Figure 5 Trans-repression be- tween integrated transgenes. (A) Confocal z projection of a wander- ing third instar larval VNC from an animal driving UAS-mCD8::RFP with R23G07-GAL4. Expression is found in many neuronal cell types, including lineage 11 (cell bodies indicated with white arrows; projections with magenta arrowheads). Note the stochastic expression in this individual: no expression is found in lineage 11 on the right side of the VNC. (B) Confocal z projection of a wander- ing third instar larval VNC from an animal driving UAS-mCD8::RFP with R11G08-GAL4. Expression in the VNC is largely limited to hemilineages 11A (cell bodies in- dicated with arrows) and 12A. Ro- bust expression is found in every 11A/12A hemilineage in seg- ments T1 and T2. (C–D99) Two examples of VNCs from w; Lex- Aop-rCD2::GFP/UAS-mCD8::RFP (attP40); R23G07-LexA (attP2)/ R11G08-GAL4(attP2) animals. Ex- pression of R23G07-LexA (C and D) is stochastic in lineage 11, as expected. Expression of R11G08- GAL4 (C9 and D9) in hemilineage 11A is also stochastic, but in the same pattern as R23G07-LexA. Merged images are shown in C99 and D99. (E) Two possible models for trans-repression. Red shading and asterisks represent repressive epigenetic modiﬁcations and red arrows indicate recruitment of chromatin silencing proteins. In model 1, R23G07 recruits chro- matin silencers that modify both paired transgenes. In model 2, only R23G07 is epigenetically modiﬁed, but these modiﬁcations directly or indirectly lead to the re- pression of both transgenes. studies of transvection depend on intragenic complementa- tion as the readout of transvection efﬁciency. To further investigate the molecular mechanisms of transvection, it will be useful to have a robust, controlled genetic system that enables both the molecular dissection of key sequence elements as well as a highly detailed and quantitative analysis of the transvection effect itself. One attractive approach is to use the phiC31 integrase system to insert transgenes into deﬁned integration sites (attP sites) and test for their ability to support transvection. This system (1) allows for the use of transgenes that contain the minimal number of sequence elements sufﬁcient to support transvec- tion, (2) inserts transgenes into a common chromosomal location to allow for testing with multiple pairwise combi- nations while minimizing position effects, and (3) allows for distinct readouts for the transcriptional activity of both alleles (e.g., two ﬂuorescent reporters) to minimize the am- biguity that is often present when attempting to quantify the Transvection at phiC31 Integration Sites 1137