Gammaretroviruses
The Gammaretrovirus is a genus in the Retroviridae family (subfamily Orthoretroviridae; Genus: Gammaretrovirus). These viruses contain a single-stranded RNA encapsulated in a nucleocapsid and enveloped by a plasma membrane derived from infected host cells during virus budding.
Overview
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 lentiviruses, however there are a few important differences between these two genera. 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 transduce only dividing cells, as they lack the nuclear import mechanisms that allow entry into the nucleus of non-dividing cells.
As with lentiviruses, gammaretroviruses have some limitations and requirements when used for experimental and/or clinical purposes:
- Require BSL-2 containment
- Can theoretically carry payload up to 10 kb
- Safety concerns regarding insertional mutagenesis
- Use of vesicular stomatitis virus G glycoprotein (VSV-G) allows a broad host range, ultracentrifugation concentration, and high titers. However, it can have cytotoxic effects on producing cells, so viruses are obtained by transient transfection.
- Most used for in vivo animal experiments using an alternative ecotropic envelop.
- Production is achieved through trans-complementation, using 2nd and 3rd generation vectors
- Gammaretroviruses transduce most dividing 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, except for the envelop plasmid, which ultimately defines the tropism of these viruses: Amphotropic with a wide range of infectivity since the envelop proteins bind to membrane lipids, e.g., VSV-G envelope. Or ecotropic with a narrow host range that can infect one or a small group of species or cell culture lines, e.g., Moloney Murine Leukemia Virus envelope for mouse and rats.
Production and services
Packaging and production of Gamma-retroviruses. The plasmids of 2nd and 3rd generation (provided by two or three different plasmids, which are capable of packaging 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 titers adequate for in vivo experiments.
Packaging and production of Gamma-retroviruses. The production is performed by transient transfection of HEK293-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 gag, pol, and iii) env genes (containing an ecotropic envelop that infects mouse and rat cells but not human) or alternatively envelop plasmid encoding the VSV-G protein for production of amphitropic retroviruses (widest tropism). Gamma-retroviruses produced using the VSV-G envelop are very stable and resistant to concentration by ultracentrifugation to obtain high titers adequate for in vivo experiments.
Services include
- Transfection and supernatant collection
- Concentration by two-step centrifugation, to suitable volumes specified by the customer.
- Quality control assay includes a physical titer (vRNA copies/mL) by qRT-PCR. Functional titer (IFU/mL) by transduction of cell lines and analysis by FACS when reporter genes (e.x., GFP) are present in the expression vector. Alternatively, titration 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 transforming the expression plasmids into E. coli strains recommended for use when cloning unstable inserts such as lentiviral DNA containing repeat elements. Such strains can be obtained from different companies such One Shot™ Stbl3™ #C737303 (Thermo Fisher Scientific) or NEB Stable competent E. coli #C3040I (NEB).
Price list
Higher titer production of up to ≥ 1*1010 IFU/ml (1*1012 vg/ml) is possible, please contact us for consultation.
| Product (KI user) | Prices (1) in SEK | Volume | Titers in IFU/mL (vg/mL) | Timeline (2) |
|---|---|---|---|---|
| Non-concentrated supernatant | 4700 | 120 ml | ≥ 1*105 - 1*106 | 3 weeks |
| Concentrated | 5700 | 500 - 1000 µl | ≥ 1*108 (1*1010) | 4 weeks |
| Ultraconcentrated | 7400 | 50 - 70 µL | ≥ 1*109 (1*1011) | 4 weeks |
| Product (external user) | Prices (1) in SEK | Volume | Titers in IFU/mL (vg/mL) | Timeline (2) |
|---|---|---|---|---|
| Non-concentrated supernatant | 5500 | 120 ml | ≥ 1*105 - 1*106 | 3 weeks |
| Concentrated | 8000 | 500 - 1000 µl | ≥ 1*108 (1*1010) | 4 weeks |
| Ultraconcentrated | 8900 | 50 - 70 µL | ≥ 1*109 (1*1011) | 4 weeks |
| Titration method | Prices (internal) in SEK (1) | Prices (external) in SEK (1) |
|---|---|---|
| Transduction + FACS (functional) | 3300 | 4100 |
| Transduction + Provirus qPCR (functional) | 3900 | 4700 |
| qRT-PCR (physical) | 3100 | 3600 |
(1) Prices include INDI
(2) From the time the plasmid is received depending on the current queue.
Ordering retrovirus production
Download the Gammaretrovirus order form. Fill it out an submit to virusfacility@ki.se.