Jun 07, 2024

Public workspaceProduction of lentiviruses in Lenti-X cells

DOI
dx.doi.org/10.17504/protocols.io.n92ldmdyol5b/v1
  • Cole S Sitron1,
  • F Ulrich Hartl1
  • 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Cole Sitron
Open access
DOI: dx.doi.org/10.17504/protocols.io.n92ldmdyol5b/v1
Protocol Citation: Cole S Sitron, F Ulrich Hartl 2024. Production of lentiviruses in Lenti-X cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldmdyol5b/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: January 16, 2024
Last Modified: June 07, 2024
Protocol Integer ID: 94535
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This is a protocol to produce lentivirus in a HEK cell line selected to produce high-titer virus.

Attachments
958-2489.docx
958-2489.docx
15KB
Materials

  • ReagentOpti-MEM™ Reduced Serum MediumThermo Fisher ScientificCatalog #31985062
  • ReagentX-tremeGENE™ HP DNA Transfection ReagentMerck MilliporeSigma (Sigma-Aldrich)Catalog #6366244001
  • ReagentLenti-X™ ConcentratorTakara Bio Inc.Catalog #631231
  • ReagentLenti-X™ 293T Cell LineTakara Bio Inc.Catalog #632180
Equipment
Mlllex®-GP Filter Unit (Sterile)
NAME
Filter
TYPE
Mlllex®
BRAND
SLGP033RB
SKU
https://www.merckmillipore.com/IN/en/product/Mlllex-GP-Filter-Unit-Sterile,MM_NF-SLGP033RB
LINK

  • ReagentpsPAX2addgeneCatalog #12260
  • ReagentpMD2.GaddgeneCatalog #12259
  • ReagentDMEM, high glucose, pyruvateThermo FisherCatalog #11995073 supplemented with 10% FBS
(ReagentFetal Bovine SerumGibco - Thermo FischerCatalog #10270106 ) and 1X Pen/Strep
(ReagentPenicillin-Streptomycin (10,000 U/mL)Gibco - Thermo FisherCatalog #15140122 )
  • 1X PBS ReagentPBS, pH 7.2Thermo Fisher ScientificCatalog #20012068
  • Appropriate transfer vector










Transfection
Transfection
Plate Lenti-X cells 2.5 x 10^6/10 cm Duration24:00:00 before transfection.

1d
Warm OptiMEM, X-tremeGENE HP DNA Transfection Reagent, and DNA to warm to TemperatureRoom temperature .

Add Amount1 mL OptiMEM to a tube.

Pipetting
Add plasmid DNA to the OptiMEM, gently pipetting to mix.

ABC
psPAX20.65 pmol4600 ng
pMD2.G0.36 pmol1400 ng
Transfer vector0.82 pmol

Pipetting
Mix
Add X-tremeGENE HP DNA Transfection Reagent to OptiMEM/DNA (Amount4 µL per Amount1 µg of DNA).

Pipetting
Incubate for Duration00:15:00 at TemperatureRoom temperature .

15m
Incubation
Add OptiMEM/DNA/X-tremeGENE to the plate of cells dropwise, then gently swirl the plate.
Pipetting
The day after transfection, replace the medium with
Collection
Collection
3d 1h 35m
Duration72:00:00 after transfection, collect the medium in a 15 ml conical.

3d
Centrifuge Centrifigation500 x g x Duration00:05:00 , then pass the supernatant through a Thikness.22 µm filter.

5m
Centrifigation
Add Amount3 mL of LentiX concentrator to the supernatant, invert 6 times to mix, then incubate DurationOvernight at Temperature4 °C .

45m
Incubation
Pipetting
Mix
Centrifuge at Centrifigation1500 x g for Duration00:45:00 at Temperature4 °C .

45m
Centrifigation
Remove the supernatant and resuspend the pellet in Amount120 µL ice cold PBS.

Pipetting
Make aliquots in multiples of Amount14 µL and freeze at Temperature-70 °C .

Pipetting