LentiPAK™ lentiviral packaging DNA mix is a pre-optimized formulation of plasmid mixture for efficient packaging of a lentivector into viral particles in the packaging cells. This pre-optimized DNA mix will express all the key structural components (in trans with optimal ratios) required for lentiviral packaging, as well as a heterologous envelope protein, Vesicular Stomatitis Virus G-protein (VSV-G) for pseudo-typing.  When co-transfected with a lentivector that contains the sequence of interest (e.g., an shRNA, miRNA or protein-coding gene) and all the cis elements necessary for viral RNA genome production and packaging, this product will yield high titers of replication-incompetent and self-inactivating lentiviral particles for transducing target cells. For the packaging cells, we recommend HEK293T cells that can be readily transfect with QuickFectin™ transfection reagent to consistently produce lentivirus with high titer and purity. The pseudo-typing with VSV-G broadens the viral tropism, allowing for the delivery of the sequence of interest to a wide variety of cell types, including hard-to-transfect primary and non-dividing cells. 

LentiPAK™ lentiviral packaging DNA mix is a pre-optimized formulation of plasmid mixture for efficient packaging of a lentivector into viral particles in the packaging cells. This pre-optimized DNA mix will express all the key structural components (in trans with optimal ratios) required for lentiviral packaging, as well as a heterologous envelope protein, Vesicular Stomatitis Virus G-protein (VSV-G) for pseudo-typing.  When co-transfected with a lentivector that contains the sequence of interest (e.g., an shRNA, miRNA or protein-coding gene) and all the cis elements necessary for viral RNA genome production and packaging, this product will yield high titers of replication-incompetent and self-inactivating lentiviral particles for transducing target cells. For the packaging cells, we recommend HEK293T cells that can be readily transfect with QuickFectin™ transfection reagent to consistently produce lentivirus with high titer and purity. The pseudo-typing with VSV-G broadens the viral tropism, allowing for the delivery of the sequence of interest to a wide variety of cell types, including hard-to-transfect primary and non-dividing cells. 

 

Product Details

 

Reagents Supplied:

LentiPAK™ lentiviral packaging DNA mix (endotoxin-free DNA), 150 μL/vial, each vial sufficient for 10 co-transfection with a lentivector into a 10-cm dish of packaging HEK293T cells using the standard protocol.   

 

Quality Control Assays:

LentiPAK™ lentiviral packaging DNA mix is lot tested in HEK293T cells with a control lentivector that express GFP to assure consistently high packaging efficiency (≥10⁷ IFU/ml).

 

Product Storage:

LentiPAK™ lentiviral packaging DNA mix is shipped at 4°C.  It is recommended to store the product at –20°C or lower for long-term storage. Aliquot the product if desired. Avoid multiple freeze thaw cycles. If stored properly, the product is stable for 12 months.

 

Reagents Required, But Not Provided:

• Lentivector (endotoxin-free DNA) encoding the sequence of interest, e.g., control lentivector that express GFP (pLTC-GFP, SKU#LTP001), Luciferase (pLTC-Luc, SKU#LTP002), or LacZ (pLTC-LacZ, SKU#LTP003).
• Transfection Reagent: QuickFectin™ (SKU#QTF001) is recommended.
• Packaging HEK293T cells (ATCC#CRL-11268): Follow the distributor’s instruction for culturing and maintaining the cell line. Use the lowest passage # of cells as possible for transfection experiments.          
• Serum-free or serum-containing growth medium for HEK293T packaging cells.

 

 

 

FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMAN.

 

Important Safety Information: With the safety features in place, our lentiviral vectors and viral particles can be employed in standard Biosafety Level 2 tissue culture facilities and should be treated with the same level of caution as any other potentially infectious agent. Any investigator who purchases our lentiviral/retroviral products & services is responsible for following Biosafety Level 2 requirements on the handling of viral particles. For more information on Biosafety Level 2 agents and practices, please refer to NIH’s “Biosafety Considerations for Research with Lentiviral Vectors”.

 

Restriction: LentiPAK™ lentiviral packaging DNA mix is developed and sold exclusively for research purposes. Neither the products, nor any individual components, have been tested for use in humans or animals. This product is not transferable or re-sellable.  Customer obtains no right to transfer, assign, or sublicense its use rights, or to transfer, resell, package, or otherwise distribute the product, or to use the product for the benefit of any third party or for any commercial purpose.  Customer may only use the product in compliance with applicable local, state and federal laws, regulations and rules.  Customer may not directly or indirectly use the product or allow the transfer, transmission, export or re-export of all or any part of the product in violation of any export control law or regulation of the united states or any other relevant jurisdiction.  Your use of this product constitutes acceptance of the terms of this limited use agreement.  Please refer to our “terms & conditions” for details.  If you are not willing to accept the limitation of this agreement, G&P Biosciences will accept return of the product for a full/partial refund.

