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.
A GAL4(DBD)::Zip- hemidriver (component of the 'Split GAL4' system, contains the GAL4 DNA-binding domain) has been inserted into the first, non-sex specific coding exon of dsx, such that the hemidriver is expressed under the control of endogenous dsx sequences.
HsapRELAGad1-MI09277-Tp65AD.2 in combination with ScerGAL4dsx-GAL4-DBD drives expression specifically in a subset of GABAergic neurons. These include ~1 adult fruitless pIP-e (male) neuron, ~3 adult doublesex SN (male) neurons and, adult GABAergic doublesex AbN (male) neurons and adult GABAergic doublesex AbN (female) neurons. These latter neurons aborize locally, except for two in males that project to the adult mesothoracic and prothoracic neuromeres. pIP-e neurons and adult doublesex SN (male) neurons project within the superior medial protocerebrum and to the adult subesophageal zone. In males, all pIP-e neurons are positive for fru-XP, as is a single adult doublesex SN (male) neuron and ~30 adult GABAergic doublesex AbN (male) neurons.
ScerGAL4dsx-GAL4-DBD drives expression specifically in neurons when combined with HsimVP16AD.elav in a split-GAL4 system including mechanosensory neurons in the terminalia associated with the bristles of the clasper tooth, lateral plates and anal plates in males, and the anal and vaginal plates in females. ScerGAL4dsx-GAL4-DBD in combination with HsimVP16AD-VGlut-OK371 drives expression specifically in a subset of VGlut positive glutamatergic neurons. These include; adult doublesex aDN neurons, motor neurons in the reproductive system and abdomen that arborize onto muscles of the aedeagus, dorsal and central muscles of the sixth abdominal segment, motor neurons that innervate via the abdominal nerve trunk the phallic and periphallic musculature in males and the muscles of the uterus, spermathecal and parovarian ducts in females and prothoracic leg taste bristle chemosensory neurons that project to the prothoracic neuromere and cross the midline in males, but not females. Also included are adult glutamatergic doublesex AbN (male) neurons, adult glutamatergic doublesex AbN (female) neurons and, in males ~8 glutamatergic motor neurons in the abdominal neuromeres that are positive for fru-XP, including those that innervate the muscles of the sixth abdominal segment. ScerGAL4dsx-GAL4-DBD in combination with HsapRELAGad1-MI09277-Tp65AD.2 drives expression specifically in a subset of GABAergic neurons. These include ~1 adult fruitless pIP-e (male) neuron, ~3 adult doublesex SN (male) neurons and, adult GABAergic doublesex AbN (male) neurons and adult GABAergic doublesex AbN (female) neurons. These latter neurons aborize locally, except for two in males that project to the adult mesothoracic and prothoracic neuromeres. pIP-e neurons and adult doublesex SN (male) neurons project within the superior medial protocerebrum and to the adult subesophageal zone. In males, all pIP-e neurons are positive for fru-XP, as is a single adult doublesex SN (male) neuron and ~30 adult GABAergic doublesex AbN (male) neurons.
Labels a subset of GABAergic neurons including ~1 adult fruitless pIP-e (male) neuron, ~3 adult doublesex SN (male) neurons and, adult GABAergic doublesex AbN (male) neurons and adult GABAergic doublesex AbN (female) neurons.
