A novel technique for large-fragment knock-in animal production without ex vivo handling of zygotes.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
08 02 2023
08 02 2023
Historique:
received:
16
09
2022
accepted:
06
02
2023
entrez:
9
2
2023
pubmed:
10
2
2023
medline:
11
2
2023
Statut:
epublish
Résumé
CRISPR/Cas-based genome editing has dramatically improved genetic modification technology. In situ electroporation called genome editing via oviductal nucleic acid delivery (GONAD), which eliminates the need for ex vivo embryo handling, is technically the simplest method for gene transfer and can be performed in laboratories without developmental engineering expertise including micromanipulation techniques. However, the use of this method remains challenging in the case of large-fragment knock-in, such as gene expression cassettes. Adeno-associated viruses (AAV) act as donor DNA for homologous recombination in infected cells, including rodent embryos. In this study, we demonstrated simultaneous electroporation of AAV donors and CRISPR/Cas9 components into embryos to create knock-in animals, and successfully generated knock-in rats carrying a gene cassette with a length of 3.0 kb using a small number of animals and in situ electroporation. These findings indicate that this technique is an efficient high-throughput strategy for producing genetically modified rodents and may be applicable to other animal species.
Identifiants
pubmed: 36755180
doi: 10.1038/s41598-023-29468-1
pii: 10.1038/s41598-023-29468-1
pmc: PMC9908863
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2245Informations de copyright
© 2023. The Author(s).
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