Abstract
Studies on Caenorhabditis elegans would benefit from the introduction of new selectable markers to allow more complex types of experiments to be conducted with this model animal. We established a new antibiotic selection marker for C. elegans transformation based on nourseothricin (NTC) and its resistance-encoding gene, streptothricin-acetyl transferase 2 (Sat2). NTC was able to efficiently prevent worm development at very low concentrations, and the worms expressing Sat2 were able to survive on the selection plates without any developmental defects. Using CRISPR/Cas9 and NTC selection, we were able to easily insert a 13-kb expression cassette into a defined locus in C. elegans. The structure and spectrum of NTC differs from other antibiotics like hygromycin B and geneticin, making it possible to use NTC alongside them. Indeed, we confirmed NTC-sat2 selection could work with the hygromycin B selection system simultaneously. Thus, the new NTC–Sat2 system can act as a useful dominant marker for gene transfer and genome editing in C. elegans.
Highlights
The nematode Caenorhabditis elegans has been used to study a broad spectrum of biological problems in many fields
In the search for alternative antibiotics and antibiotic resistance genes that can be applied to C. elegans genetics, we found that NTC can kill wild-type worms efficiently (Fig 1)
The NTC–streptothricin-acetyl transferase 2 (Sat2) selection system was established as a dominant selection tool for C. elegans transgenesis and genome editing
Summary
The nematode Caenorhabditis elegans has been used to study a broad spectrum of biological problems in many fields. Transgene expression in C. elegans is accomplished by microinjection of DNA into the gonads [1]. Several selectable genetic markers have been used [2]. Transformed animals are identified by rescuing non-lethal mutations [3, 4] via the expression of a dominant phenotype marker [1] or a fluorescent marker [5, 6]. C. elegans can inherit DNA molecules injected into it as extrachromosomal arrays [7]. Because extrachromosomal arrays tend to be inherited in only a subset of the progeny worms, it is difficult to grow large populations of transgenic strains. To maintain the extrachromosomal array-carrying strains, researchers need to pick up individual worms one by one using readily distinguishable markers [1, 7]
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