4.7 Article

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

期刊

DEVELOPMENT
卷 148, 期 19, 页码 -

出版社

COMPANY BIOLOGISTS LTD
DOI: 10.1242/dev.199994

关键词

Zebrafish; Knock-in; Epithelial; Morphogenesis; CRISPR; Quantitative imaging

资金

  1. National Institutes of Health [DK121007, DK113123, NS102322]
  2. Duke University Training Grant in Digestive Diseases and Nutrition [DK007568]
  3. New York State StemCell Science institutional training grant [C322560GG]
  4. American Heart Association fellowship [20PRE35180164]

向作者/读者索取更多资源

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 has been developed, enabling the generation of endogenously tagged alleles for crucial genes in epithelial biology and organ development. This approach facilitates the generation of knock-in lines in zebrafish, paving the way for accurate quantitative imaging studies.
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 beta 1b. Our approach facilitates the generation of knock-in lines in zebrafish, opening the way for accurate quantitative imaging studies.

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