I have expressed a RNAi construct in a subset of cells in the drosophila ovary using c306 GAL4, which gives me a severe phenotype, probably worth characterizing.
But to validate the specificity of the RNAi construct I need to do a RT-PCR with primers against my target gene.
The problem I am facing here is that, I can not find a suitable model to test on. When driving with Act5C GAL4, I see lethality at the larval stage. So I thought of trying to express it in the larval salivary gland using c306 Gal4, but then the salivary glands do not develop.
So, I actually need a not very strong GAL4, but would show ubiquitous expression in some tissue that can be used for isolating RNA?
Could you suggest me some GAL4 lines that can be used?
You may try with C765-GAL4, which is expressed ubiquitously in most the wing disc. Probably in other imaginal tissues but I think you would bypass the lethality problem. You can check Schwank et al 2008 to see the expression pattern in Fig 4A. I have seen people also using escargot-GAL4 to drive ubiquitous expression in imaginal tissues. scalloped-GAL4, nubbin-GAL4, rotund-GAL4 are expressed in the wing as well, not the entire imaginal disc but aproximately in a big domain that encircles the wing pouch, the domain that will give rise to the adult wing blade. Another option is to use compartment-specific drivers such as engrailed-GAL4 or ci-GAL4 together with UAS-GFP (they are available in Bloomington recombined) and sort the GFP vs non-GFP cells before qRT-PCR and compare the mRNA levels of interest of the GFP domain vs the non-GFP. This way you would have an internal control in the same animal.
If you want to try with the eye disc. You may try GMR-GAL4, which is expressed in all eye cells disc committed to neural fate, once the morphogenetic furrow is quite advanced. Otherwise, you could use the combination eyeless-FLP actin-FRT-stop-FRT-GAL4 to flip a GAL4 cassette and drive permanent GAL4 expression in all the cells that ever expressed the gene eyeless even though these cells eventually lose ey-FLP expression once committed to neural fate.
All these stocks are available on Bloomington. Just be sure that en-GAL4 you order is the e16E construct for instance #25752 stock
I hope it helps
Carles,
Thank you for your suggestions. We do not have a facility to sort cells based on GFP expression. Except for that, I can probably try what you have suggested.
I would also suggest using a heatshock GAL4. Cross the heatshock GAL4 with the uas-RNAi line you want, place adults (or whatever stage you want) at 37 degrees C for an hour, and then take out whatever tissue you want for the RT-PCR. I just did this with the line HS-GAL4 #2077 from bloomington and found that my RNAi line knocked down my target gene by about 7 fold in fly heads.
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