Tagging the tjp1a gene in zebrafish with mRFP using biotin homology arms
Date
2024-04-11
Authors
Davison, Connor
Harzman, Hamelynn
Nicholson, Jessie
Entriken, Seth
Mobley, Kierinn
Krull, Abigail
Singhal, Manik
Skow, Caleb
Matthews, Nathan
Kopp, Lindsey
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Mary Ann Liebert
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 red fluorescent tag (mRFP) using CRISPR/Cas9-mediated targeted integration of biotin-labeled 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 prior to 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.
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article
Comments
This is a manuscript of an article published as Davison, Connor, Hamelynn Harzman, Jessie Nicholson, Seth Entriken, Kierinn Mobley, Abigail Krull, Manik Singhal et al. "Tagging the tjp1a Gene in Zebrafish with Monomeric Red Fluorescent Protein Using Biotin Homology Arms." Zebrafish 21, no. 2 (2024): 191-197. doi: https://doi.org/10.1089/zeb.2023.0096.
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Copyright 2024, Mary Ann Liebert, Inc., publishers