After making a stock with DD1 and Yolk-G4 together, I found that dipt-lacZ was strongly activated in adult fatbody (female), under unchallenged conditions. Individual stocks were negative, only the combination was positive. Any thoughts as to why?
these AMP reporters (and genes) are also up-regulated with age. Are you comparing animals of similar age?
Thanks.
Typically yes, we try to use animals that are 3-5 days old. Also we tried fed versus starved and that didn't make much difference.
As this response appears to be sex specific, an important consideration is the physiological state of the female, i.e. naive (unmated) compared with mature (& mated). Copulation, specifically the transmission of the seminal package, is seen by the female fly as a potential xenobiotic challenge and as such their immune system is upregulated (also activity and feeding patterns are altered, which again affects immune responses). In fact there are some indications to say that there may be 'anticipatory' immune response upregulation in females in response to con-specific courtship song.
that's very interesting, I'll have to look back to see if we tried virgins, non virgins etc. Thank you
No worries, though I have to admit I didn't read the question clearly enough. A Yolk-Gal4 driver should obviously in and of itself be sex-specific in expression. The fact that you're not seeing driven expression of the dipt-LacZ reporter in the adult male fatbody should be unsurprising. So again there also exists the small chance that the combination of the driver and reporter is somewhat 'leaky' resulting in a higher level of expression of the reporter (have you tried the driver out with another reporter?). Also do you see any increase in dipt-LacZ expression if you challenge the flies immune system?
So we tried the r4-G4 driver, thinking it was some sort of response to high levels of Gal4, but this wasn't the explanation. diptLZ was not upregulated in fat body with r4G4. As for inducibility, E.coli challenge gave a mild up regulation of the diptLZ reporter in both male and female of the DD1 stock. We haven't yet tried rtPCR for endogenous dipt transcripts vs the lacZ reporter, maybe its just something weird with the construct.
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