Generation and Analysis of hTERT-RPE1 VPS54 Knock-Out and Rescued Cell Lines.
Methods Mol Biol
; 2557: 349-364, 2023.
Article
en En
| MEDLINE
| ID: mdl-36512226
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas de Transporte Vesicular
/
Aparato de Golgi
Límite:
Humans
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2023
Tipo del documento:
Article
País de afiliación:
Estados Unidos