A new miniMOS tool kit capable of visualizing single copy insertion in C. elegans.

Abstract

The miniMOS technique has been widely used in the C. elegans community to generate single copy insertions. A worm is considered as a potential insertion candidate if it is resistant to G418 antibiotics and does not express a co-injected fluorescence marker. If the expression of the extrachromosomal array is very low, it is possible for a worm to be mistakenly identified as a miniMOS candidate, as this low expression level can still confer resistance to G418 without producing a detectable fluorescence signal from the co-injection marker. This may increase the workload for identifying the insertion locus in the subsequent steps. In the present study, we modified the plasmid platform for miniMOS insertion by incorporating a myo-2 promoter-driven TagRFP or a ubiquitous H2B::GFP expression cassette into the targeting vector and introducing two loxP sites flanking the selection cassettes. Based on this new miniMOS tool kit, the removable fluorescence reporters can be used to visualize the single copy insertions, greatly reducing insertion locus identification efforts. In our experience, this new platform greatly facilitates the isolation of the miniMOS mutants.