Generation of Tfap2c-T2A-tdTomato knock-in reporter rats via adeno-associated virus-mediated efficient gene targeting.
CRISPR/Cas9
Tfap2c
gene targeting in zygote
genetically modified rat
primordial germ cell
Journal
Molecular reproduction and development
ISSN: 1098-2795
Titre abrégé: Mol Reprod Dev
Pays: United States
ID NLM: 8903333
Informations de publication
Date de publication:
03 2022
03 2022
Historique:
revised:
20
12
2021
received:
18
07
2021
accepted:
25
01
2022
pubmed:
17
2
2022
medline:
12
4
2022
entrez:
16
2
2022
Statut:
ppublish
Résumé
Gene editing in mammalian zygotes enables us to generate genetically modified animals rapidly and efficiently. In this study, we compare multiple gene targeting strategies in rat zygotes by generating a novel knock-in reporter rat line to visualize the expression pattern of transcription factor AP-2 gamma (Tfap2c). The targeting vector is designed to replace the stop codon of Tfap2c with T2A-tdTomato sequence. We show that the combination of electroporation-mediated transduction of CRISPR/Cas9 components with adeno-associated virus-mediated transduction of the targeting vector is the most efficient in generating the targeted rat line. The Tfap2c-T2A-tdTomato fluorescence reflects the endogenous expression pattern of Tfap2c in preimplantation embryo, germline, placenta, and forebrain during rat embryo development. The reporter line generated here will be a reliable resource for identifying and purifying Tfap2c expressing cells in rats, and the gene targeting strategy we used can be widely applied for generating desired animals.
Substances chimiques
Luminescent Proteins
0
Types de publication
Letter
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
129-132Informations de copyright
© 2022 Wiley Periodicals LLC.
Références
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Mizuno, N., Mizutani, E., Sato, H., Kasai, M., Ogawa, A., Suchy, F., Yamaguchi, T., & Nakauchi, H. (2018). Intra-embryo gene cassette knockin by CRISPR/Cas9-mediated genome editing with adeno-associated viral vector. iScience, 9, 286-297.
Weber, S., Eckert, D., Nettersheim, D., Gillis, A. J. M., Schafer, S., Kuckenberg, P., Ehlermann, J., Werling, U., Biermann, K., Looijenga, L. H. J., & Schorle, H. (2010). Critical function of AP-2 gamma/TCFAP2C in mouse embryonic germ cell maintenance. Biology of Reproduction, 82, 214-223.