利用CRISPR-Cas9介导的敲入技术生成周细胞报告基因斑马鱼系Ki(pdgfrb-P2A-GAL4-VP16)的方案。
Protocol for generating a pericyte reporter zebrafish line Ki(pdgfrb-P2A-GAL4-VP16) using a CRISPR-Cas9-mediated knockin technique.
作者信息
Zi Huaxing, Peng Xiaolan, Du Jiulin, Li Jia
机构信息
Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China; University of Chinese Academy of Sciences, 19A Yu-Quan Road, Beijing 100049, China; Dongguan Innovation Institute, Guangdong Medical University, 1 Xin-Cheng Road, Dongguan, Guangdong 523808, China.
Institute of Neuroscience, State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
出版信息
STAR Protoc. 2025 Mar 21;6(1):103490. doi: 10.1016/j.xpro.2024.103490. Epub 2024 Dec 13.
Pericytes, the mural cells that envelop small blood vessels, play crucial roles in the formation of the blood-brain barrier (BBB). Here, we present a protocol for generating a pericyte reporter zebrafish line Ki(pdgfrb-P2A-GAL4-VP16) using a CRISPR-Cas9-mediated knockin technique. We describe steps for identifying efficient single guide RNA (sgRNA), constructing donor plasmid, and generating and maintaining the knockin line. We then detail procedures for in vivo imaging of brain pericytes. This protocol is adaptable for creating other knockin lines for specific cell labeling. For complete details on the use and execution of this protocol, please refer to Zi et al..
周细胞是包裹小血管的壁细胞，在血脑屏障（BBB）的形成中起关键作用。在这里，我们展示了一种使用CRISPR-Cas9介导的敲入技术生成周细胞报告基因斑马鱼品系Ki(pdgfrb-P2A-GAL4-VP16)的方案。我们描述了鉴定高效单向导RNA（sgRNA）、构建供体质粒以及生成和维持敲入品系的步骤。然后，我们详细介绍了脑周细胞体内成像的程序。该方案适用于创建用于特定细胞标记的其他敲入品系。有关本方案使用和执行的完整详细信息，请参考Zi等人的研究。
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