Basic Steps for Developing Genetic Technologies in a Non-Model Organism

Transformation-based applications, such as transposon tagging, enhancer trapping, and genome-wide mutagenesis, are powerful technologies that have greatly facilitated the genetic dissection of model species such as Drosophila melanogaster. However, the vast majority of economically-important insects are not model organisms, thus posing the challenge of how to extended these technologies to new species. Here we use our work with the western corn rootworm (WCR), a major pest of maize, as a guide for the basic steps required for adapting genetic technologies to a non-model pest. For us, the first step in this process was the development of a small-scale rearing system for WCR, one specifically designed to enable efficient phenotypic screening of offspring from single-pair matings. The next steps involved obtaining high-quality precellular embryos, determining optimal microinjection conditions and assessing available marker genes. Because both Minos and piggyBac are known to work in another coleopteran, the red flour beetle, Tribolium castaneum, we chose to produce transgenic WCR using a 3xP3-DsRed marked Minos element, which also carried an hsp70-driven piggyBac transposase gene. This choice was motivated by the desire to increase downstream transformation efficiencies. For example, microinjection of marked-piggyBac elements into embryos that already express piggyBac transposase can result in transformation rates that are 10-fold higher than those obtained by standard coinjection of plasmid DNAs into wild-type embryos. We will also discuss challenges faced when using eye-specific fluorescent markers, and how we used CRISPR/Cas9 to overcome these. We expect similar strategies can be used to extend genetic technologies to additional non-model organisms.