Generation of Flp-In™ T-REx™ 293 cells stably expressing VPS13C^mClover under a tetracycline inducible promoter
Pietro De Camilli, Will Hancock-Cerutti, Shujun Cai, Xinbo Wang
Abstract
This method describes the generation of Flp-In™ T-REx™ 293 cells stably expressing internally mClover tagged VPS13C under a tetracycline inducible promoter.
Steps
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
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
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 2µg total DNA (0.2µg pcDNA5/FRT/TO-VPS13C^mClover, 1.8µg pOG44) in 100µL opti-MEM.
Add 8µL Fugene HD to DNA mixture. Let sit 0h 15m 0s.
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).
At 48h post-transfection, split cells and plate at <25% confluence in selection medium containing Blasticidin and 200μg/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.
Pooled cells can then be split, frozen, and/or used for experiments.
Freeze cells according to Gibco manual.
Expression of VPS13C
Plate cells at desired confluence in format appropriate for experiment in expression media containing 0.1μg/ml-1μg/ml tetracycline.
After 24 hours, cells may be lysed for western blot or used for microscopy.
