Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization

Honda, Arata; Tachibana, Ryoma; Hamada, Kazuya; Morita, Kohtaro; Mizuno, Naoto; Morita, Kento; Asano, Masahide

doi:10.1038/s41598-019-47964-1

Cited by 30 publications

(31 citation statements)

References 46 publications

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“…Over the past ten years, many transgenic and targeted mutant rats have been produced worldwide 1 . Moreover, even more genetically modified rats will be produced using genome-editing techniques in the near future 18 . However, rats are approximately ten times larger than mice, and the number of rats that can be kept in an animal room is limited.…”

Section: Discussionmentioning

confidence: 99%

Establishment of sperm cryopreservation and in vitro fertilisation protocols for rats

Nakagata

Mikoda

Nakao

et al. 2020

Sci Rep

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Recently, genome-editing tools have come into common use in the field of rat research, and consequently, many genetically modified rat strains have been preserved and archived as frozen embryos. Although there have been many reports published on the topic of rat sperm cryopreservation, no report has yet provided satisfactory and acceptable protocols for the cryopreservation of rat sperm. in this study, we developed methods for both the cryopreservation of transgenic rat sperm and in vitro fertilisation using frozen sperm, which yielded high fertilisation rates.

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Section: Discussionmentioning

confidence: 99%

Establishment of sperm cryopreservation and in vitro fertilisation protocols for rats

Nakagata

Mikoda

Nakao

et al. 2020

Sci Rep

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“…Gene editing of zygotes/embryos using the CRISPR/Cas9 system poses a risk of mosaicism [ 17 , 31 ]. In one study using rats, the CRISPR/Cas9 system introduced into in vitro-fertilized embryos using electroporation disrupted genes with 100% efficiency [ 32 ]. In the case of marmoset embryos manipulated by the cytoplasmic microinjection of gene editors, optimized conditions using transcription activator-like effector nucleases (TALENs) achieved highly efficient gene disruption with low or no mosaicism [ 33 ], whereas higher mosaicism was observed when the CRISPR/Cas9 system was used [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

“…A high electroporation voltage to introduce large molecules has harmful effects on embryonic viability [ 41 ]. To deliver the knock-in donor DNA into zygotes, an adeno-associated viral (AAV) vector has been applied in mice [ 42 ] and rats [ 32 ] without removing the zona pellucida, requiring no sophisticated techniques. In pigs, the development of new, efficient delivery techniques of large molecules for zygotes and embryos is crucial.…”

Section: Discussionmentioning

confidence: 99%

One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

Tanihara

Hirata

Nguyen

et al. 2021

IJMS

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Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

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“…Owing to the greater sensitivity of in vitro- fertilized porcine zygotes to electricity compared with that of in vivo -derived mouse embryos [ 52 , 56 ], the size of molecules that can be introduced into zygotes/embryos is limited. To efficiently deliver knock-in donor DNA into zygotes without mechanical injury, an adeno-associated viral (AAV) vector has been applied in mice [ 111 ] and rats [ 112 ] without removing the zona pellucida. Although AAV vector-mediated gene modification in porcine cells has been adapted to generate mutant pigs by combining it with SCNT techniques [ 113 ], the investigation of gene modification during embryogenesis via an AAV vector is insufficient.…”

Section: Current Status and Future Prospects Of Gene-edited Pigsmentioning

confidence: 99%

Current status of the application of gene editing in pigs

2021

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Genetically modified animals, especially rodents, are widely used in biomedical research. However, non-rodent models are required for efficient translational medicine and preclinical studies. Owing to the similarity in the physiological traits of pigs and humans, genetically modified pigs may be a valuable resource for biomedical research. Somatic cell nuclear transfer (SCNT) using genetically modified somatic cells has been the primary method for the generation of genetically modified pigs. However, site-specific gene modification in porcine cells is inefficient and requires laborious and time-consuming processes. Recent improvements in gene-editing systems, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) system, represent major advances. The efficient introduction of site-specific modifications into cells via gene editors dramatically reduces the effort and time required to generate genetically modified pigs. Furthermore, gene editors enable direct gene modification during embryogenesis, bypassing the SCNT procedure. The application of gene editors has progressively expanded, and a range of strategies is now available for porcine gene engineering. This review provides an overview of approaches for the generation of genetically modified pigs using gene editors, and highlights the current trends, as well as the limitations, of gene editing in pigs.

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Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization

Cited by 30 publications

References 46 publications

Establishment of sperm cryopreservation and in vitro fertilisation protocols for rats

Establishment of sperm cryopreservation and in vitro fertilisation protocols for rats

One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

Current status of the application of gene editing in pigs

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