Fluorescent markers are useful for identifying transgenic C. elegans after injection. In some cases, fluorophores are used to identify transgenics and to propagate animals with extra-chromosomal or integrated arrays (e.g.,
sur-5::gfp) (Gu et al. 1998). In other cases, fluorescent markers are used as visual markers to identify and later select against array animals. Such negative selection is used generate single-copy transgene insertions by plasmid injection, e.g., Mos1-mediated single-copy insertion (MosSCI)(Frkjr-Jensen et al. 2008) or CRISPR/Cas9 (Dickinson et al. 2013). These methods generate targeted double-strand breaks, and transgenes are inserted by homologous recombination into specific locations. Selection schemes that rely on positive (e.g., cbr-
unc-119, NeoR, HygroR)(Maduro and Pilgrim 1995; Giordano-Santini et al. 2010; Radman et al. 2013) and negative (e.g., fluorophores or the
peel-1 toxin) (Frkjr-Jensen et al. 2012) selection markers are frequently used to identify single-copy insertions. Negative
peel-1 selection is under control of a heat-shock promoter (Phsp-16.41); after heat-shock, animals with arrays or dual insertions with the
peel-1 transgene rapidly die (Seidel et al. 2011).
peel-1 is convenient, but this selection has two significant drawbacks: the transgene is toxic in the absence of heat shock, resulting in fewer F1 progeny (Frkjr-Jensen et al. 2012), and the induced lethality is often not fully penetrant resulting in false positives. Regardless of whether the
peel-1 selection is used, we have advocated for the inclusion of all three red fluorescent markers expressed in neurons (Prab-3), body-wall muscle (Pmyo-3), and pharynx (Pmyo-2) because a single or even two markers were inefficient at avoiding false positives. These three selection markers are widely used and have been requested more than 300 times each from Addgene. However, adding three fluorescent markers to every injection mix is increasingly inconvenient as the mixes also contain plasmids encoding enzymes (Mos1 transposase or Cas9), sgRNAs, repair templates, and sometimes additional markers. Pharyngeal expression of the Pmyo-2 fluorophore is the easiest to identify on a fluorescence dissection microscope due to early embryonic expression and brightness, but the transgene is frequently toxic and is therefore not commonly injected at high concentrations. For these reasons, we sought to identify an improved co-injection marker. An ideal marker would be bright, non-toxic, expressed in a clearly identifiable tissue, and only require a single marker in the injection mix. Here we demonstrate that fluorescent transgenes containing the pan-muscular promoter from myosin light chain 1 (
mlc-1) fulfill these criteria. The
mlc-1 and the related
mlc-2 promoter may also be generally useful to identify all (body wall, pharynx, vulval, stomato-intestinal, and anal depressor) muscles or for robust expression of transgenes (e.g., optogenetic sensors).We focused on promoters from genes expected to have muscle-specific expression and manually checked their relative expression levels based on RNA-seq from the ModENCODE project (Gerstein et al. 2010). We identified two putative muscle-specific myosin light chain genes,
mlc-1 and
mlc-2, that are highly expressed from a single divergent locus. RNA expression levels based on Fragments Per Kilobase Million reads (FPKM) are comparatively high for these two genes in young adults, with 500 FPKM (
mlc-1) and 1159 FPKM (
mlc-2) compared to 230 FPKM (
unc-54), 14 FPKM (
myo-2), and 29 FPKM (
myo-3). We selected
unc-54,
mlc-1, and
mlc-2 as candidates and amplified 1.9 kb, 1.3 kb, and 1.2 kb promoters, respectively. Due to an early splice site (immediately after the start codon), Pmlc-1 also included the first six amino acids of
mlc-1. To quantify the relative expression level of the three promoters, we generated otherwise identical transgenes with a codon-optimized gfp under each promoters control and made single-copy insertions at the
ttTi5605 site using MosSCI. All promoters drove GFP expression in the body wall, stomato-intestinal, and anal depressor muscles (Figure 1A-C). However, only Punc-54 was not expressed in pharyngeal (Figure 1A) and vulval muscles (Figure 1B) in agreement with previous promoter analysis (Okkema et al. 1993). Using a Copas large-particle flow cytometer, we quantified GFP expression in young adults from a mixed population of the MosSCI strains and found that Pmlc-1:gfp resulted in the highest expression, with approximately two-fold more expression than Punc-54 (Figure 1D). The fluorescence signal matched our subjective visual impression from transgenic animals carrying extra-chromosomal arrays. We verified that the Pmlc-1 and Pmlc-2 strains harbored only single transgene insertions (Figure 1E), excluding the possibility that transgene copy-number caused the observed difference in fluorescence.