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

Methods Mol Biol. 2024:2725:147-166. doi: 10.1007/978-1-0716-3507-0_9.

Authors

Stefanie Kuhns¹, Alice Dupont Juhl¹, Zeinab Anvarian², Daniel Wüstner¹, Lotte B Pedersen², Jens S Andersen³

Affiliations

¹ Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
² Department of Biology, University of Copenhagen, Copenhagen Ø, Denmark.
³ Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark. [email protected].

PMID: 37856023
DOI: 10.1007/978-1-0716-3507-0_9

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.

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

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biological Transport
Cilia* / metabolism
Humans
Protein Transport / genetics
Proteins* / metabolism

Substances

Proteins