Jun 27, 2023
Generation of stable ATG3-mCherry wild-type or mutans HeLa cells with HaloTag-LC3B using lentivirus
- 1Laboratory of James H. Hurley, University of California, Berkeley, CA
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, mutant hela cells with halotag, hela cells with halotag, using lentivirus, mutant hela cell, generation of stable atg3, lentivirus this protocol detail, hela cell, stable atg3, lc3b, cell
Funders Acknowledgements:
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.
Attachments
737-1853.pdf
117KB
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.
pVSV-GaddgeneCatalog ##138479
pCMVR8.74addgeneCatalog #22036
pCDH1-CMV-HT-LC3-SV40-HygroaddgeneCatalog #182045
Q5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
NheI-HF - 5,000 unitsNew England BiolabsCatalog #R3131L
BamHI-HF - 50,000 unitsNew England BiolabsCatalog #R3136L
EZ-10 Spin Column DNA Gel Extraction KitBio Basic Inc.Catalog #BS654.SIZE.250preps
EZ-10 Spin Column Plasmid DNA Min-preps KitBio Basic Inc.Catalog #BS614.SIZE.250preps
Qiagen Hi‐Speed MidiPrep kitQiagenCatalog #12643
DMEM, high glucose, GlutaMAX™ SupplementThermo FisherCatalog #10566016
Gibco™ Fetal Bovine Serum value heat inactivated (formerly USDA-approved in North America or qualiFisher ScientificCatalog #A5256801
Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122
Opti-MEM™ I Reduced Serum MediumThermo FisherCatalog #31985070
TransIT®-LT1 Transfection ReagentMirus BioCatalog #MIR 2300
Lenti-X™ ConcentratorTakara Bio Inc.Catalog #631231
15-Dimethyl-15-diazaundecamethylene polymethobromide PolybreneMerck MilliporeSigma (Sigma-Aldrich)Catalog #H9268
Hygromycin B (50 mg/mL)Thermo FisherCatalog #10687010
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 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).
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 37 °C for 16:00:00 .
16h
Inoculate one single colony in 5 mL LB medium with ampicillin at 37 °C Overnight .
16h
Miniprep plasmids (Bio Basic) and send for sequencing.
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), 5 µg each in 1.5 mL warm Opti-MEM medium.
Add 45 µL of TransIT-LT1 transfection reagent (Mirus) and swirl.
Incubate at Room temperature for 00:15:00 .
15m
Add 1.5 mL dropwise into 10 cm HEK293T plate.
At 72 hours post-transfection, collect retroviral supernatant into a falcon tube.
Concentrate retroviral supernatant to 1 mL using Lenti-X concentrator (Takara) with the manufacturer instruction.
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 100 µL , 200 µL , 400 µL of concentrated retroviral solution with 8 µg/mL Polybrene (Sigma) into target HeLa cells.
At 24 hours post-transduction, remove retroviral supernatant and replace with fresh DMEM complete medium with 0.5 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).