使用crispr - cas9介导的敲入技术生成斑马鱼周细胞报告细胞系Ki（pdgfrb-P2A-GAL4-VP16）的方案。

IF 1.3 Q4 BIOCHEMICAL RESEARCH METHODS

STAR Protocols Pub Date : 2025-03-21 Epub Date: 2024-12-13 DOI:10.1016/j.xpro.2024.103490

Huaxing Zi, Xiaolan Peng, Jiulin Du, Jia Li

{"title":"使用crispr - cas9介导的敲入技术生成斑马鱼周细胞报告细胞系Ki（pdgfrb-P2A-GAL4-VP16）的方案。","authors":"Huaxing Zi, Xiaolan Peng, Jiulin Du, Jia Li","doi":"10.1016/j.xpro.2024.103490","DOIUrl":null,"url":null,"abstract":"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.1.","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103490"},"PeriodicalIF":1.3000,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11699729/pdf/","citationCount":"0","resultStr":"{\"title\":\"Protocol for generating a pericyte reporter zebrafish line Ki(pdgfrb-P2A-GAL4-VP16) using a CRISPR-Cas9-mediated knockin technique.\",\"authors\":\"Huaxing Zi, Xiaolan Peng, Jiulin Du, Jia Li\",\"doi\":\"10.1016/j.xpro.2024.103490\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"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.1.\",\"PeriodicalId\":34214,\"journal\":{\"name\":\"STAR Protocols\",\"volume\":\"6 1\",\"pages\":\"103490\"},\"PeriodicalIF\":1.3000,\"publicationDate\":\"2025-03-21\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11699729/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"STAR Protocols\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1016/j.xpro.2024.103490\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/12/13 0:00:00\",\"PubModel\":\"Epub\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"STAR Protocols","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.xpro.2024.103490","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/12/13 0:00:00","PubModel":"Epub","JCR":"Q4","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}

引用次数: 0

摘要

周细胞是包裹小血管的壁细胞，在血脑屏障（BBB）的形成中起着至关重要的作用。在这里，我们提出了一种使用crispr - cas9介导的敲入技术生成斑马鱼周细胞报告细胞系Ki（pdgfrb-P2A-GAL4-VP16）的方案。我们描述了识别有效的单导RNA (sgRNA)，构建供体质粒，以及产生和维持敲蛋白线的步骤。然后，我们详细说明了脑周细胞的体内成像程序。该协议适用于创建用于特定细胞标记的其他敲蛋白系。有关本协议使用和执行的完整细节，请参阅Zi等人1。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

查看原文本刊更多论文

Protocol for generating a pericyte reporter zebrafish line Ki(pdgfrb-P2A-GAL4-VP16) using a CRISPR-Cas9-mediated knockin technique.

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.¹.

求助全文

通过发布文献求助，成功后即可免费获取论文全文。去求助

来源期刊