Endogenous Tagging of Ciliary Genes in Human RPE1 Cells for Live-Cell Imaging

Stefanie Kuhns; Alice Dupont Juhl; Zeinab Anvarian; Daniel Wüstner; Lotte B. Pedersen; Jens S. Andersen

doi:10.1007/978-1-0716-3507-0_9

Endogenous Tagging of Ciliary Genes in Human RPE1 Cells for Live-Cell Imaging

Stefanie Kuhns, Alice Dupont Juhl, Zeinab Anvarian, Daniel Wüstner, Lotte B. Pedersen, Jens S. Andersen^*

^*Corresponding author for this work

Cell Biology and Physiology

Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review

Abstract

CRISPR-mediated endogenous tagging of genes provides unique possibilities to explore the function and dynamic subcellular localization of proteins in living cells. Here, we describe experimental strategies for endogenous PCR-tagging of ciliary genes in human RPE1 cells and how image acquisition and analysis of the expressed fluorescently tagged proteins can be utilized to study the dynamic ciliary processes of intraflagellar transport and vesicular trafficking.

Original language	English
Title of host publication	Methods in Molecular Biology
Number of pages	20
Publisher	Humana Press
Publication date	2024
Pages	147-166
Chapter	9
ISBN (Print)	978-1-0716-3506-3
ISBN (Electronic)	978-1-0716-3507-0
DOIs	https://doi.org/10.1007/978-1-0716-3507-0_9
Publication status	Published - 2024

Series	Methods in Molecular Biology
Volume	2725
ISSN	1064-3745

Bibliographical note

Funding Information:
We thank Joel Pomerantz, Kazuhisa Nakayama, Michael Knop, Keith Joung, and Benjamin Kleinstiver for reagents and the Danish Molecular Biomedical Imaging Center, University of Southern Denmark, for use of imaging equipment, supported by Novo Nordisk Foundation (NNF18SA0032928). This work was supported by grants from Independent Research Fund Denmark (grant 8021-00425B) to J.S.A. and Novo Nordisk Foundation (NNF18OC0053024, NNF15OC0016886 and NNF14OC0011535), The Danish Cancer Society (R146-A9590) and Hartmann Foundation (A31662) to L.B.P.

Publisher Copyright:
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024.

Keywords

Cas12a
Cas9
Cilia
CRISPR
Fluorescently tagged proteins
Image analysis
Intraflagellar transport
Live-cell imaging
Vesicle trafficking

Access to Document

10.1007/978-1-0716-3507-0_9

FulltextFinal published version, 2.92 MB

Cite this

Endogenous Tagging of Ciliary Genes in Human RPE1 Cells for Live-Cell Imaging. / Kuhns, Stefanie; Juhl, Alice Dupont; Anvarian, Zeinab; Wüstner, Daniel; Pedersen, Lotte B.; Andersen, Jens S.

Methods in Molecular Biology. Humana Press, 2024. p. 147-166 (Methods in Molecular Biology, Vol. 2725).

Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review

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