The VirusTech Core Facility - user guidelines

It is always important to plan the production of viral particles ahead, and therefore some documentation will be needed prior to starting the production of the viral particles.

These documents are designed to establish the proper work routines, as well as a safe work environment and inform the relevant and appropriate authorities to establish work and environmental responsibilities.

Before starting a project

You need to

  • Obtain the work permits for the viruses to be produced (read more about Laboratory Biosafety on the Staff portal), which you will be using when the core facility delivers them to you. Make sure you are 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. Please note the VirusTech Core Facility can help with that. We also offer packaging plasmid systems for lenti-and adeno-associated viruses.

The core offers DNA-preparations and cloning services to ease the workload of the investigator.

What to provide to the core

  1. 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, for the VirusTech Core Facility to work with those plasmids.
  2. Download, fill and submit the order form:
  3. Download the L–A Anmälan (Permits and notifications for Genetic Modified Microorganisms work) documents in Swedish and/or in English (word).

Once the documentation has been revised and approved by the VirusTech Core Facility and the KI biosafety administrator, the investigator must:

  1. Provide the expression vector (plasmid) at the right concentration: ≥ 150 µg of AAV transfer vector plasmid and ≥ 200 µg of the LV transfer plasmid at a concentration of at least 0.30 µg/µl (preferably ≥ 1 µg/µl). The DNA should have been purified using an endotoxin-free protocol (e.g., endo-free Maxi/Mega/Giga plasmids purification kits or equivalent).
  2. High Quality DNA: Plasmid DNA should be checked for purity and have an A260/280 ratio not lower than 1.8 (the actual value of this ratio must be provided to the VirusTech Core Facility).
  3. To ensure the quality of AAV transfer vector plasmids, it is essential to conduct an additional SmaI/SrfI digestion analysis. The results of this analysis should then be submitted to the VirusTech Core Facility, as outlined in the AAV section. For more rigorous quality control of the AAV inverted terminal repeat (ITR) sites, consider utilizing Eurofins’ whole plasmid sequencing service. This service uses Oxford Nanopore technology, which is capable of reading GC-rich and repetitive sequences, such as ITRs.”
MP
Content reviewer:
12-02-2024