is detected by a probe set containing one or more DNA probes, with each probe carrying two HCR initiators. For multiplexed experiments, probe sets for different target mRNAs carry orthogonal initiators that trigger orthogonal DNA HCR amplification cascades labeled by spectrally distinct fluorophores. As a result, in situ amplification is performed for all targets simultaneously, and the duration of the experiment is independent of the number of target mRNAs. We have used multiplexed fluorescent in situ HCR and advanced imaging technologies to address questions of cell heterogeneity and tissue complexity in craniofacial patterning and anterior neural development. In the sample protocol presented here, we detect three different mRNA targets: Tg(egfp), encoding the enhanced green fluorescent protein (GFP) transgene (typically used as a control); Twist1, encoding a transcription factor involved in cell lineage determination and differentiation; and Pax2, encoding a transcription factor expressed in the mid-hindbrain region of the mouse embryo.
多重荧光杂交链式反应（HCR）和先进的成像技术可用于以前所未有的空间分辨率评估完整小鼠胚胎中的组合基因表达模式。使用HCR时，与mRNA靶标互补的DNA探针会引发链式反应，其中亚稳态荧光团标记的DNA HCR发夹会自组装成连接的荧光扩增聚合物。每个靶标mRNA通过一个包含一个或多个DNA探针的探针组进行检测，每个探针携带两个HCR引发剂。对于多重实验，不同靶标mRNA的探针组携带正交引发剂，这些引发剂会触发由光谱不同的荧光团标记的正交DNA HCR扩增级联反应。结果，所有靶标同时进行原位扩增，并且实验持续时间与靶标mRNA的数量无关。我们已经使用多重荧光原位HCR和先进的成像技术来解决颅面模式形成和前神经发育中细胞异质性和组织复杂性的问题。在本文介绍的示例方案中，我们检测三种不同的mRNA靶标：编码增强型绿色荧光蛋白（GFP）转基因的Tg(egfp)（通常用作对照）；编码参与细胞谱系确定和分化的转录因子的Twist1；以及编码在小鼠胚胎中脑后脑区域表达的转录因子的Pax2。
