Methodology and theoretical basis of forward genetic screening for sleep/wakefulness in mice

Chika Miyoshi, Staci J. Kim, Takahiro Ezaki, Aya Ikkyu, Noriko Hotta-Hirashima, Satomi Kanno, Miyo Kakizaki, Mana Yamada, Shigeharu Wakana, Masashi Yanagisawa, Hiromasa Funato

Research output: Contribution to journalArticle

Abstract

The regulatory network of genes and molecules in sleep/wakefulness remains to be elucidated. Here we describe the methodology and workflow of the dominant screening of randomly mutagenized mice and discuss theoretical basis of forward genetics research for sleep in mice. Our high-throughput screening employs electroencephalogram (EEG) and electromyogram (EMG) to stage vigilance states into a wake, rapid eye movement sleep (REMS) and non-REM sleep (NREMS). Based on their near-identical sleep/wake behavior, C57BL/ 6J (B6J) and C57BL/6N (B6N) are chosen as mutagenized and counter strains, respectively. The total time spent in the wake and NREMS, as well as the REMS episode duration, shows sufficient reproducibility with small coefficients of variance, indicating that these parameters are most suitable for quantitative phenotype-driven screening. Coarse linkage analysis of the quantitative trait, combined with whole-exome sequencing, can identify the gene mutation associated with sleep abnormality. Our simulations calculate the achievable LOD score as a function of the phenotype strength and the numbers of mice examined. A pedigree showing a mild decrease in total wake time resulting from a heterozygous point mutation in the Cacna1a gene is described as an example.

Original languageEnglish (US)
Pages (from-to)16062-16067
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume116
Issue number32
DOIs
StatePublished - Aug 6 2019

Fingerprint

Wakefulness
Genetic Testing
Sleep
REM Sleep
Exome
Phenotype
Genetic Research
Workflow
Gene Regulatory Networks
Electromyography
Pedigree
Point Mutation
Genes
Electroencephalography
Mutation

Keywords

  • C57BL/6 substrains
  • Cacna1a
  • Dominant screening
  • ENU mutagenesis
  • Linkage analysis

ASJC Scopus subject areas

  • General

Cite this

Methodology and theoretical basis of forward genetic screening for sleep/wakefulness in mice. / Miyoshi, Chika; Kim, Staci J.; Ezaki, Takahiro; Ikkyu, Aya; Hotta-Hirashima, Noriko; Kanno, Satomi; Kakizaki, Miyo; Yamada, Mana; Wakana, Shigeharu; Yanagisawa, Masashi; Funato, Hiromasa.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 116, No. 32, 06.08.2019, p. 16062-16067.

Research output: Contribution to journalArticle

Miyoshi, C, Kim, SJ, Ezaki, T, Ikkyu, A, Hotta-Hirashima, N, Kanno, S, Kakizaki, M, Yamada, M, Wakana, S, Yanagisawa, M & Funato, H 2019, 'Methodology and theoretical basis of forward genetic screening for sleep/wakefulness in mice', Proceedings of the National Academy of Sciences of the United States of America, vol. 116, no. 32, pp. 16062-16067. https://doi.org/10.1073/pnas.1906774116
Miyoshi, Chika ; Kim, Staci J. ; Ezaki, Takahiro ; Ikkyu, Aya ; Hotta-Hirashima, Noriko ; Kanno, Satomi ; Kakizaki, Miyo ; Yamada, Mana ; Wakana, Shigeharu ; Yanagisawa, Masashi ; Funato, Hiromasa. / Methodology and theoretical basis of forward genetic screening for sleep/wakefulness in mice. In: Proceedings of the National Academy of Sciences of the United States of America. 2019 ; Vol. 116, No. 32. pp. 16062-16067.
@article{c0b8ec62ef0446b0bbf430ee644f2e9f,
title = "Methodology and theoretical basis of forward genetic screening for sleep/wakefulness in mice",
abstract = "The regulatory network of genes and molecules in sleep/wakefulness remains to be elucidated. Here we describe the methodology and workflow of the dominant screening of randomly mutagenized mice and discuss theoretical basis of forward genetics research for sleep in mice. Our high-throughput screening employs electroencephalogram (EEG) and electromyogram (EMG) to stage vigilance states into a wake, rapid eye movement sleep (REMS) and non-REM sleep (NREMS). Based on their near-identical sleep/wake behavior, C57BL/ 6J (B6J) and C57BL/6N (B6N) are chosen as mutagenized and counter strains, respectively. The total time spent in the wake and NREMS, as well as the REMS episode duration, shows sufficient reproducibility with small coefficients of variance, indicating that these parameters are most suitable for quantitative phenotype-driven screening. Coarse linkage analysis of the quantitative trait, combined with whole-exome sequencing, can identify the gene mutation associated with sleep abnormality. Our simulations calculate the achievable LOD score as a function of the phenotype strength and the numbers of mice examined. A pedigree showing a mild decrease in total wake time resulting from a heterozygous point mutation in the Cacna1a gene is described as an example.",
keywords = "C57BL/6 substrains, Cacna1a, Dominant screening, ENU mutagenesis, Linkage analysis",
author = "Chika Miyoshi and Kim, {Staci J.} and Takahiro Ezaki and Aya Ikkyu and Noriko Hotta-Hirashima and Satomi Kanno and Miyo Kakizaki and Mana Yamada and Shigeharu Wakana and Masashi Yanagisawa and Hiromasa Funato",
year = "2019",
month = "8",
day = "6",
doi = "10.1073/pnas.1906774116",
language = "English (US)",
volume = "116",
pages = "16062--16067",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "32",

