Work

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

Year: 2024

Type: article

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 • Detailed protocol for generating a knockin zebrafish line • Instructions for generating the donor plasmid for pdgfrb knockin • Steps to generate Ki(pdgfrb-P2A-Gal4-VP16) zebrafish lines for pericyte labeling Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.

Source: STAR Protocols

DOI: 10.1016/j.xpro.2024.103490

Authors: Huaxing Zi, Xiaolan Peng, Jiulin Du, Jia Li

Institutions: Chinese Academy of Sciences, Center for Excellence in Brain Science and Intelligence Technology, University of Chinese Academy of Sciences, State Key Laboratory of Neuroscience, ShanghaiTech University

Language: English

Topic: Zebrafish Biomedical Research Applications

Subfield: Cell Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,500