Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation

Mariko Kinoshita-Kawada; Hiroshi Hasegawa; Tsunaki Hongu; Shigeru Yanagi; Yasunori Kanaho; Ichiro Masai; Takayasu Mishima; Xiaoping Chen; Yoshio Tsuboi; Yi Rao; Junichi Yuasa-Kawada; Jane Y. Wu

doi:10.21769/BioProtoc.3373

Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation

Mariko Kinoshita-Kawada, Hiroshi Hasegawa, Tsunaki Hongu, Shigeru Yanagi, Yasunori Kanaho, Ichiro Masai, Takayasu Mishima, Xiaoping Chen, Yoshio Tsuboi, Yi Rao, Junichi Yuasa-Kawada^*, Jane Y. Wu^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

Developing axons change responsiveness to guidance cues during the journey to synapse with target cells. Axon crossing at the ventral midline serves as a model for studying how axons accomplish such a switch in their response. Although primary neuron culture has been a versatile technique for elucidating various developmental mechanisms, many in vivo characteristics of neurons, such as long axon-extending abilities and axonal compartments, are not thoroughly preserved. In explant cultures, such properties of differentiated neurons and tissue architecture are maintained. To examine how the midline repellent Slit regulated the distribution of the Robo receptor in spinal cord commissural axons upon midline crossing and whether Robo trafficking machinery was a determinant of midline crossing, novel explant culture systems were developed. We have combined an "open-book" spinal cord explant method with that devised for flat-mount retinae. Here we present our protocol for explant culture of embryonic mouse spinal cords, which allows flexible manipulation of experimental conditions, immunostaining of extending axons and quantitative analysis of individual axons. In addition, we present a modified method that combines ex vivo electroporation and "closed-book" spinal cord explant culture. These culture systems provide new platforms for detailed analysis of axon guidance, by adapting gene knockdown, knockout and genome editing.

Original language	English (US)
Article number	e3373
Journal	Bio-protocol
Volume	9
Issue number	18
DOIs	https://doi.org/10.21769/BioProtoc.3373
State	Published - Sep 20 2019

Funding

This work was supported by grants from the National Institutes of Health and the Japan Society for the Promotion of Science. We thank all of present and previous members of the Wu and Rao labs for advice, Drs. Fujio Murakami and Atsushi Tamada for generously providing anti-Robo antibodies, Dr. Chen-bing Guan for generously providing his Slit2 purification protocol, Animal/Biology Resources Sections at OIST, Yasutomo Kubota (Molecular Devices) and Nepa Gene for their valuable support. We are grateful to Dr. Hiroyuki Ichijo for introducing us retinal explant culture techniques that were originally developed by Dr. Friedrich Bonhoeffer and colleagues. Our dSC (-FP)/SC (+FP) explant culture protocols were adapted from the previously published protocols (Bonhoeffer and Huf, 1982; Halfter et al., 1981 and 1983; Ichijo and Bonhoeffer, 1998; Walter et al., 1987a and 1987b; Zou et al., 2000). Our ex vivo RNAi assay system was adapted based on the previously published protocols (Parra and Zou, 2010; Wolf et al., 2008). Finally, we would like to thank Dr. Zhao Chen for her kind invitation to publish our protocols here.

Keywords

Axon guidance
Commissural axons
Explant culture
Floor plate
Gene knockdown
Midline
Mouse embryos
Spinal cord

ASJC Scopus subject areas

General Neuroscience
General Biochemistry, Genetics and Molecular Biology
General Immunology and Microbiology
Plant Science

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title = "Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation",

abstract = "Developing axons change responsiveness to guidance cues during the journey to synapse with target cells. Axon crossing at the ventral midline serves as a model for studying how axons accomplish such a switch in their response. Although primary neuron culture has been a versatile technique for elucidating various developmental mechanisms, many in vivo characteristics of neurons, such as long axon-extending abilities and axonal compartments, are not thoroughly preserved. In explant cultures, such properties of differentiated neurons and tissue architecture are maintained. To examine how the midline repellent Slit regulated the distribution of the Robo receptor in spinal cord commissural axons upon midline crossing and whether Robo trafficking machinery was a determinant of midline crossing, novel explant culture systems were developed. We have combined an {"}open-book{"} spinal cord explant method with that devised for flat-mount retinae. Here we present our protocol for explant culture of embryonic mouse spinal cords, which allows flexible manipulation of experimental conditions, immunostaining of extending axons and quantitative analysis of individual axons. In addition, we present a modified method that combines ex vivo electroporation and {"}closed-book{"} spinal cord explant culture. These culture systems provide new platforms for detailed analysis of axon guidance, by adapting gene knockdown, knockout and genome editing.",

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AU - Kinoshita-Kawada, Mariko

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AU - Yanagi, Shigeru

AU - Kanaho, Yasunori

AU - Masai, Ichiro

AU - Mishima, Takayasu

AU - Chen, Xiaoping

AU - Tsuboi, Yoshio

AU - Rao, Yi

AU - Yuasa-Kawada, Junichi

AU - Wu, Jane Y.

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KW - Gene knockdown

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Explant Culture of the Embryonic Mouse Spinal Cord and Gene Transfer by ex vivo Electroporation

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