Isolation and analysis of a genome-edited single-hepatocyte from a Cas9 transgenic mouse line

Takayuki Sakurai, Akiko Kamiyoshi, Masato Ohtsuka, Channabasavaiah B Gurumurthy, Masahiro Sato, Takayuki Shindo

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

The primary cells isolated from the freshly dissected organ are thought to be different from those cultured for a long time in vitro. For instance, hepatocytes isolated in situ from the liver, display the ability to produce albumin, cultured for about a week often tend to cease production of albumin, including loss of proliferation capability. Thus, it is difficult to perform genome editing (i.e., production of genome-edited hepatocytes by in vitro gene delivery) in such cultured cells. Furthermore, hepatic cell lines available so far do not produce albumin and they would also have lost several characteristics of native liver cells. This poses a serious disadvantage when researchers want to study gene expression profiles under specific experimental settings, for example before and after genome editing. However, this demerit can be overcome if genome-editing is performed in situ in liver and single hepatocytes (both genome-edited and wild-type) can be isolated for analysis immediately following transient gene editing. Previously, we demonstrated successful isolation of genome-edited single hepatocytes, using mice expressing systemic Cas9 transgene (called “sCAT” mouse) and by tail-vein-mediated hydrodynamics-based gene delivery of gRNA targeted to Albumin gene (Sakurai et al., Sci Rep 6:20011, 2016). Here, we describe the detailed protocols for collection and analysis of single genome-edited hepatocytes, which will be useful for many types of hepatocyte functional studies.

LanguageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages257-271
Number of pages15
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1874
ISSN (Print)1064-3745

Fingerprint

Transgenic Mice
Hepatocytes
Genome
Albumins
Liver
Guide RNA
Genes
Hydrodynamics
Transgenes
Transcriptome
Tail
Veins
Cultured Cells
Research Personnel
Cell Line
Gene Editing

Keywords

  • CRISPR/Cas9
  • gRNA
  • Hepatocyte
  • Hydrodynamics-mediated gene delivery
  • In vivo genome editing
  • Single-cell isolation
  • Surveyor assay
  • T7 endonuclease I assay
  • Transgenic mouse
  • Whole genome amplification

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Sakurai, T., Kamiyoshi, A., Ohtsuka, M., Gurumurthy, C. B., Sato, M., & Shindo, T. (2019). Isolation and analysis of a genome-edited single-hepatocyte from a Cas9 transgenic mouse line. In Methods in Molecular Biology (pp. 257-271). (Methods in Molecular Biology; Vol. 1874). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8831-0_15

Isolation and analysis of a genome-edited single-hepatocyte from a Cas9 transgenic mouse line. / Sakurai, Takayuki; Kamiyoshi, Akiko; Ohtsuka, Masato; Gurumurthy, Channabasavaiah B; Sato, Masahiro; Shindo, Takayuki.

Methods in Molecular Biology. Humana Press Inc., 2019. p. 257-271 (Methods in Molecular Biology; Vol. 1874).

Research output: Chapter in Book/Report/Conference proceedingChapter

Sakurai, T, Kamiyoshi, A, Ohtsuka, M, Gurumurthy, CB, Sato, M & Shindo, T 2019, Isolation and analysis of a genome-edited single-hepatocyte from a Cas9 transgenic mouse line. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1874, Humana Press Inc., pp. 257-271. https://doi.org/10.1007/978-1-4939-8831-0_15
Sakurai T, Kamiyoshi A, Ohtsuka M, Gurumurthy CB, Sato M, Shindo T. Isolation and analysis of a genome-edited single-hepatocyte from a Cas9 transgenic mouse line. In Methods in Molecular Biology. Humana Press Inc. 2019. p. 257-271. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-8831-0_15
Sakurai, Takayuki ; Kamiyoshi, Akiko ; Ohtsuka, Masato ; Gurumurthy, Channabasavaiah B ; Sato, Masahiro ; Shindo, Takayuki. / Isolation and analysis of a genome-edited single-hepatocyte from a Cas9 transgenic mouse line. Methods in Molecular Biology. Humana Press Inc., 2019. pp. 257-271 (Methods in Molecular Biology).
@inbook{bd84ce30a64d4722bf4651f60c975589,
title = "Isolation and analysis of a genome-edited single-hepatocyte from a Cas9 transgenic mouse line",
abstract = "The primary cells isolated from the freshly dissected organ are thought to be different from those cultured for a long time in vitro. For instance, hepatocytes isolated in situ from the liver, display the ability to produce albumin, cultured for about a week often tend to cease production of albumin, including loss of proliferation capability. Thus, it is difficult to perform genome editing (i.e., production of genome-edited hepatocytes by in vitro gene delivery) in such cultured cells. Furthermore, hepatic cell lines available so far do not produce albumin and they would also have lost several characteristics of native liver cells. This poses a serious disadvantage when researchers want to study gene expression profiles under specific experimental settings, for example before and after genome editing. However, this demerit can be overcome if genome-editing is performed in situ in liver and single hepatocytes (both genome-edited and wild-type) can be isolated for analysis immediately following transient gene editing. Previously, we demonstrated successful isolation of genome-edited single hepatocytes, using mice expressing systemic Cas9 transgene (called “sCAT” mouse) and by tail-vein-mediated hydrodynamics-based gene delivery of gRNA targeted to Albumin gene (Sakurai et al., Sci Rep 6:20011, 2016). Here, we describe the detailed protocols for collection and analysis of single genome-edited hepatocytes, which will be useful for many types of hepatocyte functional studies.",
keywords = "CRISPR/Cas9, gRNA, Hepatocyte, Hydrodynamics-mediated gene delivery, In vivo genome editing, Single-cell isolation, Surveyor assay, T7 endonuclease I assay, Transgenic mouse, Whole genome amplification",
author = "Takayuki Sakurai and Akiko Kamiyoshi and Masato Ohtsuka and Gurumurthy, {Channabasavaiah B} and Masahiro Sato and Takayuki Shindo",
year = "2019",
month = "1",
day = "1",
doi = "10.1007/978-1-4939-8831-0_15",
language = "English (US)",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "257--271",
booktitle = "Methods in Molecular Biology",

