adenovirus vs. retrovirus cell infection--pros and cons? - infecting PC12 cells and overall general toxicity (Jul/20/2006 )
I am having tons of trouble efficiently transfecting PC12 cells--in order to avoid making a stable cell line, I am strongly considering infecting the cells with either an adenovirus or retrovirus. I have been forewarned that some cell lines are not very tolerant to adenovirus infection, and retro. might work better. Does anyone have experience with adenovirus and/or retro. infection of PC12 cells, or the toxicity of adenoviruses towards other cell types in general??? I plan on using Tet-off cells from Clontech and purchasing either their Tet inducible adeno. or retro. vectors for regulated expression of my protein.
BTW, has anyone ever infected a cell with two different viral vectors--I am wondering about using a tet-inducible expression system in primary neurons by infecting with a tTA construct as well as my gene of interest.
Base on my limited experience with Adv on cells that do not support Adv replication, I think toxicity only shows up when you use high MOI, like over 1:1000 and higher, and is generally less than lipid methods. You can certainly get a ratio that gives you good transfection and reasonable toxicity.
I donot work with adenovirus but my old lab used to make them. They have a large trangene capacity and the third generation adeno virus (gutless), expression is 10x or more that of the old adenovirus with the same titres. unfortunately, it induces immune reaction and is more cytotoxic to cells. Therefore its not advisable to use them for longer term or invivo experiments.
For the tTA system. . . I have been told that you have to transfect in the tTA plasmid first and select for positive cells and test the tTA system. Then you transfect/infect with the plasmid that has the tet-responsive element and screen for positive cells. We use retroviruses-MSCV based to introduce genes into PC12 cells. I have also heard of groups using a Sindibis (sp?) based viral system. Good luck.