Generation of endogenous BMP transcriptional reporter cells through CRISPR/Cas9 genome editing

Luke D. Hutchinson; Polyxeni Bozatzi; Thomas Macartney; Gopal P. Sapkota

doi:10.1007/978-1-4939-8904-1_4

Generation of endogenous BMP transcriptional reporter cells through CRISPR/Cas9 genome editing

Luke D. Hutchinson, Polyxeni Bozatzi, Thomas Macartney, Gopal P. Sapkota (Lead / Corresponding author)

MRC PPU

Research output: Chapter in Book/Report/Conference proceeding › Chapter (peer-reviewed) › peer-review

1 Citation (Scopus)

238 Downloads (Pure)

Abstract

Transcriptional reporter systems allow researchers to investigate the function and regulation of transcription factors. Conventional systems employ artificial cDNA overexpression vectors containing either a promoter fragment or specific nucleotide sequence repeats upstream of firefly luciferase or fluorescent reporters, such as green fluorescence protein (GFP) cDNA. These systems suffer mainly from the lack of chromatin context. Here, we describe the rapid generation of endogenous transcriptional reporter cells for the bone morphogenetic protein (BMP) pathway using CRISPR/Cas9 genome editing. In principle, our methodology can be applied to any cell line. The endogenous reporters will provide a robust system for the investigation of BMP transcriptional activity in the context of native chromatin landscape and facilitate chemical and genetic screens.

Original language	English
Title of host publication	Bone Morphogenetic Proteins
Subtitle of host publication	Methods and Protocols
Editors	Melissa B. Rogers
Place of Publication	New York
Publisher	Humana Press
Pages	29-35
Number of pages	7
Volume	1891
ISBN (Electronic)	9781493989041
ISBN (Print)	9781493989034
DOIs	https://doi.org/10.1007/978-1-4939-8904-1_4
Publication status	Published - 2018

Publication series

Name	Methods in Molecular Biology
Publisher	Springer
Volume	1891
ISSN (Print)	1064-3745

Keywords

Transcription
Reporter vectors
CRISPR/Cas9
Genome editing

Access to Document

10.1007/978-1-4939-8904-1_4

Author Accepted Manuscript
This is a post-peer-review, pre-copyedit version of an article published in Bone Morphogenetic Proteins. The final authenticated version is available online at: https://doi.org/10.1007/978-1-4939-8904-1_4.
Accepted author manuscript, 376 KB

Cite this

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abstract = "Transcriptional reporter systems allow researchers to investigate the function and regulation of transcription factors. Conventional systems employ artificial cDNA overexpression vectors containing either a promoter fragment or specific nucleotide sequence repeats upstream of firefly luciferase or fluorescent reporters, such as green fluorescence protein (GFP) cDNA. These systems suffer mainly from the lack of chromatin context. Here, we describe the rapid generation of endogenous transcriptional reporter cells for the bone morphogenetic protein (BMP) pathway using CRISPR/Cas9 genome editing. In principle, our methodology can be applied to any cell line. The endogenous reporters will provide a robust system for the investigation of BMP transcriptional activity in the context of native chromatin landscape and facilitate chemical and genetic screens.",

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author = "Hutchinson, {Luke D.} and Polyxeni Bozatzi and Thomas Macartney and Sapkota, {Gopal P.}",

note = "G.P.S. is supported by the UK MRC (MC_UU_12016/3) and the DSTT companies (Boehringer Ingelheim, GlaxoSmithKline, and Merck Serono); L.D.H. and P.B. are funded by the MRC Ph.D. studentships.",

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Generation of endogenous BMP transcriptional reporter cells through CRISPR/Cas9 genome editing. / Hutchinson, Luke D.; Bozatzi, Polyxeni; Macartney, Thomas et al.
Bone Morphogenetic Proteins: Methods and Protocols. ed. / Melissa B. Rogers. Vol. 1891 New York: Humana Press, 2018. p. 29-35 (Methods in Molecular Biology; Vol. 1891).

Research output: Chapter in Book/Report/Conference proceeding › Chapter (peer-reviewed) › peer-review

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AU - Hutchinson, Luke D.

AU - Bozatzi, Polyxeni

AU - Macartney, Thomas

AU - Sapkota, Gopal P.

N1 - G.P.S. is supported by the UK MRC (MC_UU_12016/3) and the DSTT companies (Boehringer Ingelheim, GlaxoSmithKline, and Merck Serono); L.D.H. and P.B. are funded by the MRC Ph.D. studentships.

PY - 2018

Y1 - 2018

N2 - Transcriptional reporter systems allow researchers to investigate the function and regulation of transcription factors. Conventional systems employ artificial cDNA overexpression vectors containing either a promoter fragment or specific nucleotide sequence repeats upstream of firefly luciferase or fluorescent reporters, such as green fluorescence protein (GFP) cDNA. These systems suffer mainly from the lack of chromatin context. Here, we describe the rapid generation of endogenous transcriptional reporter cells for the bone morphogenetic protein (BMP) pathway using CRISPR/Cas9 genome editing. In principle, our methodology can be applied to any cell line. The endogenous reporters will provide a robust system for the investigation of BMP transcriptional activity in the context of native chromatin landscape and facilitate chemical and genetic screens.

AB - Transcriptional reporter systems allow researchers to investigate the function and regulation of transcription factors. Conventional systems employ artificial cDNA overexpression vectors containing either a promoter fragment or specific nucleotide sequence repeats upstream of firefly luciferase or fluorescent reporters, such as green fluorescence protein (GFP) cDNA. These systems suffer mainly from the lack of chromatin context. Here, we describe the rapid generation of endogenous transcriptional reporter cells for the bone morphogenetic protein (BMP) pathway using CRISPR/Cas9 genome editing. In principle, our methodology can be applied to any cell line. The endogenous reporters will provide a robust system for the investigation of BMP transcriptional activity in the context of native chromatin landscape and facilitate chemical and genetic screens.

KW - Transcription

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M3 - Chapter (peer-reviewed)

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VL - 1891

T3 - Methods in Molecular Biology

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BT - Bone Morphogenetic Proteins

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Generation of endogenous BMP transcriptional reporter cells through CRISPR/Cas9 genome editing

Abstract

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Elucidating the biological function of the uncharacterised protein FAM83G/PAWS1: Role in Wnt signalling through its interaction with Caseine Kinase 1α

Identification of novel protein kinase regulators of TGFβ signalling

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Generation of endogenous BMP transcriptional reporter cells through CRISPR/Cas9 genome editing

Abstract

Publication series

Keywords

Access to Document

Other files and links

Fingerprint

Student theses

Elucidating the biological function of the uncharacterised protein FAM83G/PAWS1: Role in Wnt signalling through its interaction with Caseine Kinase 1α

Identification of novel protein kinase regulators of TGFβ signalling

Cite this