4.1. General Lentiviral Packaging Protocol

The following protocol describes the general procedure for generation of pseudoviral packaged lentiviral constructs using ThermoFisher’s Invitrogen Lipofectamine™ and PLUS Reagent (see Additional Materials for Production of Lentivirus). Other transfection reagents may be used, but the protocol should be adjusted to fit the manufacturer’s protocol. This protocol can be used to package individual lentiviral plasmid constructs expressing shRNA, sgRNA, Cas9/dCas9, barcodes, cDNA, promoter reporters, and sgRNA, shRNA, or barcode libraries in 3rd generation lentiviral vectors.

• The yield of recombinant lentiviral particles produced under these optimized conditions is typically between 1 × 10⁶ TU/ml and 5 × 10⁶ TU/ml for individual lentiviral constructs with a viral transcript less than 7kb (i.e. the region from the beginning of 5’LTR to the end of 3’LTR).
  

  Plate Size	Surface Area	Volume of Media	Approximate Virus Yield *
  10 cm	55 cm2	10 ml	2 × 107 TU
  15 cm	150 cm2	30 ml	6 × 107 TU

  * NOTE: The yield of lentiviral particles will be significantly lower for lentiviral constructs and libraries with viral transcripts larger than 7kb (e.g., approximately 1 × 10⁵ TU/ml for Cas9 constructs with viral transcripts around 8kb).

• If using tissue culture plates or flasks of other sizes, please scale amounts in the protocol based on surface area.

 

*Cellecta offers Lentiviral packaging services. Please contact us at sales@cellecta.com or visit Cellecta’s Lentiviral Packaging Services web page.

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2-3 Days Prior to Packaging

1. Start growing 293T cells in D-MEM medium plus glutamine* supplemented with 10% FBS without antibiotics, and expand until you have sufficient cells to package at the scale desired.

* NOTE: ADD FRESH GLUTAMINE (1X) to Dulbecco’s Modified Eagle Medium (D-MEM) at the time a sealed bottle of D-MEM is opened, even if the label indicates glutamine has already been added. Glutamine in solution at 4°C has a half-life of 1-2 months, so glutamine(+) D-MEM purchased “off-the-shelf” from a supplier is to be regarded as glutamine(-). If D-MEM comes supplemented with stable L-Alanyl-L-Glutamine dipeptide, addition of fresh glutamine is not necessary. In our experience, the addition of glutamine increases titer approximately 2-fold.

Day 0 – Plate Cells

1. Twenty four (24) hours prior to transfection, plate 12.5 × 10⁶ 293T cells per 15-cm plate (or 150 cm² flask) and use 30 ml of media per plate. If you are using 10-cm plates, plate 4 × 10⁶ cells and use 10 ml of media per plate. Disperse the cells and ensure even distribution.

2. Incubate at 37°C in a CO₂ incubator for 24 hours.

NOTE: The goal is to have the 293T cells reach ~80% confluency by Day 1. You may want to calculate the number of cells seeded empirically since cell counts can vary.

Day 1 – Transfection into 293T Cells

1. Using the volumes in the table below, mix Ready-to-use Lentiviral Packaging Plasmid Mix and your Plasmid Lentiviral construct in a sterile, appropriately-sized polypropylene tube. Add the D-MEM medium without serum or antibiotics to the plasmid mixture, then mix. Add the PLUS Reagent, mix, and incubate at room temperature for 15 minutes.
   

   1 × 10-cm plate	1 × 15-cm plate	4 × 15-cm plates	8 × 15-cm plates	16 × 15-cm plates	24 × 15-cm plates	Component
   20 μl	60 μl	240 µl	480 μl	960 µl	1440 µl	Packaging Plasmid Mix (0.5 μg/μl)
   2 μl	6 μl	24 µl	48 μl	96 µl	144 µl	Plasmid / Library (1 μg/μl)* see NOTE
   1,000 μl	1,200 μl	4,800 µl	9,600 μl	19,200 µl	28,800 µl	D-MEM, no FBS, no antibiotics
   20 μl	60 μl	240 µl	480 μl	960 µl	1,440 µl	PLUS Reagent
   1,042 μl	1,326 μl	5,304 µl	10,608 μl	21,216 µl	31,824 µl	Total volume

NOTE: The volume of plasmid DNA assumes the DNA is suspended at a 1 μg/μl concentration. For plasmid DNA at other concentrations, adjust the volume accordingly (e.g., for 0.5 μg/μl, use twice the indicated μl).

