Jun 29, 2023

Public workspaceGeneration of ATG3 KO Hela cells stably expressing HaloTag-LC3B

DOI
dx.doi.org/10.17504/protocols.io.5qpvo3xq9v4o/v1
  • 1Laboratory of James H. Hurley, University of California, Berkeley, CA
Xuefeng Ren
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DOI: dx.doi.org/10.17504/protocols.io.5qpvo3xq9v4o/v1
Protocol Citation: Xuefeng Ren 2023. Generation of ATG3 KO Hela cells stably expressing HaloTag-LC3B. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo3xq9v4o/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: June 12, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 83216
Keywords: ATG3 KO Hela cells, HaloTag-LC3B, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000350
Abstract
This protocol details generation of ATG3 KO Hela cells stably expressing HaloTag-LC3B.
Attachments
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Materials
Buffers and reagents:

  • Growth broth: LB broth
  • Stbl3 competent E. coli (MacroLab, UC Berkeley)
  • DMEM medium with GlutaMAX containing 10% FBS and 10% Pen-Strep.

ReagentpVSV-GaddgeneCatalog ##138479

ReagentpBS-CMV-gagpoladdgeneCatalog #35614

ReagentpMRX-IP-HaloTag7-LC3addgeneCatalog #184899

ReagentQiagen Hi‐Speed MidiPrep kitQiagenCatalog #12643

ReagentDMEM, high glucose, GlutaMAX™ SupplementThermo FisherCatalog #10566016

ReagentGibco™ Fetal Bovine Serum value heat inactivated (formerly USDA-approved in North America or qualiFisher ScientificCatalog #A5256801

ReagentPenicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122

ReagentOpti-MEM™ I Reduced Serum MediumThermo FisherCatalog #31985070

ReagentTransIT®-LT1 Transfection ReagentMirus BioCatalog #MIR 2300

ReagentLenti-X™ ConcentratorTakara Bio Inc.Catalog #631231

Reagent15-Dimethyl-15-diazaundecamethylene polymethobromide PolybreneMerck MilliporeSigma (Sigma-Aldrich)Catalog #H9268

ReagentPuromycin DihydrochlorideGold BiotechnologyCatalog #P-600

Procedures
Procedures
15m
15m
Transform each plasmid into Stbl3 competent cells for the propagation. Next day, pick up one colony to inoculate DurationOvernight culture in LB medium with ampicillin.

Overnight
Midi prep the cultures to purify plasmids (Qiagen).
Plate 5 x 106 HEK 293T cells on a 10 cm plate in DMEM medium.
HEK293T transfection:
Add retroviral packaging plasmids (pVSV-G, pBS-CMV-gagpol) and pMRX-IP-HaloTag7-LC3, Amount5 µg each in Amount1.5 mL warm Opti-MEM medium.

Pipetting
Add Amount45 µL of TransIT-LT1 transfection reagent (Mirus) and swirl.

Pipetting
Incubate at TemperatureRoom temperature for Duration00:15:00 .
15m
Incubation
Add Amount1.5 mL dropwise into 10 cm HEK293T plate.

Pipetting
At 72 hours post-transfection, collect retroviral supernatant into a falcon tube.
Concentrate retroviral supernatant to Amount1 mL using Lenti-X concentrator (Takara) with the manufacturer instruction.

Retroviral transduction:
Plate 1 x 105 ATG3 KO HeLa cells into 12-well plate one day before.
Next day, titrate Amount100 µL , Amount200 µL , Amount400 µL of concentrated retroviral solution with Amount8 µg/mL Polybrene (Sigma) into target HeLa cells.

At 24 hours post-transduction, remove retroviral supernatant and replace with fresh DMEM complete medium with Amount2 µg/mL puromycin (GoldBio).

Perform Puromycin selection for two weeks. Confirm Halo-LC3B positive cells by FACS sorting.