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TZM-bl Assay Problem

HIV TZM-Bl

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#1 mjl8ce

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Posted 03 March 2012 - 09:46 AM

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

#2 bob1

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Posted 04 March 2012 - 11:54 AM

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

#3 mjl8ce

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Posted 04 March 2012 - 02:42 PM

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.





Also tagged with one or more of these keywords: HIV, TZM-Bl

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