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.
Knock-in tagging in zebrafish facilitated by insertion into non-coding regions
- Award Group:
- Funder(s): National Institutes of Health
- Award Id(s): DK121007
- Funder(s):
- Award Group:
- Funder(s): New York State Stem Cell Science
- Award Id(s): C322560GG
- Funder(s):
- Award Group:
- Funder(s): American Heart Association
- Award Id(s): 20PRE35180164
- Funder(s):
Currently Viewing Accepted Manuscript - Newer Version Available
- Split-screen
- Views Icon Views
- Open the PDF for in another window
-
Article Versions Icon
Versions
- Version of Record 04 October 2021
- Accepted Manuscript 08 September 2021
- Share Icon Share
-
Tools Icon
Tools
- Search Site
Daniel S. Levic, Naoya Yamaguchi, Siyao Wang, Holger Knaut, Michel Bagnat; Knock-in tagging in zebrafish facilitated by insertion into non-coding regions. Development 2021; dev.199994. doi: https://doi.org/10.1242/dev.199994
Download citation file:
Advertisement
Call for papers: Uncovering Developmental Diversity
Development invites you to submit your latest research to our upcoming special issue: Uncovering Developmental Diversity. This issue will be coordinated by our academic Editor Cassandra Extavour (Harvard University, USA) alongside two Guest Editors: Liam Dolan (Gregor Mendel Institute of Molecular Plant Biology, Austria) and Karen Sears (University of California Los Angeles, USA).
Choose Development in 2024
In this Editorial, Development Editor-in-Chief James Briscoe and Executive Editor Katherine Brown explain how you support your community by publishing in Development and how the journal champions serious science, community connections and progressive publishing.
Journal Meeting: From Stem Cells to Human Development
Register now for the 2024 Development Journal Meeting From Stem Cells to Human Development. Early-bird registration deadline: 3 May. Abstract submission deadline: 21 June.
Pluripotency of a founding field: rebranding developmental biology
This collaborative Perspective, the result of a workshop held in 2023, proposes a set of community actions to increase the visibility of the developmental biology field. The authors make recommendations for new funding streams, frameworks for collaborations and mechanisms by which members of the community can promote themselves and their research.
Read & Publish Open Access publishing: what authors say
We have had great feedback from authors who have benefitted from our Read & Publish agreement with their institution and have been able to publish Open Access with us without paying an APC. Read what they had to say.