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.
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Acknowledgements
This work was supported by the National Institutes of Health (R01GM083144) and UAMS Easy Win Early Victory grant program.
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Khakurel, A., Kudlyk, T., Lupashin, V.V. (2023). Generation and Analysis of hTERT-RPE1 VPS54 Knock-Out and Rescued Cell Lines. In: Wang, Y., Lupashin, V.V., Graham, T.R. (eds) Golgi. Methods in Molecular Biology, vol 2557. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2639-9_22
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DOI: https://doi.org/10.1007/978-1-0716-2639-9_22
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