retroviral infection of lymphoblastoid cells

[Kimberly Ann Winkeler]

Hi All!

I am looking for some help with my retroviral infections.  I have various 
luciferase constructs which I have transfected into BING cells and have  
frozen the virus stocks (at least I think I have virus--the BING cells showed 
luciferase activity when they were assayed following the final viral 
harvest).  I am then attempting to infect two different lymphoblastoid 
cell lines with these constructs, but with no success thus far.  I have 
tried all kinds of things including centrifugation at 2500rpm/32¡C, 
phosphate depletion prior to infection, and/or simply adding the viral 
sup to the cells.  After selecting my cells for 3 weeks or more, I have 
no luciferase activity. I know that these cells aren't the easiest to 
infect, but this is getting ridiculous!  Does anyone have a tried-and-true 
protocol for infecting lymphoblastoid cells that you could share with 
me?  Also, what is the best way to change the media on these cells after 
infecting them?  Any and all suggestions would be greatly appreciated!

Thanks in advance

Kim Winkeler
Emory University
kwinkel at emory.edu