Figure 1. Using a pan-muscular promoter for single-copy transgene selection: AC. Characterization of the fluorescence expression pattern from MosSCI insertions of transgenes with Punc-54:: gfp, Pmlc-2:: gfp, or Pmlc-1:: gfp. We acquired images at 20x magnification from immobilized young adult animals. Scale bar= 20 microns. A. Expression in the head region. All transgenes were expressed in head muscles, but only Pmlc-1 and Pmlc-2 were expressed in pharyngeal muscles (indicated by white arrowheads). B. In the vulval region, all promoters expressed GFP in body wall muscles. Only Pmlc-1 and Pmlc-2 were expressed in vulval muscles. C. In the tail region, all promoters expressed GFP in body wall muscles, stomato-intestinal and anal depressor muscles, D. Quantification of GFP expression in young adult animals by flow cytometry (Copas) in MosSCI strains expressing Punc-54:: gfp, Pmlc-2:: gfp and Pmlc-1:: gfp. One-way ANOVA with Sidak's multiple comparison test (N2 (N= 293), Punc-54 (N= 277), Pmlc-1 (N= 301), Pmlc-2 (N= 346)). E. Screen for transgene copy number. PCR primers were designed to amplify (1) the vector backbone which is commonly duplicated for dual inserts (950 bp),(2) the transgene promoters (1.5 kb for Pmlc-2 and 1.0 kb for Pmlc-1),(3) the gfp fluorophore (344 bp),(4) the gfp and promoter junctions (1.8 kb for Pmlc-2 and 945 bp for Pmlc-1), and (5) the junction between gfp and 3'UTR (1.4 kb). Green boxes indicate expected bands, and red boxes indicate controls with no expected PCR amplification. The band in lane 3 from wildtype DNA is likely due to minor contamination. No dual or" complex" inserts were detected. F. Transgenes were tested for toxicity by injection at increasing concentrations in ten animals (2.5, 10, or 25 ng/uL) using Pmlc-1 or Pmyo-2 with an mCherry fluorescent marker. G. Picture at 40x magnification of a Psmu-1: gfp MosSCI insertion used to test Pmlc-1 as a single co-injection marker. As expected, GFP was expressed strongly in the germline and less in somatic cells (not shown). Scale bar= 20 microns.