VirusTech Core Facility
Viral vectors are a key technology for researchers, since it allows the delivery in an efficient and reproducible manner of genes into cells in order to label specific cohorts, control neural activity, as well as gain/loss-of –function experiments to test the role of different genes in cellular processes. The production of viruses requires special expertise and installations, as well as work permits specifically designed for each lab and virus.
The core facility specializes in the production and concentration of Lentiviral and Adeno Associated Viruses (AAVs), but it aims to develop and optimize the production of other types of viruses such as Adenovirus (AV), retroviruses, rabies virus and Herpes simplex viruses (HSV).
The VirusTech Facility is a newly created core facility directed by Dr. Emma R. Andersson and Dr. Jens Hjerling-Leffler, and managed by Dr. Albert Blanchart, located at the Department of Cell and Molecular Biology (CMB), C5.
Its primary purpose is to produce and distribute quality viral vectors to our fellow researchers within Karolinska Institutet and investigators world-wide. The VirusTech facility is a non-profit facility hosted and sponsored by StratNeuro, that produces viral vectors on a fee-for-service basis, in accordance with KI policies for core facilities. Its primary goal is to serve the investigators at KI, but services are also available to other academic centers within Sweden and world-wide.
Type of Viruses offered by us to your research
The VirusTech Facility offers both Lentiviruses and Adeno Associated Viruses (AAV), each of which has different capabilities, weakness and strengths (see table below).
The core facility also offers consulting in order to help researchers to decide which type would fit their need for a specific project.
|Packaging capacity (kB)||7-9||4-5|
|Infects post-mitotic cells||Yes||Yes|
|Chance of oncogene activation||Yes||No|
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 envelop (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.
Lentivirus Production by co-transfection. Virions are produced by transfecting three different plasmid 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, 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.
Adeno Associated Viruses (AAVs)
Small viruses that can carry relatively small payload, but they do not exhibit pathogenicity nor cytotoxicity in the host organism. They can mediate gene transfer to both mitotic and non-mitotic cells, they lack the risk of oncogene activation, since they can exist stably in an episomal state with a low rate of chromosomal integration. AA virions are small (20-25 nm diameter) and have no enveloped, they carry ssDNA and they are defective virus dependent on the presence of a helper virus for replication.
- Require BSL2 safety level lab
- They carry a small payload up to 4.5 Kb
- No pathogenic and replicant-defective.
- AAV dsDNA in host cells is maintained in an “episomal” state (not integrated in the host genome).
- Transgene expression mediate by recombinant AAV vectors can persist for months and up to several years.
- Can be package with different serotypes that confer different infectious spectrum (tropism):
AAV viruses and production. A) AAV are non-enveloped, icosahedral and small. They package ~4.7 kB, flanked by ITR singled stranded DNA containing two Open Reading Frames encoding for Replication (rep) and capside (cap) proteins. B) We currently work with recombinant AAV, meaning that AAV ssDNA no longer contains the cap and rep genes but the transgene which will be introduced in the host cells. Therefore, production of these viruses is achieved by co-transfection of the transgene vector and an AAV helper plasmids that contains the necessary genes for the packaging and virus production within the host cells.
|AAV1||CNS, skeletal muscle, Heart|
|AAV2||CNS, photoreceptor cells||Neurons, general tropism in all brain areas|
|AAV5||CNS, photoreceptor cells, Lung||Astrocytes, hippocampal neurons, Selective retrogade Transport|
|AAV6||skeletal muscle, Lung||Retrogade transduction|
|AAV7||skeletal muscle, Liver|
|AAV8||CNS, photoreceptor cells, skeletal muscle, Heart, Liver, Pancreas||High specificity in Auditory Cortex|
|AAV9||CNS, skeletal muscle, Heart, Liver, Lung||Neurons, hippocampus, auditory cortex|
AAV pseudotyping: Define as the mix of a capsid and genome from different viral serotypes. We currently have in stock some plasmids for AAV production purchased from PasmidFactory including pDM and pDG (Wild type AAV2 serotype), and a series of pDP including rep2 (structural protein from AAV2 serotype) and different cap proteins (capsid protein) conferring different tropisms depending which cap proteins are coded.
Before Starting a Project
1. Obtain the work permits for the viruses to be produced (more information can be found in the biosafety website from Karolinska Institutet), that you will be using when the core facility delivers them to you. Be sure to be familiar with the institutional guidelines for virus use and disposal.
2. Obtain the desired expression vector to package in the type of virus you have decided to use (The core facility can help with that, and we also offer packaging plasmid systems for lenti and AA viruses).
3. Obtain the necessary Material Transfer Agreement (MTA) if needed. If the customer provides a plasmid purchased from a company or a non-profit organization (i.e. AddGene), it is the customer obligation to contact with the provider (laboratory or company which produced the plasmid), to find out if some MTA agreement must be fulfilled, in order for the VirusTech core facility to work with those plasmids.
4. Provide the core facility with the expression vector (plasmid) at the right concentration (varies depend on the system chosen), and with a A260/280 ratio no lower than 1.8.
Services and Ordering
Production of Lentivirus: 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.
Price List for Lentiviral production: Contact us for a quotation
Production of AAV: Production and packaging available from different pseudotypes. Purchased from PlasmidFactory which holds the license from the German Cancer Research Center, we provide the user with several serotypes to choose from, depending on their needs:
- AAV helper plasmid pDP1 (rep2/cap1)
- AAV helper plasmid pDG (rep2/cap2)
- AAV helper plasmid pDP5 (rep2/cap5)
- AAV helper plasmid pDP8.ape (rep2/cap8)
Price List for AAV production: Contact us for a quotation
The facility is located at the Department of Cell and Molecular Biology (CMB), von Eulers väg 1-3, SE-171 65 Solna, Sweden