Nov 07, 2024

Public workspaceStable cell line generation via retrovirus

 Forked from Stable cell line generation via retrovirus
DOI
dx.doi.org/10.17504/protocols.io.3byl4w2o8vo5/v1
  • 1University of California, Berkeley;
  • 2Imperical College London
Victor Velecela
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DOI: dx.doi.org/10.17504/protocols.io.3byl4w2o8vo5/v1
Protocol Citation: Dan Tudorica, Victor Velecela, Victor Velecela 2024. Stable cell line generation via retrovirus. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4w2o8vo5/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: November 06, 2024
Last Modified: November 07, 2024
Protocol Integer ID: 111689
Abstract
Production of pseudotyped virus an subsequent transduction.
Day 0: Thaw fresh cryovial of cells to be transduced
Thaw ~ 1 week prior to planned transduction day, use low passage number if possible. Aim for cells to be 80% confluent on day of transduction. Recommend seeding four wells of a 12-well plate (100,000 cells/well) the day before transduction (after collecting virus).
Day 1: HEK293T Transfection
  1. Prepare 2 80% confluent 10 cm plates of HEK293T for transfection
  1. Prepare 3 mL of warm Opti-mem solution. Add 10 µg of retro/lentiviral transfection plasmid, 10 µg VSV-G plasmid, and 10 µg pCMV-MLV (if retroviral)[DT1] [DT2] or 10 µg pCMV R8.74 (if lentiviral)
  1. Add 90 µL of LT-1 reagent and swirl.
  1. Incubate at room temperature for 15 minutes.
  1. Add 1.5 mL dropwise to each 10 cm HEK293T plate.
  1. Incubate for 3 days in incubator before harvesting viral particles. If a fluorescent marker was included, it should begin to be visible on days 1,2 post transfection.
SAFETY: After this point all media and plasticware is to be treated as potentially containing viral particles. Decontaminate using 10% bleach for 10 minutes before discarding as red bag waste. Rinse pipettes with 10% bleach before discarding as biohazard waste.
Day 4: Harvest viral particles
  1. Collect media from both HEK293T plates, combine into one 20 mL portion in a 50 mL falcon tube. Bleach and discard used HEK293T plate.
  1. Spin 50 mL tube containing harvested viral particles in Sorvall for 2 min at 2,000 rpm[DT3] [DT4] to clarify media and pellet stray cells
  1. Aspirate off media without disturbing pellet.
  1. Add 6 mL Lenti-X concentrator solution to 18 mL of recovered media (concentrator is 4x). Invert to mix.
  1. Incubate in refrigerator 1 H – O/N[DT5]
  1. Spin 50 mL conical containing viral particles in centrifuge at 1,500 rcf for 45 min
  1. Remove 2 mL of supernatant from top of pelleted solution and save.
  1. Aspirate off remaining media to recover viral pellet. Bleach excess media.
  1. Resuspend retro/lenti pellet in 2 mL of saved medium.
  1. Titrate concentrated retro/lenti solution into prepared 12-well containing target cells. Recommend 100-200-400-800 µL titration for 4 total attempts at transfection. Typically 400 or 800 uL will yield ~100% transfection efficiency
  1. Return 12-well plate containing transduced cells to incubator.
Day 5: Check on transduction
  1. May be able to see fluorescence today.
  1. Swap media to fresh.
Day 6-7: Check on transduction
Day 8-:
  1. Allow cells to grow until almost confluent on 12-well plate. Trypsinize and replate in a 6-well, allow to grow until confluent, then replate in a 10 cm plate.
  1. Split into two 10 cm plates, Freeze one plate down into 4 cryovials the next day to save progress. Label with estimated transduction efficiency if able to measure.
  1. Take other plate and select with antibiotics if able to/necessary, or clonally isolate populations if able to/necessary.