Gamma-Retroviruses

Overview

Gammaretrovirus is a genus in the retroviridae family (subfamily Orthoretrovirinae; Genus: Gammaretrovirus). these viruses contain a single stranded RNA encapsulated in a nucleocapsid and enveloped by a plasma membrane derived from host infected cells during virus budding. Besides viral encoded nucleocapsid (Gag) and envelope (Env) proteins, each virion contains two identical single-stranded RNAs and enzymes including reverse transcriptase and integrase essential for virus replication. Viral cell entry is mediated through the interaction between viral Env protein and its cell surface receptor. They belong to the same family as Lentivirus, however there are few important differences between these two Geni. First, these viruses are derived from different genomes (Moloney Murine Leukemia Virus and Murine Stem Cell Virus in the case of retrovirus and the human immunodeficiency virus for Lentiviruses), second and most important difference is that in contrast to Lentiviruses Gammaretroviruses can transduced only dividing cells, since their Integrase complex is too big to be transpoted in the nuclei of the host cells. As with Lentivirus, gamma-retroviruses have some limitation and requirements when used for experimental and/or clinical purposes:

• Require BL2 Containment
• Can theoretically carry payload up to 10 Kb
• 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.
• Most commonly used for in vivo animal experiments using an alternative ecotropic envelop.
• Production is achieved through trans-complementation, using second and third generation vectors.
• Gamma-Retroviruses transduce most cell types within the central nervous system (CNS) in vivo.

The production of these viruses follows the same principles as with Lentiviruses. However the plasmids cannot be used interchangeably, but for the envelop plasmid, which ultimately defines the tropism of these viruses: Amphitropic (wide range of infectivity since the envelop proteins bind to membrane lipids) or Ecotropic (narrow host range and can infect one or a small group of species or cell culture lines).

Production, services and prices

Packaging and production of Gamma-retroviruses. The production is performed by transient transfection of HEK 293-T Lenti-X cells with either 2 or 3 plasmids: i) Expression vector (plasmid containing the desired vector flanked by LTR sequences), ii) Packaging Plasmid with the gag, pol,and env genes (this plasmids contains an ecotropic envelop that infects mammalian cells such as mouse, rat cells but not human) or alternatively the packaging plasmids contains gag and pol genes and iii) envelop plasmid encoding the VSV-G protein for production of amphitropic retroviruses. Gamma-retroviruses produced using the VSV-G envelop are very stable and resistant to concentration by ultracentrifugation to 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 gamma-retroviral 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:

ProductPrices⁽¹⁾ SEKVolumeTiters in IFU/mL (vg/mL)Timeline⁽²⁾

Non-Concentrated supernatant: 7.500 - 8.500 100 mL~10⁶ 3 weeks

Concentrated small volumes: 9.800 - 10.700 50 - 80 µL≥ 10⁹ (10¹¹) 4 weeks

Concentrated big volumes: 9.800 - 10.700 500 - 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.

2. External users:

ProductPrices⁽¹⁾ SEKVolumeTiters in IFU/mL (vg/mL)Timeline⁽²⁾

Non-concentrated supernatant: 9.500 - 10.800 100 mL~105-10⁶ 3 weeks

Concentrated small volumes: 11.000 - 13.800 50-80 µl≥10⁹ (10¹¹) 4 weeks

Concentrated big volumes: 11.000 - 13.800 500-1000 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.

Ordering Retrovirus production

1. Download the order form
2. Fill it out an submit to: hongyan.xia@ki.se