Early Development and Genetic Engineering in the Lepidopteran model organism Galleria mellonella

There is a rapidly growing list of publications validating Galleria mellonella’s use as an in-vivo animal partial replacement model in the fields of infection, immunology, and inflammation. This is because Galleria mellonella larvae exhibit an easily identifiable, but qualitative, biological read-out of such challenges – they produce melanin pigment, turning the larvae from cream-coloured to black. They possess broad susceptibility to microbial pathogens, with pharmacodynamics of drug clearance showing remarkably similar patterns of drug clearance to humans. Moreover, individual larvae can be precisely dosed by injection, their maintenance is straightforward and, in contrast with competing non-mammalian systems, such as zebrafish, C. elegans and Drosophila, they can be reared at 37°C, facilitating research into both normal cellular kinetics of biological processes and host-pathogen interactions. Unlike these other model organisms however, Galleria is not currently genetically tractable and lacks detailed protocols for molecular tools or in depth knowledge about its biology. This thesis project describes work done to develop an embryonic microinjection pipeline and better the understanding of preblastodermal development for this organism. In addition, robust protocols for the insertion of new genetic material via PiggyBac mediated transposition, and gene knock-out via CRISPR/Cas9 mediated mutagenesis are described for the first time in Galleria.Defence Science and Technology Laboratory (DSTL)Defence Science and Technology Laboratory (DSTL)Defence Science and Technology Laboratory (DSTL