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Construction and Packaging Service of virus Vector

Construction and Packaging Services of AAV

Large scale AAV production

Recombinant LV Production Protocol
Lentiviral vectors can transduce a wide range of cell types and integrate into the host genome in both dividing and post-mitotic cells, resulting in long-term expression of the transgene both in vitro and in vivo.
Transient transfection method is now widely used for generation of recombinant lentivirus. The third-generation lentiviral vector system consists transfer vector, pMDL (Gag/Pol), pREV and pVSVG. 
The experimental process of recombinant lentivirus production is as follows.
Day 1: Cell seeding
The day previous to transfection, prepare 15-cm platess by seeding each platess with HEK-293. Be sure cells should be at 70%~80% confluence the next day.
Day 2: Transfection with plasmid mix
● Prepare the plasmid mix by aliquoting the four plasmids into a 50ml tube. For a 15 cm dish preparation, use 20 µg of transfer vector, 15 µg of pMDL (Gag/Pol), 8 µg of pVSVG (vesicular stomatitis virus glycoprotein) and 6 µg of pREV. Add 147ul PEI to the plasmid mix. Mix gently by inverting several times and incubate 15 min at room temperature.
● Add the transfection mixture (spreading in drops) to plate. Swirl the plates gently and incubate at 5% CO2, 37 °C overnight.
Day 3: Observe the cells and change the media
If a visible marker (such as GFP) is present in the lentivector plasmid, transfection efficiency may be assessed visually. Ideally, transfection efficiency should be >80%. Remove media, add 15 ml of fresh DMEM + 2% FBS to each dish and transfer to 10% CO2, 37 °C. Incubate overnight.
Day 4: Collect first harvest of supernatant 
Collect and pool supernatant and suspensions, which contains infectious lentiviral particles. Add 15ml of fresh DMEM + 2% FBS to dish. Incubate dishes at 10% CO2, 37 °C overnight.
Day 5: Collect second harvest of supernatant
Pool supernatant from first and second harvests. Repeat viral harvesting every 12-24 h, typically collect a total of 2-3 time points. Viral titer tends to decrease in later harvests. After the final harvest, discard the packaging cells.

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