Turner, W. D. | bioRxiv Science

bioRxiv2024

Multisite Assembly of Gateway Induced Clones (MAGIC): a flexible cloning toolbox withdiverse applications in vertebrate model systems.

Here we present the Multisite Assembly of Gateway Induced Clones (MAGIC) system, which harnesses site-specific recombination-based cloning via Gateway technology for rapid, modular assembly of between 1 and 3 "Entry" vector components, all into a fourth, standard high copy "Destination" plasmid backbone. The MAGIC toolkit spans a range of in vitro and in vivo uses, from directing tunable gene expression, to driving simultaneous expression of microRNAs and fluorescent reporters, to enabling site-specific recombinase-dependent gene expression. All MAGIC system components are directly compatible with existing multisite gateway Tol2 systems currently used in zebrafish, as well as existing eukaryotic cell culture expression Destination plasmids, and available mammalian lentiviral and adenoviral Destination vectors, allowing rapid cross-species experimentation. Moreover, herein we describe novel vectors with flanking piggyBac transposon elements for stable genomic integration in vitro or in vivo when used with piggyBac transposase. Collectively, the MAGIC system facilitates transgenesis in cultured mammalian cells, electroporated mouse and chick embryos, as well as in injected zebrafish embryos, enabling the rapid generation of innovative DNA constructs for biological research due to a shared, common plasmid platform.

molecular biology↗

bioRxiv2022

EZ Clear for simple, rapid, and robust mouse whole organ clearing

Tissue clearing for whole organ cell profiling has revolutionized biology and imaging for exploration of organs in three-dimensional space without compromising tissue architecture. But complicated, laborious procedures, or expensive equipment, as well as the use of hazardous, organic solvents prevents the widespread adoption of these methods. Here we report a simple and rapid tissue clearing method, EZ Clear, that can clear whole adult mouse organs in 48 hours in just three simple steps. Samples stay at room temperature and remain hydrated throughout the clearing process, preserving endogenous and synthetic fluorescence, without altering sample size. After wholemount clearing and imaging, EZ Cleared samples can be further processed for downstream embedding and cryosectioning followed by standard histology or immunostaining, without loss of endogenous or synthetic fluorescence signal. Overall, the simplicity, speed, and flexibility of EZ Clear make it easy to adopt and apply to diverse approaches in biomedical research.

developmental biology↗