The attP cassette in the Mi{MIC}Gad1MI09277 insertion has been replaced with a Trojan p65AD cassette that contains a splice acceptor site followed by the T2A peptide, the Hsap\RELA activation domain fused to the T:Zzzz\Zip+ component of the SplitGAL4 system ("p65AD") and an Hsp70 transcription termination signal. The insertion is in the correct orientation to gene trap Gad1. The splice acceptor ensures that the T2A-p65AD open reading frame is incorporated into the Gad1 mRNA, while the T2A sequence is expected to truncate the native Gad1 gene product and promotes the separate translation of the p65AD open reading frame. Thus expression of the Hsap\RELA activation domain fused to the T:Zzzz\Zip+ component of the SplitGAL4 system is driven by the native Gad1 regulatory sequences.
Comment: when combined with TI{GAL4(DBD)::Zip-}dsxGAL4-DBD
Comment: when combined with TI{GAL4(DBD)::Zip-}dsxGAL4-DBD
Comment: when combined with TI{GAL4(DBD)::Zip-}dsxGAL4-DBD
Blocking synaptic vesicle exocytosis (using Ctet\TeTxLCTNT.Scer\UAS) in neurons targeted by the split-GAL4 combination of Scer\GAL4dsx-GAL4-DBD and Hsap\RELAGad1-MI09277-Tp65AD.2 does not significantly affect male courtship behavior (they display the normal complement of courtship behaviors including attempts to copulate and relatively normal levels of courtship toward target females), but less than 20% of males successfully copulate (during a 1hr period) and those that do copulate take significantly longer than controls and 80% could not uncouple from the female ('stuck' phenotype); male fertility is also significantly reduced compared to genetic controls. Blocking synaptic vesicle exocytosis (using Ctet\TeTxLCScer\FRT.stop.Scer\UAS) in neurons targeted by the split-GAL4 combination of Scer\GAL4dsx-GAL4-DBD and Hsap\RELAGad1-MI09277-Tp65AD.2 along with Scer\FLP1FLPo.oc.Tag:NLS(SV40-largeT) results in a significant reduction of male courtship behavior but no copulation defects (successful matings in 1hr and copulation duration are not significantly different compared to genetic controls).
Thermogenetic activation (using TrpA1Scer\UAS.(B).cKa at 31[o]C) of neurons targeted by the Split-GAL4 combination of Scer\GAL4dsx-GAL4-DBD and Hsap\RELAGad1-MI09277-Tp65AD.2 does not significantly affect male courtship behavior, but completely blocks genital attachment (no successful matings) in the 1hr observation period. 90% of Scer\GAL4dsx-GAL4-DBD/Hsap\RELAGad1-MI09277-Tp65AD.2 males dismount females and terminate copulation within one minute of thermogenetic activation in copulo; matings truncated in this way never produce progeny. Thermogenetic activation (using TrpA1Scer\FRT.stop.Scer\UAS.T:Hsap\MYC at 31[o]C) of neurons targeted by the Split-GAL4 combination of Scer\GAL4dsx-GAL4-DBD and Hsap\RELAGad1-MI09277-Tp65AD.2 along with Scer\FLP1FLPo.oc.Tag:NLS(SV40-largeT) (both pre-copulation and in copulo) does not significantly impair male courtship (time spent in courtship behavior is similar to genetic controls) or copulation (successful matings in 1hr and copulation duration is similar to genetic controls).
