PUBLICATION

Tagging the tjp1a Gene in Zebrafish with Monomeric Red Fluorescent Protein Using Biotin Homology Arms

Authors
Davison, C., Harzman, H., Nicholson, J., Entriken, S., Mobley, K., Krull, A., Singhal, M., Skow, C., Matthews, N., Kopp, L., Gillette, B., Weide, T.J., Hukvari, J.R., Stumpf, S.C.P., Feldmann, O.M., McGrail, M., Srivastava, R., Essner, J.J.
ID
ZDB-PUB-240416-13
Date
2024
Source
Zebrafish   21: 191197191-197 (Journal)
Registered Authors
Essner, Jeffrey, McGrail, Maura
Keywords
CRISPR/Cas9, biotin homology arms, targeted integration, zebrafish
MeSH Terms
  • Animals
  • Biotin/genetics
  • CRISPR-Cas Systems*
  • Endothelial Cells
  • RNA, Guide, CRISPR-Cas Systems
  • Red Fluorescent Protein
  • Zebrafish*/genetics
(all 7)
PubMed
38621205 Full text @ Zebrafish
Abstract
Tjp1a and other tight junction and adherens proteins play important roles in cell-cell adhesion, scaffolding, and forming seals between cells in epithelial and endothelial tissues. In this study, we labeled Tjp1a of zebrafish with the monomeric red fluorescent protein (mRFP) using CRISPR/Cas9-mediated targeted integration of biotin-labeled polymerase chain reaction (PCR) generated templates. Labeling Tjp1a with RFP allowed us to follow membrane and junctional dynamics of epithelial and endothelial cells throughout zebrafish embryo development. For targeted integration, we used short 35 bp homology arms on each side of the Cas9 genomic target site at the C-terminal of the coding sequence in tjp1a. Through PCR using 5' biotinylated primers containing the homology arms, we generated a double-stranded template for homology directed repair containing a flexible linker followed by RFP. Cas9 protein was complexed with the tjp1a gRNA before mixing with the repair template and microinjected into one-cell zebrafish embryos. We confirmed and recovered a precise integration allele at the desired site at the tjp1a C-terminus. Examination of fluorescence reveals RFP cell-cell junctional labeling using confocal imaging. We are currently using this stable tjp1a-mRFPis86 line to examine the behavior and interactions between cells during vascular formation in zebrafish.
Genes / Markers
Figures
Figure Gallery (5 images)
Show all Figures
Expression
No data available
Phenotype
No data available
Mutations / Transgenics
Allele Construct Type Affected Genomic Region
is86TgTransgenic Insertion
y1TgTransgenic Insertion
    1 - 2 of 2
    Show
    Human Disease / Model
    No data available
    Sequence Targeting Reagents
    Target Reagent Reagent Type
    tjp1aCRISPR6-tjp1aCRISPR
    1 - 1 of 1
    Show
    Fish
    No data available
    Antibodies
    Name Type Antigen Genes Isotypes Host Organism
    Ab1-tjp1monoclonalIgG1Mouse
    1 - 1 of 1
    Show
    Orthology
    No data available
    Engineered Foreign Genes
    Marker Marker Type Name
    EGFPEFGEGFP
    mRFPEFGmRFP
    1 - 2 of 2
    Show
    Mapping
    No data available
    Your Input Welcome
    We welcome your input and comments. Please use this form to recommend updates to the information in ZFIN. We appreciate as much detail as possible and references as appropriate. We will review your comments promptly.
    Citation
    Davison, C., Harzman, H., Nicholson, J., Entriken, S., Mobley, K., Krull, A., Singhal, M., Skow, C., Matthews, N., Kopp, L., Gillette, B., Weide, T.J., Hukvari, J.R., Stumpf, S.C.P., Feldmann, O.M., McGrail, M., Srivastava, R., Essner, J.J. (2024) Tagging the tjp1a Gene in Zebrafish with Monomeric Red Fluorescent Protein Using Biotin Homology Arms. Zebrafish. 21:191197191-197.