}

TY - CHAP

T1 - Isolation and analysis of a genome-edited single-hepatocyte from a Cas9 transgenic mouse line

AU - Sakurai, Takayuki

AU - Kamiyoshi, Akiko

AU - Ohtsuka, Masato

AU - Gurumurthy, Channabasavaiah B

AU - Sato, Masahiro

AU - Shindo, Takayuki

PY - 2019/1/1

Y1 - 2019/1/1

N2 - The primary cells isolated from the freshly dissected organ are thought to be different from those cultured for a long time in vitro. For instance, hepatocytes isolated in situ from the liver, display the ability to produce albumin, cultured for about a week often tend to cease production of albumin, including loss of proliferation capability. Thus, it is difficult to perform genome editing (i.e., production of genome-edited hepatocytes by in vitro gene delivery) in such cultured cells. Furthermore, hepatic cell lines available so far do not produce albumin and they would also have lost several characteristics of native liver cells. This poses a serious disadvantage when researchers want to study gene expression profiles under specific experimental settings, for example before and after genome editing. However, this demerit can be overcome if genome-editing is performed in situ in liver and single hepatocytes (both genome-edited and wild-type) can be isolated for analysis immediately following transient gene editing. Previously, we demonstrated successful isolation of genome-edited single hepatocytes, using mice expressing systemic Cas9 transgene (called “sCAT” mouse) and by tail-vein-mediated hydrodynamics-based gene delivery of gRNA targeted to Albumin gene (Sakurai et al., Sci Rep 6:20011, 2016). Here, we describe the detailed protocols for collection and analysis of single genome-edited hepatocytes, which will be useful for many types of hepatocyte functional studies.

AB - The primary cells isolated from the freshly dissected organ are thought to be different from those cultured for a long time in vitro. For instance, hepatocytes isolated in situ from the liver, display the ability to produce albumin, cultured for about a week often tend to cease production of albumin, including loss of proliferation capability. Thus, it is difficult to perform genome editing (i.e., production of genome-edited hepatocytes by in vitro gene delivery) in such cultured cells. Furthermore, hepatic cell lines available so far do not produce albumin and they would also have lost several characteristics of native liver cells. This poses a serious disadvantage when researchers want to study gene expression profiles under specific experimental settings, for example before and after genome editing. However, this demerit can be overcome if genome-editing is performed in situ in liver and single hepatocytes (both genome-edited and wild-type) can be isolated for analysis immediately following transient gene editing. Previously, we demonstrated successful isolation of genome-edited single hepatocytes, using mice expressing systemic Cas9 transgene (called “sCAT” mouse) and by tail-vein-mediated hydrodynamics-based gene delivery of gRNA targeted to Albumin gene (Sakurai et al., Sci Rep 6:20011, 2016). Here, we describe the detailed protocols for collection and analysis of single genome-edited hepatocytes, which will be useful for many types of hepatocyte functional studies.

KW - CRISPR/Cas9

KW - gRNA

KW - Hepatocyte

KW - Hydrodynamics-mediated gene delivery

KW - In vivo genome editing

KW - Single-cell isolation

KW - Surveyor assay

KW - T7 endonuclease I assay

KW - Transgenic mouse

KW - Whole genome amplification

UR - http://www.scopus.com/inward/record.url?scp=85055603973&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85055603973&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-8831-0_15

DO - 10.1007/978-1-4939-8831-0_15

M3 - Chapter

T3 - Methods in Molecular Biology

SP - 257

EP - 271

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -