Rapid and high-efficient generation of mutant mice using freeze-thawed embryos of the C57BL/6J strain

Mohamed Darwish, Hirofumi Nishizono, Hideki Uosaki, Hitomi Sawada, Taketaro Sadahiro, Masaki Ieda, Keizo Takao

Research output: Contribution to journalArticle

Abstract

Background: The CRISPR/Cas9 technique has undergone many modifications to decrease the effort and shorten the time needed for efficient production of mutant mice. The use of fresh embryos consumes time and effort during oocytes preparation and fertilization before every experiment, and freeze-thawed embryos overcome this limitation. However, cryopreservation of 1-cell embryos is challenging. New method: We introduce a protocol that combines a modified method for cryopreserving 1-cell C57BL/6J embryos with optimized electroporation conditions that were used to deliver CRISPR reagents into embryos, 1 h after thawing. Results: Freeze-thawed 1-cell embryos showed similar survival rates and surprisingly high developmental rates compared to fresh embryos. Using our protocol, we generated several lines of mutant mice: knockout mice via non-homologous end joining (NHEJ) and knock-in mice via homology-directed repair (HDR) with high-efficient mutation rates (100%, 75% respectively) and a low mosaic rate within 4 weeks. Comparison with existing method (s): Our protocol associates the use of freeze-thawed embryos from an inbred strain and electroporation, and can be performed by laboratory personnel with basic training in embryo manipulation to generate mutant mice within short time periods. Conclusion: We developed a simple, economic, and robust protocol facilitating the generation of genetically modified mice, bypassing the need of backcrossing, with a high efficiency and a low mosaic rate. It makes the preparation of mouse models of human diseases a simple task with unprecedented ease, pace, and efficiency.

Original languageEnglish
JournalJournal of Neuroscience Methods
DOIs
Publication statusPublished - 2019 Jan 1

Fingerprint

Embryonic Structures
Clustered Regularly Interspaced Short Palindromic Repeats
Electroporation
Laboratory Personnel
Inbreeding
Cryopreservation
Mutation Rate
Fertilization
Knockout Mice
Oocytes
Survival Rate
Economics

Keywords

  • CRISPR/Cas9
  • Electroporation
  • Freeze-thawed embryos
  • Inbred strain
  • Knock-in
  • Knockout

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Rapid and high-efficient generation of mutant mice using freeze-thawed embryos of the C57BL/6J strain. / Darwish, Mohamed; Nishizono, Hirofumi; Uosaki, Hideki; Sawada, Hitomi; Sadahiro, Taketaro; Ieda, Masaki; Takao, Keizo.

In: Journal of Neuroscience Methods, 01.01.2019.

Research output: Contribution to journalArticle

Darwish, Mohamed ; Nishizono, Hirofumi ; Uosaki, Hideki ; Sawada, Hitomi ; Sadahiro, Taketaro ; Ieda, Masaki ; Takao, Keizo. / Rapid and high-efficient generation of mutant mice using freeze-thawed embryos of the C57BL/6J strain. In: Journal of Neuroscience Methods. 2019.
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