Over the last 3.5 years, I have pioneered transgenic technology in Galleria, generating a microinjection pipeline that has resulted in not only the world's first transgenic Galleria but also the first strains carrying CRISPR mediated gene knockouts. During this 24-month Fellowship, these methodologies will be employed and further built upon to increase the suite of genetic tools available for this organism. A strain expressing germline specific Cas9 will be generated and used to develop and optimise CRISPR based gene knock-in techniques via both homology and end-joining repair mechanisms. These precise gene editing techniques will then be used to insert fluorescent reporters in frame with the endogenous coding sequences. Two proteins known to be involved in Galleria's humoral immune response to both bacterial and fungal infection will be tagged: hemolin and apolipophorin III. These proteins have previously been shown to be differentially expressed post infection and are known to both be involved in pattern recognition, opsonisation and phagocytosis of microbial pathogens.
Once these strains have been created, they will be used for in vivo infection assays by injecting larvae with variable doses of immunogenic substances. The qualitative and quantitative data on both localised and total larval fluorescence obtained over the time course of the experiments will provide insight not only as to the kinetics of these tagged proteins but also their suitability as reporters for larval health in automated high throughput screening systems. The combination of both these new tools and strains will provide a lasting impact in not only Galleria's ability to directly replace rodents in infection studies and drug screens but also to its applications in a wide variety of fields.