A microinjection protocol for the greater waxworm moth, Galleria mellonella

A limitation to the non-vertebrate 3Rs model Galleria mellonella has been the lack of genetic toolkit. A common requirement for genetic tractability is a method to introduce exogenous material to the unicellular embryo, the most common of which is microinjection. This short article describes a detailed method for rearing Galleria mellonella to collect large amounts of staged embryos and to dechorionate and microinject embryos with limited mortality. Research HighlightsO_ST_ABSScientific BenefitsC_ST_ABSMicroinjection allows the introduction of a wide variety of substances, such as DNA, RNA or drugs into Galleria embryos, providing the technology needed for genetic engineering, gene editing and functional studies in this important model organism. 3Rs BenefitsGalleria is being increasingly used as a partial animal replacement model, especially in the field of infection biology. However, uptake has been limited by the lack of genetic and molecular tools. This protocol takes a step towards removing these barriers by providing a means to introduce substances that can create transgenic or genetically engineered Galleria. Practical BenefitsProtocol for injecting substances into Galleria, using for the most part easily accessible equipment. Current ApplicationsGenerating stable transgenic and gene-edited Galleria lines. Potential ApplicationsAny technique requiring the introduction of substances to Galleria embryos. This includes applying existing techniques such as pBac-mediated transgenesis or CRISPR/Cas-based gene-editing to this organism,in order to generate engineered strains of Galleria. It could also include injection of synthetic mRNAs encoding proteins fused to fluorescent genes (such as GFP) in order to visualise their dynamics in living embryos; and the injection of drugs that perturb particular cell or developmental processes in order to learn more about early Galleria development.