We are working with XVE system from Chua's lab and have encountered a serious problem of induction with estradiol: inducibility of trangene expression in whole leaves from adult plants is impossible (spray/injection at 10uM) and the inducibility is lost across generations of transgenics. Could you share your experience/ideas/tricks with me please? Many thanks in advance!
Hi Delphine,
we are working with a derivative of the XVE system and are to some extent also experiencing issues with inducibility of the expression of the transgene, particularly with T3 or later generations. I also have the feeling that the XVE system is leaky to a certain extent? Have you noticed this in your transgenics at all? thank for sharing
may be a bit leaky but this is not a major problem for us. Dagmar in our lab has also this impression, she is working with a very toxic effector and has problem to isolate proper expressing lines... let s hope we get more feedback from researchgate!
Hi Delphine,
we are using the XVE system as well, but generally only induce the transgene in seedlings (works well when grown on plates or spraying (10 µM f.c. in both assays).
We also observe leakiness and have to screen many many T1's to find a good line. We also observed that dissolving the 10 mM Est stocks in methanol (as suggested by Sigma where we ordered Est) does not work for us (it dissolves, but we can't induce the transgene when using dilutions from this stock). However, if we dissolve the stocks in DMSO, induction works fine.
Alternatively, try the ethanol inducible system. That system definitely works well in adult plants, but be careful when handling seedlings in sterile culture, since a little amount of ethanol used in the sterile hood will efficiently induce your transgene.
Hope that helps.
Ok that is a good point to mention the solvent, thanks! I am used to dissolve estradiol in 80% ethanol but I can try as well DMSO. Unfortunately we do not time anymore for the current project to change the inducible system but we may think of it in the future! Thanks to both of you for your comments.
Good Delphine, Frank and Robin, keep posting for any more developments; such discussions do help us a lot
Hi Delphine! Did you finally solved the problem? Seems that Silwet 0,01% can help to penetrate in the tissues, and if the test are made in seedlings growing on plates, DMSO seems to be the best solvent. We are gonna try it soon. I let you know if this parameters might help. We had also problems in our first try to get the plants induced. Cheers!
Hi everyone! What is the recommend concentration of estradiol that can be use to induce transgene in Arabidopsis? I used 100 uM. Is it too high concentration? Thank you.
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