2 replies to this topic

[mjl8ce]

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!

MJ

[bob1]

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

[mjl8ce]

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.