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Lentivirus packaging

JG Jakub Gemperle

Last updated date: Jan 2, 2023 Views: 599 Forks: 0

original An abbreviated version of this protocol was published in eLife
On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members
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Lentivirus Protocol 

Protocol for a T75

 

Day 0: split 293T on T75 (cells should be 70-80% transfect at day1)

 

Day 1: Transfection

               -Change medium on 293T, 5ml per T75 (no serum?)

               -DNA mix: In 250μl OptiMEM (total volume)

                               -6μg virus construct (e.g. pLenti or pCDH)

                               -4.5μg psPAX2 (Note: specific for lentiviruses vs retroviruses) 

                               -3μg pMD2G (or known as pVSVG)

               - PEI mix : dilute 100x PEI into 150mM NaCl (i.e. 1μl 100x PEI & 99μl NaCl)

                               -Add 27μl 1x PEI/NaCl into 223μl DMEM pure (no serum?) (per transfection)

                               -2min RT

               - Add PEI solution to DNA solution and pipette to mix

                               -15-20 min at RT

               - Add to cells

               - Change medium 6-8h afterwards (10ml) (normal growth media, i.e. DMEM + 10% FCS + AA + G)

 

Day 2: Sodium butyrate treatment (OPTIONAL to increase efficiency)

               -Add 100μl of sodium butyrate per 10ml (1M stock solution) (add to existing media)

               -Change the media 8h after (Nikki treats steps from this point on as virus)

 

Day 4: Virus collection 

               -collect the media of the transfected 293T cells

               -Spin 5min at 1000rpm

               -filter through 0.45 um 

               -use for infection or store at -80

 

Optional

               -Virus concentration

               -Ultracentrifugation 25 000 rpm for 2.5h

               -keep pellet in F12 overnight at 4C

               - Aliquot virus

 

PEI: 100x 100mg.ml-1 Polyethylenimine from sigma ( #408727-100ML)

Sodium Butyrate 1M stock millipore #19-137

Opti-Mem Thermofisher #31985062

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Gemperle, J(2023). Lentivirus packaging. Bio-protocol Preprint. bio-protocol.org/prep2098.
  2. On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members. eLife. DOI: 10.7554/eLife.76651

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