Apr 03, 2023
Constructs and generation of stable cell lines
- 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Protocol Citation: michela.deleidi 2023. Constructs and generation of stable cell lines. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzbr98vx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 01, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 79874
Keywords: ASAPCRN
Abstract
Protocol used to generate stable Flp-In T-REx-HEK 293 cell lines expressing WT or mutant GCase (E326K or L444P) as
a V5-FLAG-tagged protein using a tetracycline-inducible system.
Constructs were purchased from IDT (gBlocks Gene Fragments) and subcloned into pcDNA5/frt/to (Thermo Fisher Scientific, # V652020).
For the generation of V5-FLAG-GCase lines, the tag was positioned at the N-terminus, three aa after the cleavage site of the leader sequence. These three amino acids are repeated after the tag to ensure that the GBA1 sequence is intact.
To ensure proper cleavage, the V5-FLAG tag was inserted 12 bp after the cleavage site, and this 9 bp were repeated after the tag. To avoid interference with proper protein folding, we employed the short V5 sequence (IPNPLLGLD).
Site-directed mutagenesis was performed according to the manufacturer’s protocol (Agilent, QuickChange II XL), and base pair exchange was confirmed by Sanger sequencing.
Flp-In 293 T-Rex cells(Thermo Fisher Scientific, #R78007) were grown in media composed of Dulbecco’s modified Eagle’s medium (DMEM, Sigma–Aldrich), 10% fetal bovine serum
(Gibco), 1% GlutaMax (Gibco) supplemented with 100 μg/ml Zeocin
(InvivoGen) and 15μg/ml blasticidin (InvivoGen).
Inducible Flp-In T-Rex 293 cells were generated according to the manufacturer’s protocol
(Thermo Fisher Scientific).
The selection was performed with DMEM supplemented with 15μg/ml blasticidin and 100
μg/ml hygromycin B Gold (InvivoGen) 48 h after transfection and continued until the
expression of the gene of interest was induced by treating the cells with 50 ng/ml doxycycline hyclate (Sigma–Aldrich) for 48 h.