A conditional tissue-specific transgene expression system using inducible GAL4 -- Data Supplement - Supporting Figure -- Proceedings of the National Academy of Sciences

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Supporting information for Osterwalder et al. (2001) Proc. Natl. Acad. Sci. USA 98 (22), 12596–12601. (10.1073/pnas.221303298)

Supporting Figure 6

Fig. 6. Schematic representation of the construction of transformation vectors pP{ELAV-GeneSwitch} and pP{MHC-GeneSwitch}.

pP{ELAV-GeneSwitch}: We used a 2,231-bp SacII–SspI fragment, including the GeneSwitch GAL4 cDNA and a synthetic Intron (IVS8) from the pSwitch vector (blue shades; Invitrogen) and cloned it into the Klenow-blunted XhoI and the SacII site of the pUAST vector (1) to generate
pP{UAS-GeneSwitch}. We then introduced the complete multiple cloning site of the pBluescript KSII(+) cloning vector (MCS, green), released by using the two flanking BssHII sites, into a unique MluI site upstream of the 5´ upstream activating sequence (UAS) in pP{UAS-GeneSwitch} to obtain pP{MCS-UAS-GeneSwitch}. A 3.5-kb fragment of the 5¢ region of the embryonic lethal abnormal vision (ELAV) gene, including the promoter, the transcription start site, and the 5¢ untranslated region (UTR) (2, 3), was cloned into pBluescriptII by using the unique NotI and EcoRI sites. pP{MCS-UAS-GeneSwitch} was linearized by a partial digest with KpnI, and the 5´ UAS was then removed by using a unique NotI site immediately upstream of the IVS8 intron and replacing it by the 3.5-kb ELAV promoter released by KpnI and NotI to get pP{ELAV-GeneSwitch}.

pP{MHC-GeneSwitch}: A 2.4-kb fragment of the Drosophila myosin heavy chain (MHC) gene comprising » 450 bp upstream of the translation start site as well as 5¢ UTR from exons 1 and 2 (4) was cloned into EcoRV restricted pBluescriptII by using Klenow-blunted XhoI and EcoRV. The MHC-promoter fragment was then released by using BssHII and NotI and cloned into the unique MluI and NotI sites of pP{UAS-GeneSwitch} to get pP{MHC-GeneSwitch}.

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