Generation and Analysis of hTERT-RPE1 VPS54 Knock-Out and Rescued Cell Lines

Amrita Khakurel; Tetyana Kudlyk; Vladimir V Lupashin

doi:10.1007/978-1-0716-2639-9_22

Generation and Analysis of hTERT-RPE1 VPS54 Knock-Out and Rescued Cell Lines

Methods Mol Biol. 2023:2557:349-364. doi: 10.1007/978-1-0716-2639-9_22.

Authors

Amrita Khakurel¹, Tetyana Kudlyk¹, Vladimir V Lupashin²

Affiliations

¹ Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
² Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA. vvlupashin@uams.edu.

Abstract

The Golgi-associated retrograde protein (GARP) complex is proposed to tether endosome-derived transport vesicles, but the exact function and mechanism of GARP action are not completely understood. To uncover the GARP function in human cells, we employ CRISPR/Cas9 strategy and knock out (KO) the unique VPS54 subunit of the GARP complex. In this chapter, we describe the detailed method of generating CRISPR/Cas9-mediated VPS54-KO in hTERT-RPE1 cells, rescue of resulting KO cells with stable near-endogenous expression of myc-tagged VPS54, and validation of KO and rescued (KO-R) cells using Western blot and immunofluorescence approaches. This approach is helpful in uncovering new functions of the GARP and other vesicle tethering complexes.

Keywords: CRISPR/Cas9; Cathepsin D; GARP; Golgi complex; Knockout; Knockout rescue; VPS54; Vesicle tethering.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Cell Line
Endosomes / metabolism
Golgi Apparatus* / metabolism
Humans
Transport Vesicles / metabolism
Vesicular Transport Proteins* / genetics
Vesicular Transport Proteins* / metabolism

Substances

Vesicular Transport Proteins

Grants and funding

R01 GM083144/GM/NIGMS NIH HHS/United States