Q-system reagents for genetic manipulations

nature methods | ADVANCE ONLINE PUBLICATION | be subdivided into three domains (Fig. 1a) that perform specific and independent functions: the DNA-binding and dimerization domain (DBD) containing a Zn2-Cys6 motif that recognizes and binds to UAS or QUAS sites; a middle domain (MD) that has no clear function but that might be involved in endogenous regulation or stability; and a transcriptional activation domain (AD) that recruits molecular machinery necessary for transcription and that also binds the Gal80 or QS suppressor. To overcome limitations of the original QF, any new QF variant should be capable of generating healthy transgenic flies when broadly expressed. In addition, it should exhibit strong transcriptional activity yet remain QS suppressible. We generated a series of constructs in which certain QF domains were modified (Fig. 1a and Supplementary Fig. 1a). In Gal4QF, QF2w(eaker) and QFe, the QF AD was mutated to reduce activity by altering the charge on the C terminus. In QF2, QF2w and LexAQF, the QF MD was completely removed; in QFf–i, the QF MD was partially removed. Finally, in Gal4QF, LexAQF, QFa–d and QFj–l, the QF DBD or QF AD were swapped for analogous Gal4 or LexA domains. To quantitatively measure activity levels, we performed luciferase assays in cultured Drosophila Schneider 2 (S2) cells (Fig. 1b). To assay for QF toxicity, we attempted to generate transgenic animals expressing each construct under the pan-neuronal neuronal Synaptobrevin (nSyb; hereafter, nsyb) promoter (Fig. 1, Supplementary Fig. 1 and Supplementary Table 1). To allow direct comparison between transgenic constructs that use the same enhancer activation sequence (UAS, QUAS or LexAop), we used the PhiC31 integrase system and targeted all transgenic insertions to the same attP2 genomic landing site (3L: 68A4). However, direct comparison between transgenic factors using different activation sequences, e.g., UAS reporters (Fig. 1c) versus QUAS reporters (Fig. 1d), cannot be made owing to differing activities of the reporters. In relative luciferase activity assays and in vivo expression analyses, the optimal QF variants exhibited high activity levels (Fig. 1b–f and Supplementary Table 1), were efficiently repressed by QS (Supplementary Table 1) and produced healthy transgenic animals. We initially hypothesized that a potent QF AD may be the source of toxicity as it may be squelching cellular transcription factor components16, but QF variants that contained the original (QF2 and LexAQF) and mutated AD of QF (QF2w and Gal4QF) were not toxic. Instead, constructs containing the MD of QF either failed to produce transgenic animals (QFd and QFg, Supplementary Fig. 1) or were extremely unhealthy (QFf), thereby implicating the QF MD as the major source of QF toxicity. Deletion of this domain yielded two smaller QF variants, QF2 and QF2w, which exhibited strong but differing QF activities improved and expanded Q-system reagents for genetic manipulations