2. Add Lipofectamine Reagent to the D-MEM medium without serum or antibiotics in order to make a master mix according to the table below. Mix gently.
   

   1 × 10-cm plate	1 × 15-cm plate	4 × 15-cm plates	8 × 15-cm plates	16 × 15-cm plates	24 × 15-cm plates	Component
   1,000 μl	1,200 μl	4,800 µl	9,600 μl	19,200 µl	28,800 µl	D-MEM, no FBS, no antibiotics
   30 μl	90 μl	360 µl	720 μl	1,440 µl	2,160 µl	Lipofectamine Reagent
   1,030 μl	1,290 μl	5,160 µl	10,320 μl	20,640 µl	30,960 µl	Total volume

3. Add the diluted Lipofectamine (from Step 2) to the DNA / PLUS Reagent complex (from Step 1 above), mix gently by flicking the tube or vortexing and incubate at room temperature for 15 minutes.

4. Add 2.5 ml of the resulting DNA / PLUS Reagent / Lipofectamine complex to each plate (from the previous step Day 0 – Plate Cells), and mix complexes into the medium with gentle rotation. Take care not to dislodge cells from the plate.

5. Incubate at 37°C in the CO₂ incubator for 24 hours.

Day 2 – DNase I Treatment

1. At 24 hours post-transfection, replace the medium containing complexes with 30 ml (for 15-cm plates) or 10 ml (for 10-cm plates) of fresh D-MEM medium supplemented with 10% FBS, DNase I (1 U/ml), MgCl₂ (5 mM), and 20mM HEPES, pH 7.4.

2. Continue incubation in the CO₂ incubator at 37°C overnight.

NOTE: Overnight DNase I treatment before harvesting virus does not negatively affect lentiviral titer or infectivity and helps prevent undesirable carryover of plasmid DNA into the virus prep.

IMPORTANT: Failure to change the media the day after transfection results in large carryover of plasmid (free and/or Lipofectamine-bound) into your lentiviral prep. This may cause problems with most downstream molecular biology applications, especially whenever there is a PCR step involved such as during NGS sample preparation after a pooled library screen.

Day 3 – Collect Lentiviral Supernatant

1. At 48 hours post-transfection, collect the entire virus-containing medium from each plate and filter the supernatant (~30 ml per 15-cm plate or ~10 ml per 10-cm plate) through a Nalgene 0.2 μm PES filter (a low protein-binding filter) to remove debris and floating packaging cells. Failure to filter supernatant could result in carry-over of cells into your lentiviral prep.

NOTE: Usually, the peak of virus production is achieved at 48 hours post-transfection. Supernatant can also be collected again at 72 hours post-transfection—replace the collected 48-hour supernatant with 30 ml (for 15-cm plates) or 10 ml (for 10-cm plates) of fresh D-MEM medium supplemented with 10% FBS and 20mM HEPES pH 7.4, and continue incubation in the CO₂ incubator at 37°C for 24 hours. Then, repeat Step 1 above for 72 hours post-transfection.

*NOTE: Freezing and thawing lentivirus results in some loss of titer with each cycle.

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Concentration of Lentivirus (Optional)

The following procedure was optimized to concentrate virus harvested at 48 hours with high recovery. Although concentrating virus is optional, it is recommended if any of the following conditions applies:

• Very high titer virus stock is needed to achieve desired MOI in hard-to-transduce target cells.

• The virus needs to be suspended in a different media (besides D-MEM/10%FBS) that is optimal for sensitive target cells.

• If packaging an shRNA, sgRNA, or barcode library for screening, because the concentration minimizes problems that might arise from genomic DNA carryover.

Virus Concentration Protocol

1. Aliquot lentiviral supernatant in clear, sterile centrifuge tubes.

2. (Recommended) Add Cellecta’s LentiFuge™ Viral Concentration Reagent (see Additional Materials for Production of Lentivirus) according to the protocol described in the LentiFuge User Manual.

3. Centrifuge at 15,000 × g for at least 1 hour at 4°C. Mark the tubes to identify the location where the pellet will be. At the end of centrifugation, you may or may not be able to see a pellet—assume it is at the location of the mark.

4. Immediately discard the supernatant by aspirating.

5. Place the tubes on ice, resuspend the pellet (which may not be visible) in PBS, PBS/10%FBS, or PBS/1%BSA, make aliquots, and freeze at -80°C. 100-fold concentration is recommended (e.g., resuspend in 1 ml PBS if starting from 100 ml supernatant).

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