}

TY - JOUR

T1 - Methodology and theoretical basis of forward genetic screening for sleep/wakefulness in mice

AU - Miyoshi, Chika

AU - Kim, Staci J.

AU - Ezaki, Takahiro

AU - Ikkyu, Aya

AU - Hotta-Hirashima, Noriko

AU - Kanno, Satomi

AU - Kakizaki, Miyo

AU - Yamada, Mana

AU - Wakana, Shigeharu

AU - Yanagisawa, Masashi

AU - Funato, Hiromasa

PY - 2019/8/6

Y1 - 2019/8/6

N2 - The regulatory network of genes and molecules in sleep/wakefulness remains to be elucidated. Here we describe the methodology and workflow of the dominant screening of randomly mutagenized mice and discuss theoretical basis of forward genetics research for sleep in mice. Our high-throughput screening employs electroencephalogram (EEG) and electromyogram (EMG) to stage vigilance states into a wake, rapid eye movement sleep (REMS) and non-REM sleep (NREMS). Based on their near-identical sleep/wake behavior, C57BL/ 6J (B6J) and C57BL/6N (B6N) are chosen as mutagenized and counter strains, respectively. The total time spent in the wake and NREMS, as well as the REMS episode duration, shows sufficient reproducibility with small coefficients of variance, indicating that these parameters are most suitable for quantitative phenotype-driven screening. Coarse linkage analysis of the quantitative trait, combined with whole-exome sequencing, can identify the gene mutation associated with sleep abnormality. Our simulations calculate the achievable LOD score as a function of the phenotype strength and the numbers of mice examined. A pedigree showing a mild decrease in total wake time resulting from a heterozygous point mutation in the Cacna1a gene is described as an example.

AB - The regulatory network of genes and molecules in sleep/wakefulness remains to be elucidated. Here we describe the methodology and workflow of the dominant screening of randomly mutagenized mice and discuss theoretical basis of forward genetics research for sleep in mice. Our high-throughput screening employs electroencephalogram (EEG) and electromyogram (EMG) to stage vigilance states into a wake, rapid eye movement sleep (REMS) and non-REM sleep (NREMS). Based on their near-identical sleep/wake behavior, C57BL/ 6J (B6J) and C57BL/6N (B6N) are chosen as mutagenized and counter strains, respectively. The total time spent in the wake and NREMS, as well as the REMS episode duration, shows sufficient reproducibility with small coefficients of variance, indicating that these parameters are most suitable for quantitative phenotype-driven screening. Coarse linkage analysis of the quantitative trait, combined with whole-exome sequencing, can identify the gene mutation associated with sleep abnormality. Our simulations calculate the achievable LOD score as a function of the phenotype strength and the numbers of mice examined. A pedigree showing a mild decrease in total wake time resulting from a heterozygous point mutation in the Cacna1a gene is described as an example.

KW - C57BL/6 substrains

KW - Cacna1a

KW - Dominant screening

KW - ENU mutagenesis

KW - Linkage analysis

UR - http://www.scopus.com/inward/record.url?scp=85070219214&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85070219214&partnerID=8YFLogxK

U2 - 10.1073/pnas.1906774116

DO - 10.1073/pnas.1906774116

M3 - Article

VL - 116

SP - 16062

EP - 16067

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 32

ER -