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In situ genome editing method suitable for routine generation of germline modified animal models

Masato Ohtsuka, Hiromi Miura, Naomi Arifin, Shingo Nakamura, Kenta Wada, Channabasavaiah B. Gurumurthy, Masahiro Sato
doi: https://doi.org/10.1101/172718
Masato Ohtsuka
1Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan
2Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Isehara, Kanagawa, Japan
3The Institute of Medical Sciences, Tokai University, Isehara, Kanagawa, Japan
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Hiromi Miura
1Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan
2Center for Matrix Biology and Medicine, Graduate School of Medicine, Tokai University, Isehara, Kanagawa, Japan
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Naomi Arifin
1Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Japan
4Department of Applied Biochemistry, School of Engineering, Tokai University, Hiratsuka, Kanagawa, Japan
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Shingo Nakamura
5Division of Biomedical Engineering, National Defense Medical College, Tokorozawa, Saitama, Japan
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Kenta Wada
6Department of Bioproduction, Tokyo University of Agriculture, Abashiri, Hokkaido, Japan
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Channabasavaiah B. Gurumurthy
7Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA
8Developmental Neuroscience, Munroe Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha, NE, USA
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Masahiro Sato
9Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Kagoshima, Japan
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Abstract

Animal genome engineering experimental procedures involve three major steps: isolation of zygotes from pregnant females; microinjection of zygotes, and; transfer of injected zygotes into recipient females, that have been practiced for over three decades. The laboratory set ups intending to performing these procedures require to have sophisticated equipment as well as highly skilled technical personnel. Because of these reasons, animal transgenesis experiments are typically performed at centralized core facilities in most research organizations. We recently showed that all three steps, of animal transgensis, can be bypassed using a method termed GONAD (Genome-editing via Oviductal Nucleic Acids Delivery), by directly electroporating genome editing components into zygotes in situ. Although our first report demonstrated the genome-editing capability, its efficiency was lower than the standard methods using microinjection. Here we investigated critical parameters of GONAD to make it suitable for creating animal models of large genomic deletions, single nucleotide corrections and long sequence insertions. The efficiency of genome editing in the improved GONAD (i-GONAD) method reached to the levels comparable to traditional microinjection methods. The streamlined parameters, and the simplified experimental steps, in the i-GONAD method makes it suitable for routine genome editing applications performed both at centralized facilities as well as at the laboratories that lack highly skilled personnel and the sophisticated equipment.

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Posted August 04, 2017.
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In situ genome editing method suitable for routine generation of germline modified animal models
Masato Ohtsuka, Hiromi Miura, Naomi Arifin, Shingo Nakamura, Kenta Wada, Channabasavaiah B. Gurumurthy, Masahiro Sato
bioRxiv 172718; doi: https://doi.org/10.1101/172718
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In situ genome editing method suitable for routine generation of germline modified animal models
Masato Ohtsuka, Hiromi Miura, Naomi Arifin, Shingo Nakamura, Kenta Wada, Channabasavaiah B. Gurumurthy, Masahiro Sato
bioRxiv 172718; doi: https://doi.org/10.1101/172718

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