 

Technical Data

 

The data example below shows the determination of lentiviral particle titer using GFP as the marker.  Serially diluted lentiviral particles (1:30 to 1:1000) pre-packaged with LentiPAK™ DNA mix were transduced into equal amounts of target cells at the density of 0.6x10⁶/ml (1 ml each in a 12-well plate) in the presence of 8 μg/ml of polybrene. The GFP-expressing populations (% GFP+) were determined by flow cytometry and monitored by fluorescence microscope 3 days post transduction (see the images below).  The functional titers were calculated using %GFP⁺ numbers and the formula below, ranging from 1.3x10⁷ to 1.7x10⁷ IFU/ml (with an average functional titer of 1.5x10⁷ IFU/ml).

 

 

 

Standard Protocol

 

The first step in lentiviral packaging is co-transfection of LentiPAK™ packaging DNA mix with a lentivector that contains the sequence of interest into the packaging cells such as HEK293T. Packaging cells should be sub-confluence (typically 50-70%) and endotoxin-free DNA should be used to maximize the transfection efficiency. HEK293T cells are highly transfectable by calcium phosphate or many lipid-based transfection reagents.  We recommend the use of Quickfectin™ transfection reagent as it transfect 293 cells with remarkably high efficiency (>95%) using a simple procedure. It shows minimal cytotoxicity and no medium change is needed post transfection.  In addition Quickfectin™ does not interfere with downstream transduction applications. The packaging cells are allowed to produce viral particles for 48-72 hours prior to the harvest of virus-containing media for transducing target cells. Fresh media can be added back on to the packaging cells and the harvest can be repeated up to 96 hours post transfection if more virus is desired. 


 

Day 1: Cell Plating

a)     Seed the HEK293T cells in complete growth media one day prior to transfection. Cells should reach 50-70% confluency on the following day of transfection. Recommended cell seeding densities and reagent volumes for different dish/plate formats are summarized in Table 1.

b)     Incubate seeded cells at 37°C with 5% CO2 overnight

 

Day 2: Co-transfection

Perform the following reaction immediately before transfection.  Thaw the vials containing the lentivector construct DNA and LentiPAK™ packaging DNA mix at room temperature.

 

a)     Mix gently the desired amounts of the lentivector construct DNA and LentiPAK™ packaging DNA mix according to Table 1.

b)     Dilute the above DNA mixture with sterile PBS pH7.4 or growth media (with or without serum) to the final volume recommended in Table 1.

c)     Add the recommended volume of Quickfectin™  to the diluted DNA mixture above and tap gently to mix well. Incubate at room temperature for 5 minutes.

d)     Add the DNA-Quickfectin™ mixture prepared above dropwise to the cells and gently rock the dish/plate to distribute the complex evenly.

e)     Incubate cells at 37°C with 5% CO2 for 48 - 72 hours before harvesting the lentiviral particle supernatants.

Note: With Quickfectin™, no medium change is necessary after transfection. If you wish to remove the complex, remove the medium 16 - 24 hours post-transfection and replace with fresh medium.

 

 

Table 1.  Co-transfection conditions for lentiviral packaging in HEK293T cells using LentiPAK™ and Quickfectin™

 

Culture Dish/Plate	10 cm Dish	6-well plate	12-well plate	96-well plate
Surface Area (cm2)	59	9.6	3.8	0.32
Medium Volume (mL)	10	2	1.0	0.1
Number of Cells Seeded	2 - 5x106	0.4 - 1.0x106	2 - 5x105	20,000 - 50,000
Lentivector Construct DNA (μg)	5	1	0.5	0.05
LentiPAK™ DNA Mix (μL)	15	3	1.5	0.15
Diluted DNA Volume (μL)	150	30	15	1.5
Quickfectin™ (μL)	50	10	5	0.5

 

Day 4: First Viral Particle Harvest

The highest concentration of virus is typically produced between 48-72 hours. However harvest time points should be optimized depending upon cell viability, passage number, and media composition at the time of transfection. Pre-warm a sufficient amount of growth media to be used for feeding of the cells.

 

a)    48-72 hours post-transfection, collect viral particles by carefully removing the media and placing it in a collection tube. To remove cellular debris, the supernatant can be centrifuged at 1500 rpm for 5 minutes and/or filtered through a 0.45 μm filter. The viral particle supernatants can be stored at 2-8 °C for 24-48 hours. For long-term storage, aliquot and freeze at –80°C or lower.

Note: Multiple freeze thaw cycles of viral particles may reduce the infectious viral titer by 20-50% per cycle.

b)    Add an equal volume of fresh pre-warmed growth media to cells and incubate for an additional 24 hours

 

Day 5: Second Viral Particle Harvest

a)    24 hours after the 1^st collection, collect viral particles by carefully removing the media (if desired, pool with first harvest). To remove cellular debris, the supernatant can be centrifuged at 1500 rpm for 5 minutes and/or filtered through a 0.45 μm filter. The harvest of viral particles can be stored at 2-8 °C for 24-48 hours. For long-term storage, aliquot and freeze at –80 °C or lower.

Note: Multiple freeze thaw cycles of viral particles may reduce the infectious viral titer by 20-50% per cycle.

b)     If desired, the titer of the virus can be determined by various methods or proceed to the downstream transduction of target cells.  

 

 

Documentation

 

Product datasheet (pdf) can be downloaded here: LP-001-PDS.pdf

 

 

 

Additional supporting documents, including COA and MSDS are available upon request.