Large transgene arrays cause aberrant transcription and synthetic molting defects

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Abstract
Multicopy transgene arrays remain widely used in Caenorhabditis elegans research. It is usually assumed that they behave neutrally, not impacting the phenotype under investigation. Here, we reveal that a previously reported heterochronic extra-molt phenotype associated with myrf-1(mg412) depends on the presence of the integrated molting reporter mgIs49. Nanopore long-read sequencing shows that mgIs49 is a massive 8.8-Mb insertion—around 50% of the size of its host chromosome—which disrupts the prmt-9 gene. Both mgIs49 and another array, maIs105, cause dysregulation of the transcriptome and accumulation of reads mapping in sense-direction to transgene promoter sequences. We identify additional arrays exceeding 4 Mb and show that variable molting defects occur across different transgenic lines when combined with myrf-1(mg412), implicating array size or composition in the synthetic phenotype. Our results underscore the necessity of replacing multicopy reporters in developmental studies with single-copy insertions or endogenous tagging whenever possible.
Funding Information
  • Office of Research Infrastructure Programs (P40 OD010440)
  • Friedrich Miescher Institute for Biomedical Research
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (#310030_207470)
  • European Research Council (741269)
  • Novartis Research Foundation

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