Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD

Gurumurthy, Channabasavaiah B.; Sato, Masahiro; Nakamura, Ayaka; Inui, Masafumi; Kawano, Naoki; Islam, Ashraful; Ogiwara, Sanae; Takabayashi, Shuji; Matsuyama, Makoto; Nakagawa, Shinichi; Miura, Hiromi; Ohtsuka, Masato

doi:10.1038/s41596-019-0187-x

Cited by 124 publications

(158 citation statements)

References 32 publications

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“…However, further optimization of EP conditions is needed for certain genetic strains and/or electroporators used. The EP conditions we are currently using are described in our protocol paper (Gurumurthy et al, ).…”

Section: Development Of I‐gonad and Its Applicationsmentioning

confidence: 99%

“…Because sensitivity of zygotes to electric pulses varies among mice strains, particularly in inbred strains such as C57BL/6, experimental conditions used for GONAD need to be optimized in each mouse strain. We have frequently used 150 mA of pulses for the MCH(ICR) outbred strain; however, this value is harmful for zygotes of the C57BL/6 inbred strain, which performs better with 100 mA pulses (Gurumurthy et al, ). The condition may need to be optimized in other strains such as BALB/c.…”

Section: Future Challenges In I‐gonadmentioning

confidence: 99%

“…Although performing the procedure at an earlier stage (e.g., Day 0.4) could reduce the mosaicism further, the presence of cumulus cells around the zygotes hampers the successful delivery of CRIPSR components into zygotes. Although this problem could be avoided by forced dispersion of the solution throughout ampulla by gentle squeezing of ampulla after injection, this has not yet been tested (Gurumurthy et al, ).…”

Section: Future Challenges In I‐gonadmentioning

confidence: 99%

“…For example, in our laboratory, we generally use 1 μg/μl and 50 ng/μl Cas9 protein for i ‐GONAD and microinjection, respectively. The concentration of Cas9 may be reduced to 100 ng/μl without enormous reduction of genome editing efficiency (Gurumurthy et al, ). The window of working concentrations of other components such as gRNAs needs to be determined.…”

Section: Future Challenges In I‐gonadmentioning

confidence: 99%

“…Currently, we are using C57BL/6 females for i ‐GONAD experiments without superovulation. In this case, we usually mate 10 estrous C57BL/6 females (after examination for vaginal smears) with C57BL/6 males to obtain five plugged females, of which >90% tend to retain pregnancy after the i ‐GONAD procedure (Gurumurthy et al, ).…”

Section: Future Challenges In I‐gonadmentioning

confidence: 99%

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i‐GONAD: A method for generating genome‐edited animals without ex vivo handling of embryos

Ohtsuka

2019

Dev Growth Differ

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The recent development of genome editing technologies has enabled the creation of genome‐edited animals, with alterations at the desired target locus. The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for this purpose because it is simpler and more efficient than other genome editing technologies. The conventional methods for creation of genome‐edited animals involve ex vivo handling of embryos (zygotes) for microinjection or in vitro electroporation. However, this process is laborious and time‐consuming, and relatively large numbers of animals are used. Furthermore, these methods require specialized skills for handling embryos. In 2015, we reported a novel method for the creation of genome‐edited animals without ex vivo handling of embryos. The technology known as Genome‐editing via Oviductal Nucleic Acids Delivery (GONAD) involved intraoviductal instillation of genome editing components into a pregnant female and subsequent in vivo electroporation of an entire oviduct. The genome editing components present in the oviductal lumen are transferred to preimplantation embryos in situ for introducing insertion or deletion (indel) mutations at the desired loci. This technology was further improved by optimizing several parameters to develop improved GONAD (i‐GONAD) for the efficient generation of mutant or knock‐in animals. In this review, we discuss the historical background, potential applications, advantages, and future challenges of GONAD/i‐GONAD technology.

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Section: Development Of I‐gonad and Its Applicationsmentioning

confidence: 99%

Section: Future Challenges In I‐gonadmentioning

confidence: 99%

Section: Future Challenges In I‐gonadmentioning

confidence: 99%

Section: Future Challenges In I‐gonadmentioning

confidence: 99%

Section: Future Challenges In I‐gonadmentioning

confidence: 99%

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i‐GONAD: A method for generating genome‐edited animals without ex vivo handling of embryos

Ohtsuka

2019

Dev Growth Differ

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Mice deficient in the phosphatase activity of sEH show decreased levels of the endocannabinoid 2‐AG in the olfactory bulb and depressive‐like behavior

Oguro,

Kaga,

Sato

et al. 2024

FEBS Letters

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Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has epoxide hydrolase activity and phosphatase activity. Our earlier study revealed that lysophosphatidic acids are a substrate of the phosphatase activity of sEH in vitro, but its physiological function remained unknown. Herein, we used the CRISPR/Cas9 system and i‐GONAD method to generate mice that are deficient in sEH phosphatase activity. In the mouse brain, sEH was highly expressed in the olfactory bulb. Deletion of the sEH phosphatase activity resulted in decreased levels of the endocannabinoid 2‐arachidonoyl glycerol (2‐AG), which is a dephosphorylated form of 2‐arachidonoyl‐lysophosphatidic acid in the olfactory bulb. The sEH‐deficient mice showed depressive‐like behavior. These results indicate that sEH can regulate the production of 2‐AG and brain function in vivo.

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Genome Editing with CRISPR‐Cas Systems

Lotfy

Hsu

2022

Crispr

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Creation of CRISPR-based germline-genome-engineered mice without ex vivo handling of zygotes by i-GONAD

Cited by 124 publications

References 32 publications

i‐GONAD: A method for generating genome‐edited animals without ex vivo handling of embryos

i‐GONAD: A method for generating genome‐edited animals without ex vivo handling of embryos

Mice deficient in the phosphatase activity of sEH show decreased levels of the endocannabinoid 2‐AG in the olfactory bulb and depressive‐like behavior

Genome Editing with CRISPR‐Cas Systems

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