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. Author manuscript; available in PMC: 2016 Mar 3.
Published in final edited form as: Cell Rep. 2015 Feb 26;10(8):1410–1421. doi: 10.1016/j.celrep.2015.01.059

Fig. 3. Combinatorial gene targeting using Gal80, Split Gal4, QF2, and LexA::QFAD Trojan exons.

Fig. 3

A–B) Suppression of Gal4 activity in glutamatergic neurons using 3XGal80FLAG/HA/EGFP. A) vGlutMI04979- 3XGal80FLAG/HA/EGFP expresses Gal80 (left; anti-GFP immunostaining) in glutamatergic motor neurons of the larval VNC labeled with anti-vGlut antibody (middle); merged images (right). Scale bar (panels A–E): 25 μm. B) elav-Gal4 drives UAS-LacZ reporter in the glutamatergic motor neurons (rectangle: anti-LacZ immunoreactivity, magenta; anti-vGlut, green), but Gal4 activity is absent when the vGlutMI04979-3XGal80 transgene is present (right).

C) Identification of neuronal subsets within the amonMI00899-p65AD expression pattern. Left: The entire complement of amon-expressing neurons in the larval CNS, visualized using the pan-neuronal elav-VP16AD hemidriver and a UAS-EGFP reporter. Middle: The CCAP-expressing complement, visualized using a CCAP-Gal4DBD hemidriver. Right: The vGlut-expressing complement, visualized using a Trojan-MiMIC vGlutMI04979-Gal4DBD hemidriver.

D) Split Gal4 isolation of the subset of neurons that express both the Shaw K+ channel and the hormone Bursicon. Left: neurons expressing the RA, RC, and RE splice isoforms of the Shaw gene, visualized using a Trojan-MiMICShawMI01735-Gal4 driver line and a UAS-EGFP reporter. Middle: The Shaw-expressing neurons that also express Burs, isolated using a Trojan-MiMIC ShawMI01735-dVP16AD in combination with a Burs-Gal4DBD hemidriver; green, UAS-EGFP. Right: anti-Burs immunostaining (magenta) of the section shown in middle panel (double-labeled neurons are white).

E) Double-labeling of the embryonic VNC to identify subsets of cholinergic (LexAop-mCherry, magenta) and glutamatergic (QUAS-mCD8-GFP, green) neurons using orthogonal Trojan-MiMIC drivers. Left: Expression pattern of a ChaMI04508-LexA::QFAD driver in a confocal section through the ventral midline. Middle: Same section showing expression of a vGlutMI04979-QF2 driver. Right: Merged images; note the two expression patterns do not overlap.

F) Double-labeling of glutamatergic neurons in the larval CNS and body wall muscles using the orthogonal Trojan-MIMC drivers vGlutMI04979-T2A-LexA::QFAD (green, LexAop2-myr::GFP) and GluRIIBMI03631-T2A-Gal4 (magenta, UAS-CD4-tdTomato). Right panel: magnified image of the boxed region at left showing neuromuscular synapses. Scale bar: 100 μm.