Using Adenovirus to transfect cells - (Sep/20/2007 )
Dear all,
I'm doing a physiological project and would like to use Adenovirus to transfect the cell line, in hope that the gene of interest can be overexpressed. However, I'm not sure how these virus can be get rid off before i put the cells into the Ussing chamber. This is more of safety issue. I hope someone out there can help to provide some answer. Thank you.
Regards,
Tan
These adenovirus are defect in replication following infection. As long as you wash most of free adv off, the risk is low.
Thank you for the advice. However, is there a new to work within a class 3 environment or a normal laminal flow will do the job?
Thank you for the advice. However, is there a new to work within a class 3 environment or a normal laminal flow will do the job?
as there are no adenovirus detectable after infection, you may work at standard biosafety level 1
however, biosafety levels are differently defined in different countries, so in case of uncertainty ask your supervisor;
Most adenoviruses are not replication competent, especially mutants, however some are such as wildtype Ad5 and can cause mild infections and possibly a few other things. Replication competency is also dependant on the cell line e.g HEK293 have integrated into the genome of the cells the replication machinery for adenoviruses so even mutants can and do replicate in these cells .
Check the local regulations for use of adenoviruses in your country, but here in NZ we have to use them at biosafety level 2 and have specific permission to use them as well as special containment controls.
I think that you will find that there will be virus present at all time points after infection, and there is no method of washing the virus away that will ensure that there is no potential for infection.
Most recombinant viruses are 99.9% replication deficient. There is still a very small chance of getting infection from them. Adenoviruses are safety level 2 in Germany and depending on which transgene is expressed, it could be level 3.
We infect cells and if possible change media after 24 hrs to ensure we got rid of the viruses.