Fast, precise and cloning-free knock-in of reporter sequences in vivo with high efficiency.
Development
; 150(12)2023 06 15.
Article
em En
| MEDLINE
| ID: mdl-37309812
ABSTRACT
Targeted knock-in of fluorescent reporters enables powerful gene and protein analyses in a physiological context. However, precise integration of long sequences remains challenging in vivo. Here, we demonstrate cloning-free and precise reporter knock-in into zebrafish genes, using PCR-generated templates for homology-directed repair with short homology arms (PCR tagging). Our novel knock-in reporter lines of vesicle-associated membrane protein (vamp) zebrafish homologues reveal subcellular complexity in this protein family. Our approach enables fast and efficient reporter integration in the zebrafish genome (in 10-40% of injected embryos) and rapid generation of stable germline-transmitting lines.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Peixe-Zebra
/
Sistemas CRISPR-Cas
Limite:
Animals
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article