Biology subjects

Titball, R. W.

Publications and source records attributed to Titball, R. W..

3 recordsLinked to original sources

bioRxiv2024

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.

bioengineering↗

bioRxiv2024

PiggyBac mediated transgenesis and CRISPR/Cas9 knockout in the greater waxmoth, Galleria mellonella

The larvae of the greater waxmoth, Galleria mellonella, are gaining prominence as a versatile non-mammalian in vivo model to study host-pathogen interactions. Their ability to be maintained at 37{degrees}C, coupled with a broad susceptibility to human pathogens and a distinct melanisation response that serves as a visual indicator for larval health, positions Galleria as a powerful resource for infection research. Despite these advantages, the lack of genetic tools, such as those available for zebrafish and fruit flies, has hindered development of the full potential of Galleria as a model organism. In this study, we describe a robust methodology for generating transgenic Galleria using the PiggyBac transposon system and for precise gene knockouts via CRISPR/Cas9 technology. These advances significantly enhance the utility of Galleria in molecular research, opening the way to its widespread use as an inexpensive and ethically compatible animal model for infection biology and beyond.

bioengineering↗

bioRxiv2020

Bacterial dormancy: a subpopulation of viable but non-culturable cells demonstrates better fitness for revival

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two key subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. The two subpopulations displayed different abilities for revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) has also revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. The proteins that were significantly up and down regulated between the different VBNC populations were determined and significantly regulated proteins were assigned into 23 functional groups, the majority being included in metabolism functional categories. A lactate dehydrogenase (lldD) protein was significantly upregulated in all subpopulations of VBNC cells and is responsible for converting lactate to pyruvate. Deletion of the lactate dehydrogenase (RIMD2210633:{Delta}lldD) gene causes the cells to enter the VBNC state significantly faster than the wild-type, and exogenously adding lactate to VBNC cells aided resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:{Delta}lldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase plays a putative key role in regulating the VBNC state.

microbiology↗