phiC31 integration system (Groth et al. 2004). Site-speciﬁc integration of transgenes is becoming increasingly common, as it provides the opportunity to control for and minimize genomic position effects (Markstein et al. 2008; Pfeiffer et al. 2010). Given the use of multiple transgenes in many cross- ing schemes, it is common to use two transgenes that have been independently inserted into the same integration site (attP site). This raises the question of whether it is possible to elicit transvection between two transgenes by simply inserting them into the same genomic location and pairing them. Indeed, we have found that transvection occurs be- tween transgenes at attP sites, which imposes signiﬁcant restrictions on experimental design. Our analysis of transvection focuses on the ventral nerve cord (VNC) of the Drosophila larva, which contains a seg- mentally repeating, stereotyped array of stem cells (neuro- blasts). Each neuroblast generates a stereotyped lineage that is easily identiﬁable during late larval life based on the char- acteristic morphologies of its constituent neurons (Truman et al. 2004). These lineages are further subdivided into hem- ilineages based on Notch activity (Truman et al. 2010), and these hemilineages can also be recognized by their neuron position and morphology. To have a genetic handle on these populations of neurons, we have been taking advantage of a recently developed toolset composed of transgenes in which GAL4 is driven by cis-regulatory modules (CRMs) taken from various gene loci (Pfeiffer et al. 2008). These transgenes have been inserted into the commonly used attP2 integration site. We have identiﬁed GAL4 lines that are expressed in select neuroblast lineages and hemilineages and are using these as an entry point for investigations of VNC development, structure, and function. One strategy has been to use dual binary transcription systems (GAL4/UAS and LexA/LexAop) to independently label or manipulate multiple neuroblast lineages and study their physical and functional relationships (Brand and Perrimon 1993; Lai and Lee 2006). While examining multiple combinations of GAL4 and LexA lines inserted into attP2, we noted several cases in which part of the expression pattern driven by the regulatory element of one transgene was found where we had expected to see only the expression pattern of the paired transgene. Further analyses showed that this cross- talk was due to transvection. We further investigated the transvection phenomena we encountered for two reasons. First, many experimental strategies, including the reﬁnement of expression patterns using intersectional genetic approaches, require indepen- dence between the transcriptional control of multiple genetic components (Luan and White 2007; Simpson 2009). We therefore were motivated to document the ob- served transvection effects found at integration sites to pre- vent or minimize potential problems when using these tools. Second, because our genetic toolset essentially provides a simple and robust system for studying transvection, we took the opportunity to address questions regarding the na- ture and prevalence of transvection. Most importantly we 1130 D. J. Mellert and J. W. Truman ﬁnd that (1) the ability of a cis-regulatory module to support transvection is common, and (2) the only requirement for a regulatory element to drive expression in trans is its ability to bind transcriptional activators. These results provide strong evidence that transvection is common throughout the Drosophila genome and must be considered when de- signing experiments that utilize transgenes inserted into the genome by site-speciﬁc integration. Materials and Methods Fly stocks All crosses were carried out at 25 under standard condi- tions on cornmeal/molasses food. All GAL4 lines were cre- ated as described by Pfeiffer et al. (2008). CRM lines driving a transactivator composed of the LexA fused to the full- length GAL4 activation domain (LexA::GADﬂ) were created as described by Pfeiffer et al. (2010) using the pBPLexA:: GADﬂUw vector. The sequences of the primers used to clone the CRMs used are available as Supporting Information, Table S1. PhiC31 integrated pJFRC2-10XUAS-IVS-mCD8::GFP and pJFRC21-10XUAS-IVS-mCD8::RFP lines (Pfeiffer et al. 2010) were a gift from B. Pfeiffer and G. Rubin. Fly lines containing the P{LexAop-rCD2::GFP} (Lai and Lee 2006) and P{LexAop- rCD2::RFP} reporters were gifts from T. Lee. A ﬂy line con- taining a second chromosome insert of P{UAS-mCD8::mRFP. LG} was a gift of E. Gavis and is available from the Blooming- ton Stock Center (no. 27398). Preparation and examination of tissues Tissues were dissected in PBS and ﬁxed in 4% formaldehyde in PBS (pH 7.0) for 45–60 min, then rinsed in PBS-TX [phos- phate buffered saline (pH 7.2) with 1% Triton X-100]. An- tibody staining was performed as described by Truman et al. (2004) using the following antibodies: 1:1000 dilution of rabbit anti-GFP (Invitrogen), 1:500 rat antimouse CD8a (Invitrogen), 1:500 Alexa Fluor 488 conjugated goat anti- rabbit (Invitrogen), 1:500 Texas Red conjugated Donkey antirat IgG (Jackson ImmunoResearch Laboratories). Imaging was performed on a Zeiss LSM 510 confocal microscope and Z stacks were analyzed using ImageJ (http://rsbweb.nih.gov/nih-image) and Vaa3D (Peng et al. 2010). ImageJ was used for all postprocessing of images. Construction of R24B02-Ptrans-GAL4, R58G08-Ptrans-GAL4, and 24B02-ΔP-LexA To create integration vectors for R24B02-Ptrans-GAL4 and R58G08-Ptrans-GAL4, Gateway clones for the R24B02 and R58G08 CRMs were transferred into a vector derived from pBPGw (Pfeiffer et al. 2008) that contained the P-transposase promoter (pBPGPtw; a gift from B. Pfeiffer and G. Rubin). To create the integration construct for 24B02-ΔP-LexA, the Dro- sophila synthetic core promoter (DSCP) was excised with FseI and KpnI from an integration vector for 24B02-LexA::p65, a gift from B. Pfeiffer (Pfeiffer et al. 2010). The resulting