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
  1. 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.

clip_image002_thumb[1]

Figure 1. VI-Cell

  1. The culture medium is replaced with 6mL fresh DMEM/10% FBS after a period of 6-8h and the incubation is continued.
  2. 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

Rotors

Tube

Part Number

Adapter

Process Advantages

SW 55 Ti

3.2mL g–Max, konical and BioSafety with Quick-Seal

358647

355535 and 358153

Increased concentration, biosafety, reduced sample volume

SW 32.1 Ti

4.5mL g–Max and BioSafety with Quick-Seal

356562

355579

Reduced sample volume, biosafety

8.0mL g–Max and BioSafety with Quick-Seal

344621

355579

Reduced sample volume, biosafety

SW 32 Ti

15mL g–Max and BioSafety with Quick-Seal

343664

355536

Reduced sample volume, biosafety

8.4mL g–Max, konical and BioSafety with Quick-Seal

358652

355536 and 358156

Reduced sample volume, biosafety, increased concentration

TLS-55*

2.2mL Ultra-Clear

347356

Miniaturization

MLS-50*

5.0mL Ultra-Clear

344057

Miniaturization

SW 41 Ti

13.2mL Ultra-Clear

344059

Reduced sample volume

*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.

About Beckman Coulter

Beckman Coulter develops, manufactures and markets products that simplify, automate and innovate complex biomedical tests. More than a quarter of a million Beckman Coulter instruments operate in laboratories around the world, supplying critical information for improving patient health and reducing the cost of care.


Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.

Last updated: Mar 1, 2019 at 7:47 AM

Citations

Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    Beckman Coulter Life Sciences - Particle Counting and Characterization. (2019, March 01). Lentiviral Vector Production Methods. News-Medical. Retrieved on November 20, 2019 from https://www.news-medical.net/whitepaper/20160801/Lentiviral-Vector-Production-Methods.aspx.

  • MLA

    Beckman Coulter Life Sciences - Particle Counting and Characterization. "Lentiviral Vector Production Methods". News-Medical. 20 November 2019. <https://www.news-medical.net/whitepaper/20160801/Lentiviral-Vector-Production-Methods.aspx>.

  • Chicago

    Beckman Coulter Life Sciences - Particle Counting and Characterization. "Lentiviral Vector Production Methods". News-Medical. https://www.news-medical.net/whitepaper/20160801/Lentiviral-Vector-Production-Methods.aspx. (accessed November 20, 2019).

  • Harvard

    Beckman Coulter Life Sciences - Particle Counting and Characterization. 2019. Lentiviral Vector Production Methods. News-Medical, viewed 20 November 2019, https://www.news-medical.net/whitepaper/20160801/Lentiviral-Vector-Production-Methods.aspx.

Other White Papers by this Supplier