UAS-GFP and UAS-EGFP. - Strigini, M. (1999)
Subject: FlyBase query Dear Gillian, I got this message from Dirk Bohmann via Julia Zetlinger: >>Dear Dr. Bohmann, >> >>I am curating your paper for FlyBase: >> >>Zeitlinger and Bohmann, 1999, Development 126(17): 3947--3956 >> >>and I have a question about the GFP constructs you used. >> >>You used 2 constructs: >> >>UAS-GFP >>UAS-EGFP >> >>I would be grateful if you could tell me some more details about the >>UAS-EGFP construct, so that I can work out whether it corresponds to a >>UAS-GFP line we have already in FlyBase or whether it is new. >> >>Specifically, could you tell me: >> >>a. a reference where UAS-EGFP has been used. >>b. who you got the line from. >>c. presumably the 'EGFP' means that this is not the unmodified GFP - >>does EGFP have the S65T mutation, or some other mutation that enhances >>the fluorescence ? >> >>I look forward to hearing from you, >> >>Gillian It was simply done by cloning an EcoRI-NotI fragment (about 800 bp) from the pEGFP-N1 vector of Clontech into the pUAST vector (Brand and Perrimon). pEGFP-N is the one with the 'enhanced GFP', which contains two aa substitutions: Phe-64 to Leu and Ser65 to Thr. We got a few transformants, all with inserts on the second chromosome only. Maura