Lentiviral Vectors

Overview

These viruses belong the family retroviridae, subfamily Orthoretroviridae, genus Lentivirus, characterized for long incubation periods (lente-, Latin for “slow”). The virions (or virus particles) are enveloped, spherical and within a size range of 80-100 nm in diameter. Inside the envelope (a lipid membrane derived from portions of the host cell membranes (phospholipids and proteins), but include some viral glycoproteins) resides the nucleocapsids or core where the single strand of viral RNA is packaged. One of the most striking features of this virus, from the retroviridae family, is its ability to infect both dividing and non-dividing cells and its ability to integrate a significant amount of viral RNA into the DNA of the host cell, which qualifies them as one of the most efficient methods for gene delivery.

  • Require BL2 Containment
  • Can theoretically carry payload up to 9 Kb (10 and 11 Kb have also been reported)
  • Safety concerns regarding to insertional mutagenesis.
  • Use of vesicular stomatitis virus G glycoprotein (VSV-G) allows for ultracentrifugation concentration, higher titters, but proves to have cytotoxic effects on producing cells so virus are obtained by transient transfection.
  • Production is achieved through trans-complementation, using second and third generation vectors.
  • Lentiviruses transduce most cell types within the central nervous system (CNS) in vivo

Virions are produced by transfecting three different plasmids into the hosting cells (HEK293 Lenti-X®), this methods ensures high degree of biosafety, since the risk of replicant competent lentiviruses is significantly reduced due to the separation of cis-acting sequences (transgene plasmid in the figure), required for the transfer of the viral genome to target cells, from the trans-acting sequences encoding the viral proteins (gag, pol and env VSV-G). Both types of construct are introduced in the same cell to produce the virions. We are currently producing viruses based in the 2nd generation packaging systems (but 3rd generation packaging systems are also available upon customer's request), this means that all accessory genes have been deleted and the genes necessary for viral replication (pol), viral surface proteins (env: VSV-G) and viral core proteins (gag) are provided in separate construct. One advantage of this system is that the packaging and envelop vectors work with 2nd and 3rd generation transgene vectors, and thus a wider range of possibilities are available.

Production, services and prices

Packaging and production of Lentivirus. Packaging plasmid of 2nd generation (provided by two different plasmids, i) the Envelope Plasmid, coding for VSV-G, ii) Packaging Plasmid with the gag, pol, tat and rev genes), which are capable of package both 2nd and 3rd generation transfer plasmids. All our Lentivirus are produced using the VSV-G envelop to concentrate the viruses by ultracentrifugation and obtain high titters adequate for in vivo experiments.

Services include:

  • Transfection and supernatant collection
  • Concentration by double step centrifugation, to suitable volumes stated by the customer
  • Quality control assay include a physical titter (vg/mL) by RT-qPCR or functional titter (IFU/mL) by transduction of culture cells and analysis by FACS when reporter genes are present in the expression vector, alternatively PCR protocols to titrate lentiviral vectors based on proviral DNA copies present in genomic DNA extracted from transduced cells . The titration is requested by the customer.

Note: For a successful virus production the VirusTech core recommends to transform the expression plasmids into E. coli strains recommended for use when cloning unstable inserts such as lentiviral DNA containing direct repeats, prior the DNA prep and purification. Such strains can be obtained from different companies such One Shot™ Stbl3™ #C737303 (Invitrogen, ThermoFisher) or NEB Stable competen E. coli #C30540I (NEB).

Price List for Lentiviral production:

  1. KI users
Product Prices(1) SEK Volume Titers in IFU/mL(vg/mL) Timeline(2)
Non-concentrated supernatant 6.500 - 7.500 100 ml ~105-106 3 weeks
Concentrated small volumes 7.800 - 8.700 50-80 µl ≥109 (1011) 4 weeks
Concentrated big volumes 7.800 - 8.700 500-1000 µl ≥108 (1010) 4 weeks

(1) Prices vary depending in the quality control assay performed.
(2) From the time the plasmid is received depending on the current queue.

  1. External users:
Product Prices(1) SEK Volume Titers in IFU/mL (vg/mL) Timeline(2)
Non-concentrated supernatant 8.500 - 9.800 100 mL ~105-106 3 weeks
Concentrated small volumes 9.500 - 10.800 50-80 µl ≥109 (1011) 4 weeks
Concentrated big volumes 9.500 - 10.800 500-1000 µl ≥108 (1010) 4 weeks

(1) Prices vary depending on the quality control assay performed.
(2) From the time the plasmid is received depending on the current queue.

Ordering Lentivirus production

  1. Download the order form
  2. Fill it out an submit to: albert.blanchart-aguado@ki.se

Links

Cell and Molecular BiologyVirology