The stock was balanced fine, until one day we started getting Non-CyO flies.
On crossing the non-CyO flies to UAS-GFP all larvae turned out to be GFP positive.
However, a few generations later the stock starts to show white eye, indicating loss of the insertion. (Not tested with UAS-GFP)
On a side note, I have a stock Actin GAL4/CyO; PElement / Tbe which is all curly and working fine.
I agree with Marks point that a rare recombination event has occurred, this could have been due to a few reasons. The most likely causes are contamination (where you introduced new alleles into population) or a new mutation occurred in your stock where the balance has broken down (insertion has went away).
Act-GAL4/CyO is a 'workhorse' driver used in Drosophila labs throughout the world, the CyO balancer effectively precludes any HR events, else problems with this line would have occurred and been reported ad nauseum previously.
So, while I hate to dispute Mark Mayhew, the most likely answer for your problems is simply contamination by another stock, presumably one carrying another GAL4 driver. Not much consolation to say 'it happens all the time'. That's why its always best to ensure a pristine set of your necessary/precious stocks are maintained at all times and if any question arises about the experimental stock arise you dump them and revert to the original stock (or get a replacement from the stock centres).
That said you should also be aware that the expression of Cy is suppressed in highly dense populations (reported as an effect of larval crowding) and also at lower temperatures. So if a CyO stock is maintained at 18º at a high population density and/or in lower amounts of food then you could 'lose' the Cy phenotype without losing the balancer. In this case increasing the temp the flies are raised at and reducing the population density should cause the Cy expression to become apparent again.
Transgenes in flies do break down. We have had CyO flies not showing the phenotype for some strains. I do not know if its more pronounced for CyO compared to others.
I have had a similar problem recently with cyo and agree with Anthony that the problem may be due to keeping the flies in too high a density and at 18 degrees. I also had a problem where i was loosing a TM6B stock due to this in conjunction with a bad batch of food. I switched out the food and also transferred 20-40 flies into fresh vials and kept at 25 degrees and over a generation or two the problem was solved. Hope your problem is resolved soon, but "if in doubt chuck it out" and buy in a new stock. I always kept to this moto. Cheers. Niki.
I'm inclined to agree with Anthony. White eyes in the stock are something to be worried about and strongly suggest contamination. Transgenes with a CyO (as opposed to SM6a) tend to NOT recombine out. I do believe that some 'hot spots' are known for meiotic recombination to still occur (I believe Ashburner's Drosophila: A Laboratory Handbook describes the balancers quite well). I also agree that Curly is highly sensitive to temperature and crowding. Additionally, Gal4 while toxic is not 100% lethal. I remember seeing occasional homozygotes in our Actin Gal4 stock but they had visible darker eyes suggesting two copies of the transgene.
However, more of an aside, I do suggest that you check to confirm that the actin Gal4 transgene does indeed use a w+ as the positive selection marker. It is possible that this used a y+ or v+ marker and the w+ was part of the original background which may have become mutant due to a spontaneous insertion of a retrotransposon. It is important to know what really is in the stock.
I hope that helps, Nick
I agree with both Anthony and Mark. However, if the GAL4 gene is lost due to HR, it is most likely that the mini-white gene is also lost. Thus, you would see white eyes on your vials. If I were you, I would cross your homozygous flies with UAS-mCD8-GFP and put the larvae under UV light in the 488 channel. If you still have your Act-GAL4 insertion, 100% of the progeny should be glowing green since Act is a ubiquituous promoter. Good luck!
I agree with Anthony - I have CyO stocks that had straight wings when kept at 18 and the balancer should prevent recombination. Unless the stock has gone homozygous? But then white eyes in the stock indicated contamination. Better to discard and get a stock that you can trust.
Thank you for your suggestion and for your time!!
Presence of white eyes suggests contamination. However, we are maintaining the stock at 25. The line was homozygous lethal, originally. What was more surprising was that the homozygous flies carry the Actin-Gal4 insertion as indicated by a UAS-GFP cross.
Multiple copies of the stock went homozygous at the same time, which were maintained by separate persons.
Presently, I am trying to recover the insertion from the mentioned stock, that works fine.
Else reordering would be the only option left.
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