Enhancer Reporter Transgenesis Protocols

All wildtype and mutagenized enhancer-reporter transgenic constructs were made using the lacZ reporter vector pRVV54 as an acceptor vector [49 (link)]. Coordinates of the genomic fragments PCR-amplified in the enhancer bashing experiments are listed in S1 and S5 Tables. The ΦC31 system was used for transgenesis and plasmids were introduced in landing sites 51D or 86Fa [50 (link)].
Site-directed mutagenesis of the vnE and rhoE enhancers was performed according to the QuikChange II protocol (Agilent Technologies). vnE and rhoE enhancers were first introduced in pBluescript SK+ vector for site-directed mutagenesis and the resulting mutated enhancers were consequently transferred to pRVV54 for in vivo analysis in the fruit fly. Primers used for mutagenizing of putative binding site are listed in S3 Table.
Plasmids for recombinant protein production were made by introducing cDNA sequences into pET21 series vectors (Novagen-EMD Millipore) and their derivatives, resulting in C-terminally tagged His proteins. Primers used to generate Dll-His (full-length Dll), Sp1^Zn-finger-His (only the Zn-finger domains; used for confirming in vitro binding to Sp1 sites), Sp1^424AA-His (used to examine cooperativity with Dll), Mad^MH1-His (only the MH1 domain) and Pan^HMG-His (only the HMG domain) vectors are listed in S2 Table.