Reproductive technologies for the generation and maintenance of valuable animal strains

Kaneko, Takehito

doi:10.1262/jrd.2018-035

Cited by 13 publications

(7 citation statements)

References 86 publications

(114 reference statements)

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“…Since embryogenesis occurs within oviduct and uterus, there are limited options for introducing genome-editing components into murine embryos. Genome-editing technology was initially experimented on zygotes isolated from oviducts of pregnant female mice or those obtained through in vitro fertilization (IVF) via preexisting methods, e.g., microinjection or in vitro electroporation (EP), as reviewed by Lino et al [7] and Kaneko [8]. Viral vectors have also been used as alternatives to these classical methods.…”

Section: Developments In Genome-edited (Ge) Mice Production Technologymentioning

confidence: 99%

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

Takabayashi

Akasaka

Nakamura

2020

Cells

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The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) systems that occur in nature as microbial adaptive immune systems are considered an important tool in assessing the function of genes of interest in various biological systems. Thus, development of efficient and simple methods to produce genome-edited (GE) animals would accelerate research in this field. The CRISPR/Cas9 system was initially employed in early embryos, utilizing classical gene delivery methods such as microinjection or electroporation, which required ex vivo handling of zygotes before transfer to recipients. Recently, novel in vivo methods such as genome editing via oviductal nucleic acid delivery (GONAD), improved GONAD (i-GONAD), or transplacental gene delivery for acquiring genome-edited fetuses (TPGD-GEF), which facilitate easy embryo manipulation, have been established. Studies utilizing these techniques employed pregnant female mice for direct introduction of the genome-editing components into the oviduct or were dependent on delivery via tail-vein injection. In mice, embryogenesis occurs within the oviducts and the uterus, which often hampers the genetic manipulation of embryos, especially those at early postimplantation stages (days 6 to 8), owing to a thick surrounding layer of tissue called decidua. In this review, we have surveyed the recent achievements in the production of GE mice and have outlined the advantages and disadvantages of the process. We have also referred to the past achievements in gene delivery to early postimplantation stage embryos and germ cells such as primordial germ cells and spermatogonial stem cells, which will benefit relevant research.

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Section: Developments In Genome-edited (Ge) Mice Production Technologymentioning

confidence: 99%

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

Takabayashi

Akasaka

Nakamura

2020

Cells

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“…This method has been widely applied to genome editing using the ZFN, TALEN, and CRISPR-Cas systems in mice [12][13][14][15][16][17][18]. This method has also been used to produce genome-edited strains in other animals [19,20].…”

Section: Discussionmentioning

confidence: 99%

“…The TAKE method is an easy and simple method for producing genome-edited animals [ 5 , 6 , 7 ]. This method has been widely applied to genome editing using the ZFN, TALEN, and CRISPR-Cas systems in mice [ 12 , 13 , 14 , 15 , 16 , 17 , 18 ]. This method has also been used to produce genome-edited strains in other animals [ 19 , 20 ].…”

Section: Discussionmentioning

confidence: 99%

Amount of Cas9 protein introduced into mouse embryos via electroporation affects the genome-editing rate

Wake

Vakulskas

Glenn

et al. 2022

Journal of Reproduction and Development

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Genetically engineered animals can be produced quickly using genome editing technology. A new electroporation technique, technique for animal knockout system by electroporation (TAKE), aids in the production of genome-edited animals by introducing nucleases into intact embryos using electroporation instead of microinjection. It is difficult to confirm nuclease delivery into embryos after electroporation using the conventional TAKE method. We previously reported the successful visualization of fluorescently-labeled tracrRNA in embryos after electroporation Cas9 paired with the crRNA:tracrRNA-ATTO550 duplex. However, the amount of fluorescence signal from labeled tracrRNA in embryos did not correlate with the genome editing rate of the offspring.This study examined the visualization of Cas9 protein in embryos after electroporation and its correlation with the genome editing rate of the offspring using a fluorescent Cas9 fusion protein.The fluorescent Cas9 protein was observed in all embryos that survived following electroporation.We found that the efficiency of Cas9 protein delivery into embryos via electroporation depended on the pulse length. Furthermore, we demonstrated that the amount of fluorescent Cas9 protein detected in the embryos correlated with the genome editing efficiency of the embryos. These data indicate that the TAKE method using fluorescently-labeled nucleases can be used to optimize the delivery conditions and verify nuclease delivery into individual embryos prior to embryo transfer for the efficient production of genome-edited animals.

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“…We recently developed a new method, named TAKE, that could produce genome-edited animals by electroporation instead of the microinjection method [ 5 , 6 , 7 ]. Many kinds of knockout and knock-in mice and rats have already been produced by TAKE method using ZFN, TALEN, CRISPR-Cas system [ 12 , 13 , 14 , 15 , 16 , 17 ], and other nucleases [ 18 ]. Furthermore, this method has widely been applied for the production of genome-edited strains in other animals [ 19 , 20 ].…”

Section: Discussionmentioning

confidence: 99%

Production of genome-edited mice by visualization of nucleases introduced into the embryos using electroporation

Wake

Kaneko

2020

Journal of Reproduction and Development

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Genome editing technology contributes to the quick and highly efficient production of genetically engineered animals. These animals are helpful in clarifying the mechanism of human disease. Recently, a new electroporation technique (TAKE: Technique for animal knockout system by electroporation) was developed to produce genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. The aim of this study was to increase the efficiency of production of genome-edited animals using the TAKE method. In the conventional protocol, it was difficult to confirm the introduction of nucleases into embryos and energization during operation. Using only embryos that introduced nucleases for embryo transfer, it will lead to increased efficiency in the production of genome-edited animals. This study examined the visualization in the introduction of nucleases into the embryos by using nucleases fluorescent labeled with ATTO-550. The embryos were transfected with Cas9 protein and fluorescent labeled dual guide RNA (mixture with crRNA and tracrRNA with ATTO-550) targeted tyrosinase gene by the TAKE method. All embryos that survived after electroporation showed fluorescence. Of these embryos with fluorescence, 43.7% developed to morphologically normal offspring. In addition, 91.7% of offspring were edited by the tyrosinase gene. This study is the first to demonstrate that the introduction of nucleases into embryos by the TAKE method could be visualized using fluorescent-labeled nucleases. This improved TAKE method can be used to produce genome-edited animals and confirm energization during operation.

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Reproductive technologies for the generation and maintenance of valuable animal strains

Cited by 13 publications

References 86 publications

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

Recent Advances and Future Perspectives of In Vivo Targeted Delivery of Genome-Editing Reagents to Germ cells, Embryos, and Fetuses in Mice

Amount of Cas9 protein introduced into mouse embryos via electroporation affects the genome-editing rate

Production of genome-edited mice by visualization of nucleases introduced into the embryos using electroporation

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