PUBLICATION
Knock-in tagging in zebrafish facilitated by insertion into non-coding regions
- Authors
- Levic, D.S., Yamaguchi, N., Wang, S., Knaut, H., Bagnat, M.
- ID
- ZDB-PUB-210909-8
- Date
- 2021
- Source
- Development (Cambridge, England) 148(19): (Journal)
- Registered Authors
- Bagnat, Michel, Knaut, Holger
- Keywords
- CRISPR, Epithelial, Knock-in, Morphogenesis, Quantitative imaging, Zebrafish
- MeSH Terms
-
- Animals
- Gene Knock-In Techniques/methods*
- Green Fluorescent Proteins/genetics*
- Green Fluorescent Proteins/metabolism
- Mutagenesis, Insertional
- Recombinant Proteins/genetics
- Recombinant Proteins/metabolism
- Regulatory Sequences, Nucleic Acid/genetics
- Zebrafish
- Zebrafish Proteins/genetics*
- Zebrafish Proteins/metabolism
- PubMed
- 34495314 Full text @ Development
Citation
Levic, D.S., Yamaguchi, N., Wang, S., Knaut, H., Bagnat, M. (2021) Knock-in tagging in zebrafish facilitated by insertion into non-coding regions. Development (Cambridge, England). 148(19):.
Abstract
Zebrafish provide an excellent model for in vivo cell biology studies due to their amenability to live imaging. Protein visualization in zebrafish has traditionally relied on overexpression of fluorescently tagged proteins from heterologous promoters, making it difficult to recapitulate endogenous expression patterns and protein function. One way to circumvent this problem is to tag the proteins by modifying their endogenous genomic loci. Such an approach is not widely available to zebrafish researchers due to inefficient homologous recombination and the error-prone nature of targeted integration in zebrafish. Here, we report a simple approach for tagging proteins in zebrafish on their N- or C termini with fluorescent proteins by inserting PCR-generated donor amplicons into non-coding regions of the corresponding genes. Using this approach, we generated endogenously tagged alleles for several genes critical for epithelial biology and organ development including the tight junction components ZO-1 and Cldn15la, the trafficking effector Rab11a, the apical polarity protein aPKC, and the ECM receptor Integrin β1b. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping