Lentiviral Packaging Kit Protocol

This protocol gives a step-by-step guide on how to use the Lenti-vpack packaging kit for lentiviral particle production.

Note: Performing Lentiviral experiments may require special laboratory conditions and/or permissions (BSL2). Follow the guidelines and regulations of your institution. Perform the experiments with due caution to avoid exposure to infectious materials.

*For OriGene's Lenti shRNA application, we recommend a 6-well plate format. **The protocol below is a for a 10cm dish, see the table blow for different vessels and reagent requirements.

Vessel	Cells	Lenti Plasmids	Packaging Plasmids	Transfection Reagent	Opti-MEM	Reactionsper Kit
10-cm dish	2.5x106	5 ?g	6 ?g	33 ?L	1.5 mL	10
6-well plate	5x105	1 ?g	1.2 ?g	6.6 ?L	250 mL	50
12-well plate	2.5x105	0.5 ?g	0.6 ?g	3.3 ?L	100 mL	100

Day 1, Plate Cells: Plate 2.5 x 10⁶ HEK 293T cells on a 10cm dish in 10 mL complete growth media (antibiotic-free preferred) and incubate at 37?C overnight.

Day 2, Transfection:

1. In a labeled Eppendorf tube, dilute the following DNA in 1.5 mL Opti-MEM, and pipet gently to mix completely.
   1. 5 ?g of pLenti-shRNA construct or 5 ?g of pLenti-ORF expression construct
   2. 6 ?g of packaging plasmids
2. Add 33 ?L of TurboFectin transfection reagent to the diluted DNA (not the reversed order), pipet gently to mix completely.
3. Incubate for 15 min at room temperature.
4. Add the transfection mixture prepared above, dropwise to the cells. Gently rock the plate back-and-forth and from side-to-side to distribute the complex evenly. Incubate at 37?C.

   Note: With TurboFectin, no medium change is necessary, directly add the transfection mixture to cells in complete growth media.

Day 3, Change Medium: Change the culture medium after 12-18 hours of incubation.

Day 4, Harvest 1^st Batch: Harvest the first batch of viral supernatant from the culture and store at 4?C. Add 10 mL fresh culture media to the cell culture.

Day 5, Harvest 2^nd Batch:

1. Harvest the second batch of viral supernatant and combine with the first batch.
2. Filter through a 0.45?m PES filter to remove cellular debris. The viral titer at this step is usually 10⁶ -10⁷ TU/mL**. The viral supernatant is now ready for the majority of transduction applications. If necessary, further concentration can be applied ( lentiviral concentrator).

   Note: Large ORF inserts might decrease the viral titer