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

Darwish, M.; Nishizono, Hirofumi; Uosaki, Hideki; Sawada, Hitomi; Sadahiro, Taketaro; Ieda, Masaki; Takao, Keizo

doi:10.1016/j.jneumeth.2019.01.010

Cited by 4 publications

(4 citation statements)

References 28 publications

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“…Although the method for generating GM mice by electroporation in vitrified-warmed B6 embryos was previously reported [ 20 ], this method required pretreatment before freezing the embryos. The established protocol in this study is so simple because it does not require pretreatment of embryos and can efficiently deliver CRISPR/Cas9 reagents into zygotes with intact zona pellucida and edit the targeted locus.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, the application of vitrified-warmed embryos to these electroporation methods enables to carry out the gene editing of embryos and generate GM mice more efficiently and systematically. However, GM mice generation by the electroporation method using vitrified-warmed embryos showed a low development rate and/or required pretreatment of embryos before freezing [ 20 ]. In addition, there are no reports on the gene editing of inbred mouse strain embryos using electroporation, except for C57BL/6.…”

Section: Introductionmentioning

confidence: 99%

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The versatile electric condition in mouse embryos for genome editing using a three-step square-wave pulse electroporator

et al. 2022

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Technique for Animal Knockout system by Electroporation (TAKE) is a simple and efficient method to generate genetically modified (GM) mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems. To reinforce the versatility of electroporation used for gene editing in mice, the electric condition was optimized for vitrified-warmed mouse embryos, and applied to the fresh embryos from widely used inbred strains (C57BL/6NCr, BALB/cCrSlc, FVB/NJcl, and C3H/HeJJcl). The electric pulse settings (poring pulse: voltage, 150 V; pulse width, 1.0 ms; pulse interval, 50 ms; number of pulses, +4; transfer pulse: voltage, 20 V; pulse width, 50 ms; pulse interval, 50 ms; number of pulses, ±5) were optimal for vitrified-warmed mouse embryos, which could efficiently deliver the gRNA/Cas9 complex into the zygotes without zona pellucida thinning process and edit the target locus. These electric condition efficiently generated GM mice in widely used inbred mouse strains. In addition, electroporation using the electrode with a 5 mm gap could introduce more than 100 embryos within 5 min without specific pretreatment and sophisticated technical skills, such as microinjection, and exhibited a high developmental rate of embryos and genomeediting efficiency in the generated offspring, leading to the rapid and efficient generation of genome editing mice. The electric condition used in this study is highly versatile and can contribute to understanding human diseases and gene functions by generating GM mice more easily and efficiently.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The versatile electric condition in mouse embryos for genome editing using a three-step square-wave pulse electroporator

et al. 2022

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“…The efficiency of this approach has been further optimized by using Cas9 protein instead of mRNA ( Figure 3 ) (Hashimoto and Takemoto, 2015 ; Qin et al, 2015 ). The use of frozen fertilized eggs instead of fresh ones has further simplified the whole process (Darwish et al, 2019 ; Nishizono et al, 2020a , b ). The efficiency has been reported to be 50–70% for knockin, and close to 100% for knockout (Hashimoto and Takemoto, 2015 ; Darwish et al, 2019 ; Gurumurthy et al, 2019b ).…”

Section: Rapid Generation Of Transgenic Micementioning

confidence: 99%

“…The use of frozen fertilized eggs instead of fresh ones has further simplified the whole process (Darwish et al, 2019 ; Nishizono et al, 2020a , b ). The efficiency has been reported to be 50–70% for knockin, and close to 100% for knockout (Hashimoto and Takemoto, 2015 ; Darwish et al, 2019 ; Gurumurthy et al, 2019b ). These improvements hold great promise for a rapid generation of transgenic mouse models, which will allow behavioral and functional screening at the level of F0 mice, and thus circumvent the need for long periods of cross-breeding.…”

Section: Rapid Generation Of Transgenic Micementioning

confidence: 99%

Methodologies and Challenges for CRISPR/Cas9 Mediated Genome Editing of the Mammalian Brain

2020

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Neurons and glia are highly polarized cells with extensive subcellular structures extending over large distances from their cell bodies. Previous research has revealed elaborate protein signaling complexes localized within intracellular compartments. Thus, exploring the function and the localization of endogenous proteins is vital to understanding the precise molecular mechanisms underlying the synapse, cellular, and circuit function. Recent advances in CRISPR/Cas9-based genome editing techniques have allowed researchers to rapidly develop transgenic animal models and perform single-cell level genome editing in the mammalian brain. Here, we introduce and comprehensively review the latest techniques for genome-editing in whole animals using fertilized eggs and methods for gene editing in specific neuronal populations in the adult or developing mammalian brain. Finally, we describe the advantages and disadvantages of each technique, as well as the challenges that lie ahead to advance the generation of methodologies for genome editing in the brain using the current CRISPR/Cas9 system.

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Generation of novel <i>Il2rg</i>-knockout mice with clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9

et al. 2020

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X-linked severe combined immunodeficiency (X-SCID) is an inherited genetic disorder. A majority of X-SCID subjects carries point mutations in the Interleukin-2 receptor gamma chain (IL2RG) gene. In contrast, Il2rg-knockout mice recapitulating X-SCID phenotype lack a large part of Il2rg instead of point mutations. In this study, we generated novel X-SCID mouse strains with small insertion and deletion (InDel) mutations in Il2rg by using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. To this end, we injected Streptococcus pyogenes Cas9 (SpCas9) mRNA and single guide RNA targeting the exon 2, 3 or 4 of Il2rg into mouse zygotes. In the F0 generation, we obtained 35 pups and 25 out of them were positive for Surveyor assay, and most of mutants displayed dramatic reductions of T and B lymphocytes in the peripheral blood. By amplicon sequencing, 15 out of 31 founder mice were determined as monoallelic mutants with possible minor mosaicisms while 10 mice were mosaic. Finally, we established new strains with 7nucleotide deletion and 1-nucleotide insertions in the exon 2 and the exons 3 and 4, respectively. Although no IL2RG protein was detected on T cells of exons 3 and 4 mutants, IL2RG protein was unexpectedly detected in the exon 2 mutants. These data indicated that CRISPR/Cas9 targeting Il2rg causes InDel mutations effectively and generates genetically X-SCID mice. Genetic mutations, however, did not necessarily grant phenotypical alteration, which requires an intensive analysis after establishing a strain to confirm their phenotypes.

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

Cited by 4 publications

References 28 publications

The versatile electric condition in mouse embryos for genome editing using a three-step square-wave pulse electroporator

The versatile electric condition in mouse embryos for genome editing using a three-step square-wave pulse electroporator

Methodologies and Challenges for CRISPR/Cas9 Mediated Genome Editing of the Mammalian Brain

Generation of novel <i>Il2rg</i>-knockout mice with clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9

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