Jun 27, 2023

Public workspaceGeneration of stable ATG3-mCherry wild-type or mutans HeLa cells with HaloTag-LC3B using lentivirus

DOI
dx.doi.org/10.17504/protocols.io.dm6gp3exjvzp/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.dm6gp3exjvzp/v1
Protocol Citation: Xuefeng Ren 2023. Generation of stable ATG3-mCherry wild-type or mutans HeLa cells with HaloTag-LC3B using lentivirus. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3exjvzp/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: 83237
Keywords: generation of stable ATG3-mCherry wild-type, HaloTag-LC3B, lentivirus, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000350
Abstract
This protocol details the generation of stable ATG3-mCherry wild-type or mutant HeLa cells with HaloTag-LC3B using lentivirus.
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Materials
Buffers and reagents:


  • XL10-Gold competent E. coli (home made)
  • Stbl3 competent E. coli (MacroLab, UC Berkeley)
  • Growth broth: LB broth
  • DMEM medium with GlutaMAX containing 10% FBS and 10% Pen-Strep.

ReagentpVSV-GaddgeneCatalog ##138479

ReagentpCMVR8.74addgeneCatalog #22036

ReagentpCDH1-CMV-HT-LC3-SV40-HygroaddgeneCatalog #182045

ReagentQ5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L

ReagentNheI-HF - 5,000 unitsNew England BiolabsCatalog #R3131L

ReagentBamHI-HF - 50,000 unitsNew England BiolabsCatalog #R3136L

ReagentEZ-10 Spin Column DNA Gel Extraction KitBio Basic Inc.Catalog #BS654.SIZE.250preps

ReagentEZ-10 Spin Column Plasmid DNA Min-preps KitBio Basic Inc.Catalog #BS614.SIZE.250preps

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

ReagentHygromycin B (50 mg/mL)Thermo FisherCatalog #10687010

Cloning pCDH1-CMV-ATG3-mCherry-Hygro lentiviral vector (ATG3 wild-type or mutants)
Cloning pCDH1-CMV-ATG3-mCherry-Hygro lentiviral vector (ATG3 wild-type or mutants)
Amplify the coding sequence for human ATG3 (1-125), mCherry or ATG3 (126-314) fragment using Q5 High Fidelity DNA Polymerase and purify PCR products using Gel extraction kit (Bio Basic).
PCR
PCR products in step 1 are used as templates to run the second step PCR to amplify ATG3 (1- 125)-mCherry-ATG3(126-314) and gel extracted. Primers include a NheI restriction site at the 5’ end and a BamHI site at the 3’ end.
Digest the ATG3-mCherry PCR product and pCDH1-CMV-HT-LC3-SV40-Hygro plasmid using NheI and BamHI restriction enzymes (New England BioLabs).
Digestion
Purify the digested DNAs using Gel extraction kit (Bio Basic).
Ligate the digested ATG3-mCherry and linearized pCDH1-CMV-hygro backbone using T4 DNA ligase (New England BioLabs) according to manufacturer instructions.
Transform ligation product into XL10-Gold competent E. coli and plate on Ampicillin resistant plates. Incubate the plate at Temperature37 °C for Duration16:00:00 .

16h
Inoculate one single colony in Amount5 mL LB medium with ampicillin at Temperature37 °C DurationOvernight .

16h
Overnight
Miniprep plasmids (Bio Basic) and send for sequencing.
HEK293T lentiviral transfection
HEK293T lentiviral transfection
Each plasmid for lentiviral transfection is transformed into Stbl3 competent cells for the propagation. Next day, pick up one colony to inoculate overnight culture in LB medium with ampicillin.
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, pCMV R8.74) and pCDH1-CMV-ATG3-mCherryHygro (ATG3 wild-type or mutants), 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.

Transduction of HeLa cells with pCDH-CMV-ATG3-mCherry lentivirus
Transduction of HeLa cells with pCDH-CMV-ATG3-mCherry lentivirus
Plate 1 x 105 ATG3 KO/HaloTag-LC3B 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 Amount0.5 mg/mL hygromycin B (Gibco).

After one week of hygromycin B selection, sort HeLa cells by FACS to enrich for mCherry positive cells using Wolf G2 cell sorter (Nanocellect).