Knock-in tagging in zebrafish facilitated by insertion into non-coding regions.

Journal Article (Journal Article)

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 β1b. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies.

Full Text

Duke Authors

Cited Authors

  • Levic, DS; Yamaguchi, N; Wang, S; Knaut, H; Bagnat, M

Published Date

  • October 1, 2021

Published In

Volume / Issue

  • 148 / 19

PubMed ID

  • 34495314

Pubmed Central ID

  • PMC8513609

Electronic International Standard Serial Number (EISSN)

  • 1477-9129

Digital Object Identifier (DOI)

  • 10.1242/dev.199994

Language

  • eng

Conference Location

  • England