Before Starting a Project
It is always important to plan the production of viral particles ahead and therefore some documentations will be needed prior to start the production of the viral particles. These documents are designed to stablish the proper work rutines, as well as stablish a safe work enviroment and inform the relevant and appropriate authorities to stablish work and enviromental responsabilities.
- 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.
- 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).
- The core offers DNA prep and cloning services to ease the workload of the investigator.
What to provide to the core:
- 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's obligation to contact the depository entity (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.
- Download, fill and submit the order form.
- Download the L-anmälan (Permits and notifications for Genetic Modified Microorganisms work) documents in Swedish and/or in English (word). Read the Guidelines and Instructions to fill the document (pdf).
Once all these documents have been properly filled and signed they should be send to the Core facility. The core will immediately start reviewing the uploaded documents and contact the biosafety department in order to double check and send them to the pertinent authorities (www.av.se). Once the documentation has been revised and approved by the Facility and the biosafety department at KI, the investigator must:
- Provide the core facility with the expression vector (plasmid) at the right concentration: ≥150 µg of transgene AAV plasmid and ≥300 µg of the LV expression vector at a concentration of at least 0.30 µg/µl (preferably ≥ 1µg/µl). The DNA should have been purified using an endotoxin-free protocol (e.g. Endo-free maxi/mega/Giga plasmids purification kits or equivalent).
- High Quality DNA: Plamid DNA should be checked for purity and have an A260/280 ratio no lower than 1.8 (the actual value of this ratio must be provided to the core). See interested virus section for further details.
- Additional SmaI/SrfI digestion analysis on the AAV transgene plasmids should be performed and the results should be also submitted to the core (see AAV sections for further details).
The capacity to efficiently transduce nondividing cells, shuttle large genetic payloads, and maintain stable long-term transgene expression are attributes that bring lentiviral vectors to the forefront of gene delivery vehicles for research.
Adeno Associated Viruses (AAVs)
AAV serotypes have shown different tissue tropisms in vivo, improved performance in terms of transduction efficiency, and fewer problems associated with humoral and/or cellular immunity.
The Gammaretrovirus is a sister genus to the lentivirus clade. Like the lentivirus, it is a member of the Orthoretrovirinae subfamily of the retrovirus family, they share common properties but the main difference between these two viruses is that Gammaretroviruses only infect dividing cells.