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TZM-bl Assay Problem - (Mar/03/2012 )

I have TZM-bl cells stably expressing anti-HIV aptamers. TZM-bl cells express CD4/CXCR4/CCR5 and have a tat-driven luciferase or beta galactosidase reporter system. I'm using BetaGlo (Promega) for the luciferase assay. Previously tested lines expressing aptamers inhibited over 21 days while the vector control-expressing lines did not inhibit at all. Those experiments were done in August/Sept/Oct and were very reproducible. Now, I'm testing some new cell lines and my previously non-inhibitory controls go to baseline (no replication) levels after the first assay point (luciferase assay every 3 days). Has anyone had problems with TZM-bl cells losing either HIV receptors/co-receptors, or developing a defect in the reporter system? This has happened twice now. I suppose something could be funky with my incubator, or I could have some kind of mycoplasma infection. I do keep my cells without antibiotic, so I know that no other infection is present, but have not yet tested for mycoplasma. My media colors are normal, so I haven't as yet suspected a CO2 problem. Ideas? Thanks for reading!



What selection are you keeping the receptors under to make sure that they aren't deactivated?


According to the data sheet, they do not require culture in selection media to retain their receptors. I do maintain them in G418 to retain aptamer expression. Updated experiments: The funny thing is, I can re-infect them with fresh virus and get similar levels to the first infection. But on subsequent serial passage, replication/luciferase signal drops to baseline....the HIV losing infectivity for some reason? I'm having the same problem in normal TZM-bl cells not expressing any aptamer-related construct. I'm not really sure what the problem could be.