Efficient genome editing in wild strains of mice using the i-GONAD method

Imai, Yuji; Tanave, Akira; Matsuyama, Makoto; Koide, Tsuyoshi

doi:10.1038/s41598-022-17776-x

Cited by 7 publications

(5 citation statements)

References 39 publications

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“…Since i-GONAD was first described in 2015 [6], the procedure has been adopted by several research laboratories. i-GONAD has been optimized for inbred strains such as B6 [19], and it has been used to generate GM wild mice [29]. However, we showed here that the RNP treatment or the electroporation negatively impacted embryo survival even when using the optimal constant 100 mA current for electroporation.…”

Section: Discussionmentioning

confidence: 64%

Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice

Melo‐Silva

Knudson

Tang

et al. 2023

Cells

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Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice.

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Section: Discussionmentioning

confidence: 64%

Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice

Melo‐Silva

Knudson

Tang

et al. 2023

Cells

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“…Researchers at NIG applied CRISPR-based genome editing using the i-GONAD method. Using this method, they showed that it is possible to efficiently modify genes in most wild strains (seven out of the nine strains examined) [48]. These findings suggest that i-GONAD will contribute to the development of many wild GM strains in the future, which will also be helpful for future studies.…”

Section: I-gonad-based Production Of Wild Mouse Strainsmentioning

confidence: 90%

Recent Advances in the Production of Genome-Edited Animals Using i-GONAD, a Novel in vivo Genome Editing System, and Its Possible Use for the Study of Female Reproductive Systems

Sato,

Morohoshi,

Ohtsuka

et al. 2023

OBM Genet

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Gene-engineered animals created using gene-targeting technology have long been recognized as beneficial, valid, and valuable tools for exploring the function of a gene of interest, at least in early 2013. This approach, however, suffers from laborious and time-consuming tasks, such as the production of successfully targeted embryonic stem (ES) cells, their characterization, production of chimeric blastocysts carrying these gene-modified ES cells, and transplantation of those manipulated blastocysts to the recipient (pseudopregnant) females to deliver chimeric mice. Since the appearance of genome editing technology, which is now exemplified by the CRISPR/Cas9 system, in late 2013, significant advances have been made in the generation of genome-edited animals through pronuclear microinjection (MI) of genome-editing components into fertilized eggs (zygotes) or electroporation (EP) of zygotes in the presence of these reagents. However, these procedures require the transfer of genome-edited embryos into the reproductive tracts of recipient females for further development. Genome editing via oviductal nucleic acids delivery (GONAD) and its modified version, called “improved GONAD (i-GONAD),” were developed as an alternative to the MI- or EP-based genome-edited animal production and now recognized to be very convenient and straightforward as genome editing can only be performed in vivo (within the oviductal lumen where fertilized embryos exist). This system also enables the simultaneous transfection of epithelial cells lining the oviductal lumen. In this review, we summarize the recent advances in GONAD/i-GONAD and their derivatives and discuss the potential of these technologies to study various biological systems related to female reproduction.

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“…Advancements in CRISPR/Cas9 technology have expanded the horizons of genetically modified organisms, moving beyond the confines of traditional model species to now include non‐model animals and strains (Dickinson et al., 2020; Imai et al., 2022; Mendoza & Trinh, 2018). Such breakthroughs allow researchers to scrutinize gene functions in animal species and strains that are more aligned with their research objectives.…”

Section: Discussionmentioning

confidence: 99%

Monocarboxylate transporter 4 deficiency enhances high‐intensity interval training‐induced metabolic adaptations in skeletal muscle

Tamura,

Jee,

Kouzaki

et al. 2024

The Journal of Physiology

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High‐intensity exercise stimulates glycolysis, subsequently leading to elevated lactate production within skeletal muscle. While lactate produced within the muscle is predominantly released into the circulation via the monocarboxylate transporter 4 (MCT4), recent research underscores lactate's function as an intercellular and intertissue signalling molecule. However, its specific intracellular roles within muscle cells remains less defined. In this study, our objective was to elucidate the effects of increased intramuscular lactate accumulation on skeletal muscle adaptation to training. To achieve this, we developed MCT4 knockout mice and confirmed that a lack of MCT4 indeed results in pronounced lactate accumulation in skeletal muscle during high‐intensity exercise. A key finding was the significant enhancement in endurance exercise capacity at high intensities when MCT4 deficiency was paired with high‐intensity interval training (HIIT). Furthermore, metabolic adaptations supportive of this enhanced exercise capacity were evident with the combination of MCT4 deficiency and HIIT. Specifically, we observed a substantial uptick in the activity of glycolytic enzymes, notably hexokinase, glycogen phosphorylase and pyruvate kinase. The mitochondria also exhibited heightened pyruvate oxidation capabilities, as evidenced by an increase in oxygen consumption when pyruvate served as the substrate. This mitochondrial adaptation was further substantiated by elevated pyruvate dehydrogenase activity, increased activity of isocitrate dehydrogenase – the rate‐limiting enzyme in the TCA cycle – and enhanced function of cytochrome c oxidase, pivotal to the electron transport chain. Our findings provide new insights into the physiological consequences of lactate accumulation in skeletal muscle during high‐intensity exercises, deepening our grasp of the molecular intricacies underpinning exercise adaptation. imageKey points We pioneered a unique line of monocarboxylate transporter 4 (MCT4) knockout mice specifically tailored to the ICR strain, an optimal background for high‐intensity exercise studies. A deficiency in MCT4 exacerbates the accumulation of lactate in skeletal muscle during high‐intensity exercise. Pairing MCT4 deficiency with high‐intensity interval training (HIIT) results in a synergistic boost in high‐intensity exercise capacity, observable both at the organismal level (via a treadmill running test) and at the muscle tissue level (through an ex vivo muscle contractile function test). Coordinating MCT4 deficiency with HIIT enhances both the glycolytic enzyme activities and mitochondrial capacity to oxidize pyruvate.

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Efficient genome editing in wild strains of mice using the i-GONAD method

Cited by 7 publications

References 39 publications

Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice

Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice

Recent Advances in the Production of Genome-Edited Animals Using <i>i</i>-GONAD, a Novel <i>in vivo</i> Genome Editing System, and Its Possible Use for the Study of Female Reproductive Systems

Monocarboxylate transporter 4 deficiency enhances high‐intensity interval training‐induced metabolic adaptations in skeletal muscle

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