Housden, A. | Search bioRxiv

bioRxiv2024

Enhanced methods for genetic assays in Drosophila cells

Genetic assays are an invaluable tool for both fundamental biological research and translational applications. Variable Dose Analysis (VDA) is an RNAi-based method for cell-based genetic assays that offers several advantages over approaches such as CRISPR and other RNAi-based methods including improved data quality (signal-to-noise ratio) and the ability to study essential genes at sub-lethal knockdown efficiency. Here we report the development of three new variants of the VDA method called high-throughput VDA (htVDA), VDA-plus and pooled-VDA. htVDA requires 10-fold reduced reagent volumes and takes advantage of liquid handling automation to allow higher throughput screens to be performed while maintaining high data quality. VDA-plus is a modified version of VDA that further improves data quality by 4.5-fold compared to standard VDA to allow highly sensitive detection of weak phenotypes. Finally, Pooled VDA allows greatly increased throughput by analysing multiple gene knockdowns in a single population of cells. Together, these new methods enhance the toolbox available for genetic assays, which will prove valuable in both high-and low-throughput applications. In particular, the low noise and ability of VDA to study essential genes at sub-lethal knockdown levels will support identification of novel drug-targets, among which essential genes are often enriched. While these tools have been developed in Drosophila cells, the underlying principles are transferrable to any cell culture system.

genetics↗

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↗