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1. What is the required biosafety level for handling recombinant adenovirus?
The recombinant adenoviruses are replication deficient due to deletions in the E1 and E3 regions. According to the NIH Office of Biosafety, recombinant adenovirus has been classified in biosafety level II for agents considered of ordinary potential harm, and you need BL-2 level.
2. Which precautions should I take while working with adenovirus?
Work with adenovirus must be performed in a BL2 lab. There you must:
3. Is gene expression from recombinant adenovirus transient or stable?
When a gene is delivered into mammalian cells by recombinant adenovirus, the expression is transient. Unlike lentivirus, recombinant adenovirus genomes remain epichromosomal in host cells.
4. Is recombinant adenovirus toxic to host cells?
Recombinant adenovirus shows no toxicity to cells in vitro, but it may trigger immune responses in vivo.
5. Are adenoviruses replication deficient?
Yes, because the early genes necessary for replication have been deleted from the shuttle vector. Early gene E1 is provided by tansfected cell lines.
6. How can I find out whether adenovirus will work well in my cell models?
The adenovirus has a very broad host range, it can infect human and other mammalian cell lines or primary cells. There are in fact very few cell lines that cannot be infected. Some lymphoid cell lines may be more resistant to adenovirus infection, so may need high quantities of viruses to achieve sufficient infection levels. Some marker adenovirus can be used to allow you to conduct pilot testing in your systems.
7. What are the recommended storage conditions of recombinant adenoviruses?
For long-term storage, the virus should be kept at -80°C and at -80°C the virus (especially after CsCl or chromatography purification) could be stable for 6 month to a year. However if the virus is repeated freeze-and-thaw, it will cause significant decrease of titer.