Figure 2.

Methods of transgene targeting. (A) The Gal4-UAS system described in the text. Left panel: the schematic depicts the transgene construct for Gal4 (blue outline) on one fly chromosome, and the transgene effector construct (green rectangle) on another fly chromosome. The Gal4 gene lies downstream of the promoter/enhancer element, P1, which dictates its pattern of expression. The effector transgene lies downstream of five Gal4 binding sites (i.e. “UAS,” black ovals). In flies bearing both constructs, neurons that express Gal4 protein (blue shapes) also express the effector protein (green circles). Right panel: schematic of the fly CNS depicting coincident expression of Gal4 (blue outline) and effector (green oval). (B) Subtractive restriction of effector gene expression using Gal80. Left panel: In cells that express the gene encoding Gal80 (brown outline) under the control of the promoter/enhancer P2, Gal80 protein (brown circles) will inhibit Gal4 by binding to its transcription activation domain and thus block effector gene expression (red X). Right panel: if P1 and P2 have overlapping expression patterns, Gal4 activity, and therefore effector expression, is eliminated in the region of overlap (i.e. in Gal80 positive cells, brown). (C) Random restriction using the “flp-out Gal80” system. Left panel: if the Gal80 transgene is placed downstream of the ubiquitously active tubulin promoter (tub) and is flanked by sites (triangles) that permit excision by heat-shock induced flp-recombinase activity, the Gal80 gene will be deleted in random subsets of cells in animals subjected to heat shock. Only cells that express Gal4, but not Gal80, will also express the effector gene. Right panel: Effector expression (green) is limited to cells within the Gal4 expression pattern (blue outline) that lack Gal80 expression. (D) Combinatorial restriction using Split Gal4. Left panel: If the DNA-binding (blue, DBD) and transcription activation (yellow, AD) domains of the Gal4 molecule are independently targeted to different neuronal groups using promoters P1 and P2, Gal4 activity will be reconstituted and the effector transgene expressed only in cells at the intersection of the P1 (blue) and P2 (yellow) expression patterns, as depicted in the right panel.