View source for Production of LentiCRISPR Viruses

This protocol is modified from the [https://www.addgene.org/protocols/lentivirus-production/ Addgene website] protocol

== Materials==
* Use the 293T/17 cells (purchased as a stock from ATCC and aliquoted in separate box in the liquid nitrogen; [https://www.atcc.org/Products/All/CRL-11268.aspx ATCC page]).
* Plasmids (lenti-vector, psPAX2, pMD2.G)
* OptiMEM
* Chloroquine stocks (25 mM stocks).  See [[Preparation of Chloroquine Stocks]].
* PEI (1 mg/mL stocks). See [[Preparation of PEI Stocks]].
* DMEM/10% FBS/PSG and DMEM/10% FBS with no PSG

==Preparation of Cells==
* Thaw and passage cells.  Use passage <20.  These amounts are for one plasmid.
* Split cells normally each time seeding 7 x 10<sup>5</sup> cells in a 10 cm dish, in DMEM/PSG/10% FBS.
* The day before the transfection, seed 3.8 x 10<sup>6</sup> cells into a fresh plate.  Seed cells into media '''without PSG'''
* Prepare DNA according to this table in a sterile 2 mL tube.:

{| class="wikitable"
! style="text-align: center; font-weight: bold;" | Reagent
! style="font-weight: bold;" | pmoles
! style="font-weight: bold;" | ug
! style="font-weight: bold;" | Volume Added
|-
| style="text-align: center;" | psPAX2
| 1.3
| 9.2
| 
|-
| style="text-align: center;" | pMD2.G
| 0.72
| 2.77
| 
|-
| style="text-align: center;" | LentiCRISPRv2-sgRNA
| 1.64
| 16.1
| 
|-
| style="text-align: center;" | OptiMEM
| -
| -
| 500 uL
|}

* For this amount of DNA (28 ug) try using 3:1 PEI:DNA, so add 84 uL PEI into 1 mL OptiMEM.
* Gently add the PEI solution dropwise into the DNA solution.
* Incubate at room temperature for 15-20min.
* Add transfection mixture slowly to the cells.
* Incubate overnight.  The next day carefully replace with media containing PSG.
* Collect media at 96h, or at 48, 72 and 96h post infection.  Combine supernatants in a 50 mL conical tube.
* Centrifuge at 500g for 5 minutes to pellet any cells.
* Filter supernatant through the 0.45 um.  Aliquot and snap freeze in liquid nitrogen in screw capped vials.