May 31, 2024

Public workspaceGeneration of Flp-In™ T-REx™ 293 cells stably expressing VPS13C^mClover under a tetracycline inducible promoter

DOI
dx.doi.org/10.17504/protocols.io.36wgqnnnxgk5/v1
  • Xinbo Wang1,2,
  • Shujun Cai1,2,
  • Will Hancock-Cerutti1,2,3,
  • Pietro De Camilli1,2
  • 1Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
  • 3Interdisciplinary Neuroscience Program and MD-PhD Program, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Xinbo Wang
Open access
DOI: dx.doi.org/10.17504/protocols.io.36wgqnnnxgk5/v1
Protocol Citation: Xinbo Wang, Shujun Cai, Will Hancock-Cerutti, Pietro De Camilli 2024. Generation of Flp-In™ T-REx™ 293 cells stably expressing VPS13C^mClover under a tetracycline inducible promoter. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqnnnxgk5/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: May 22, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 100990
Keywords: ASAPCRN
Abstract
This method describes the generation of Flp-In™ T-REx™ 293 cells stably expressing internally mClover tagged VPS13C under a tetracycline inducible promoter.
Materials
Specialized Reagents:
ReagentFlp-In™ T-REx™ 293 Cell LineThermo Fisher ScientificCatalog #R78007
ReagentVPS13C^mClover3addgeneCatalog #118760
ReagentpcDNA™5/FRT/TO Vector KitThermo FisherCatalog #V652020
ReagentpOG44 Flp-Recombinase Expression VectorThermo FisherCatalog #V600520
ReagentBlasticidin S HCl (10 mg/mL)Thermo FisherCatalog #A1113903
ReagentZeocin™ Selection ReagentThermo FisherCatalog #R25001
Hygromycin B (ThermoFisher Catalog # J67371.8EQ)
Tetracycline (ThermoFisher Catalog # A39246)

Reagents to prepare

Culture media:

AB
Dulbecco's modified Eagle's medium (DMEM)
fetal bovine serum (FBS)10%
zeocin200 μg/ml
blasticidin S.15 μg/ml
Post transfection media:

AB
Dulbecco's modified Eagle's medium (DMEM)
fetal bovine serum (FBS)10%
blasticidin S.15 μg/ml
Selection media:

AB
Dulbecco's modified Eagle's medium (DMEM)
fetal bovine serum (FBS)10%
blasticidin S15 μg/ml
hygromycin B200 μg/ml
Expression media:

AB
Dulbecco's modified Eagle's medium (DMEM)
fetal bovine serum (FBS)10%
blasticidin S15 μg/ml
hygromycin B200 μg/ml
tetracycline0.1 μg/ml
Cloning:
  • Takara Catalog # 638947
  • Gibco manual (Catalog # R780-07)
Safety warnings
Attention
All appropriate biosafety precautions should be observed when handling Cell culture and recombinant DNA.
Cloning
Cloning
Clone gene of interest into pcDNA5/FRT/TO. For VPS13C^mClover the In-Fusion system (Takara Catalog # 638947) was used.
Sequence plasmid.
Cell culture
Cell culture
Thaw and culture Flp-In™ T-REx™ 293 cells according to Gibco manual (Catalog # R780-07) in a 10cm dish. Once cells are 90% Confluent, split.
Freeze some cells according to Gibco manual protocol for future use.
Transfection and selection of stably integrated cells
Transfection and selection of stably integrated cells
15m
Plate cells in 6 well format to ~30% confluence in media containing no antibiotics.
The next day, transfect cells using Fugene HD and a ratio of 9:1 pOG44: pcDNA5/FRT/TO-VPS13C^mClover.
Mix Amount2 µg total DNA (Amount0.2 µg pcDNA5/FRT/TO-VPS13C^mClover, Amount1.8 µg pOG44) in Amount100 µL opti-MEM.
Mix
Add Amount8 µL Fugene HD to DNA mixture. Let sit Duration00:15:00 .
15m
Add Fugene/DNA mixture to cells in a dropwise fashion.
At 24h post transfection, wash cells and add fresh post-transfection media containing Blasticidin (no Zeocin or Hygromycin).
Wash
At 48h post-transfection, split cells and plate at <25% confluence in selection medium containing Blasticidin and Amount200 μg/ml Hygromycin B (no zeocin).
Feed the cells with Hygromycin B selection medium every 3-4 days.
When foci become apparent, pool Foci and allow cells to expand.

Note
  • In our experience, at Amount200 μg/ml Hygromycin B, it may appear that no colonies remain viable, however after 1-2 weeks a small amount of colonies were observed in which the gene of interest was stably integrated.
  • If all cells are nonviable under these conditions, it may be necessary to reduce the concentration of Hygromycin B in the selection media to Amount100 μg/ml and/or optimize transfection conditions.
Per manufacturers protocol, these surviving foci should be isogenic for the inserted gene.

Pooled cells can then be split, frozen, and/or used for experiments.
Freeze cells according to Gibco manual.
Expression of VPS13C
Expression of VPS13C
Plate cells at desired confluence in format appropriate for experiment in expression media containing Amount0.1 μg/ml -Amount1 μg/ml tetracycline.

Note
In our hands, we observed no difference in expression levels of VPS13C^mClover protein between 0.1 μg/ml and 1 μg/ml and thus we used the lower concentration, however the Gibco manual suggests using 1 μg/ml.

After 24 hours, cells may be lysed for western blot or used for microscopy.