TY  - GEN
T1  - Characterizing a strong pan-muscular promoter (Pmlc-1) as a fluorescent co-injection marker to select for single-copy insertions
AU  - El Mouridi, Sonia
AU  - Peng, Yuli
AU  - Frøkjær-Jensen, Christian
DO  - 10.17912/micropub.biology.000302
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000302/
AB  - 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)(Frøkjær-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) (Frøkjær-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 (Frøkjær-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).
PY  - 2020
JO  - microPublication Biology
ER  -