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
  • Zebrafish
  • Green Fluorescent Proteins/genetics*
  • Green Fluorescent Proteins/metabolism
  • Recombinant Proteins/genetics
  • Recombinant Proteins/metabolism
  • Animals
  • Zebrafish Proteins/genetics*
  • Zebrafish Proteins/metabolism
  • Regulatory Sequences, Nucleic Acid/genetics
  • Mutagenesis, Insertional
  • Gene Knock-In Techniques/methods*
(all 11)
PubMed
34495314 Full text @ Development
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
Figures
Figure Gallery (4 images)
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Expression
No data available
Phenotype
No data available
Mutations / Transgenics
Allele Construct Type Affected Genomic Region
pd1034TgTransgenic Insertion
    pd1224TgTransgenic Insertion
    pd1244TgTransgenic Insertion
    pd1249TgTransgenic Insertion
    pd1252TgTransgenic Insertion
    pd1260TgTransgenic Insertion
    sk108TgTransgenic Insertion
    1 - 7 of 7
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    Human Disease / Model
    No data available
    Sequence Targeting Reagents
    Target Reagent Reagent Type
    cldn15laCRISPR1-cldn15laCRISPR
    cldn15laCRISPR2-cldn15laCRISPR
    itgb1bCRISPR1-itgb1bCRISPR
    prkciCRISPR1-prkciCRISPR
    rab11aCRISPR3-rab11aCRISPR
    rab11aCRISPR4-rab11aCRISPR
    tjp1aCRISPR4-tjp1aCRISPR
    tjp1aCRISPR5-tjp1aCRISPR
    1 - 8 of 8
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    Fish
    No data available
    Antibodies
    No data available
    Orthology
    No data available
    Engineered Foreign Genes
    Marker Marker Type Name
    EGFPEFGEGFP
    GFPEFGGFP
    TomatoEFGTomato
    1 - 3 of 3
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    Mapping
    No data available
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    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):.