Lentiviral Vector Production Methods

Lentiviral vector construction

1. Gene of interest is cloned into a modified Lentiviral vector
2. The Lentiviral vector plasmid and packaging plasmids constructed are then purified

Production of lentiviral particles

Production of Lentiviral particles in 10mL cell culture dish by transient transfection involves the following steps:

1. Day 0: 293T cells are placed in 10mL DMEM/10% FBS at a density of 2 x 10⁶ per 100-mm tissue culture plate for incubation one day before the transfection.
2. Day 1: Culture medium is changed with 10mL fresh medium on the day of and one hour before the transfection.
3. DNAs that are used for Lentiviral particle production are mixed in a sterile 6mL polypropylene tube.

• The volume is adjusted to 437µl with TE79/10
• 63µL of 2M CaCl₂ is added and mixed well
• 500µL of 2 x HBS is added with constant agitation
• The mixture is sat at room temperature for 30min to allow the precipitation of calcium phosphate-DNA

4. The precipitate is added by drop into tissue culture plates, wherein 293T cells are at least 80% to 90% confluent. The cell density is crucial for vector production. Best results can be obtained when the plate is 90% confluence on the day of transfection. To increase titer, the ideal cell confluence can be reached with Vi-Cell (Figure 1) for accurate cell counting.

Figure 1. VI-Cell. Image credt: Beckman Coulter

5. The culture medium is replaced with 6mL fresh DMEM/10% FBS after a period of 6-8h and the incubation is continued.
6. Day 2–4: The culture supernatant is collected and replaced by 6mL fresh culture medium. The collection is filtered through a sterile 0.4µm syringe and stored at ultra-low temperature (ULT).

Purification and concentration of lentiviral particles

1. The thawed collection is mixed with 40% PEG solution until a final PEG concentration of 10% is reached. The mixture is then incubated in ice for a period of 3-6h.
2. The mixture is centrifuged at a speed of 2,000 x g for 30min.
3. The supernatant is discarded and the viral particle pellet is dispersed by gently pipetting in 1/20 of the initial harvest volume of phosphate buffered saline (PBS) or a medium of choice.
4. The tubes are placed into buckets and weighed and balanced.
5. They are then centrifuged at a speed of 100,000 x g (24,500rpm) in a SW 32 Ti rotor in a Beckman Optima X Series ultracentrifuge, at 4ºC for 90min.
6. The supernatant is removed by inverting the tubes or pipetting. The viral pellet should not be dislodged.
7. The pellet is resuspended in PBS or the medium of choice.
8. The pellet is completely dissolved by pipetting up and down or shaking for a few minutes.
9. It is then aliquoted and stored at the desired temperature. It is recommended to use an ultra-low temperature (ULT) for long-term storage.

Improved process

*TLS and MLS rotors are used with the Optima MAX-XP tabletop ultracentrifuge.

Supporting products

• Optima XPN-100 with NVT rotors (100, 90, 65 & 65.5)
• Microfuge 16, GeXP
• Optima MAX-XP with MLA rotors (150 & 130) and TLA rotors (120.1 & 120.2)
• Avan ti J-26S XP with JA rotors (10, 14) and JLA rotors (16.250, 10.500), JA rotors (17, 20 & 25.5)

References

1. Hsin-Lung Lo and Jiing-Kuan Yee; Production of Pseudotype-Retroviral Vectors—Current Protocols in Human Genetics. John Wiley & Sons, Inc. pp. 12.7.1-11: 2007.
2. Jane C. Burns, Theodore Friedmann, Wolfgang Driever, Michelle Burrascano, and Jiing-Kuan Yee; Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: Concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells—Proc. Natl. Acad. Sci. Vol. 90: pp. 8033-8037.
3. Steven R. Bartz and Marie A. Vodicka; Production of High-Titer Human Immunodeficiency Virus Type 1 Pseudotyped with Vesicular Stomatitis Virus Glycoprotein—METHODS: A Companion to Methods in Enzymology. 12: pp. 337–342.
4. Jiing-Kuan Yee, Atsushi Miyanohara, Patricia Laporte, Kathy Bouic, Jane C. Burns, and Theodore Friedmann; A general method for the generation of high-titer, pantropic retroviral vectors: Highly efficient infection of primary hepatocytes—Proc. Nati. Acad. Sci. Vol. 91: pp. 9564-9568.
5. Richard A. Klinghoffer, Brian Roberts, James Annis, Jason Frazier, Patrick Lewis, Peter S. Linsley, and Michele A. Cleary; An Optimized Lentivirus-Mediated RNAi Screen Reveals Kinase Modulators of Kinesin-5 Inhibitor Sensitivity—ASSAY and Drug Development Technologies. Volume 6: pp. 105-119.
6. Jean-Michel Garcia, Anhui Gao, Pei-Lan He, Joyce Choi, Wei Tang, Roberto Bruzzone, Olivier Schwartz, Hugo Naya, Fa-Jun Nan, Jia Li, Ralf Altmeyer, and Jian-Ping Zuo; High-throughput screening using pseudotyped lentiviral particles: A strategy for the identification of HIV-1 inhibitors in a cell-based assay—Antiviral Research. 81: pp. 239–247.
7. H-L Lo, T Chang, P Yam, PM Marcovecchio, S Li, JA Zaia, and J-K Yee; Inhibition of HIV-1 replication with designed miRNAs expressed from RNA polymerase II promoters—Gene Therapy. 14: pp. 1503–1512.

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