Knock-in tagging in zebrafish facilitated by insertion into non-coding regions.
Development
; 148(19)2021 10 01.
Article
em En
| MEDLINE
| ID: mdl-34495314
ABSTRACT
Zebrafish provide an excellent model for in vivo cell biology studies because of 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 because of 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 that are crucial 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 ß1b. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies.
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Base de dados:
MEDLINE
Assunto principal:
Proteínas de Peixe-Zebra
/
Proteínas de Fluorescência Verde
/
Técnicas de Introdução de Genes
Limite:
Animals
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article