Biology subjects

Largoza, G.

Publications and source records attributed to Largoza, G..

3 recordsLinked to original sources

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↗

bioRxiv2024

MEK signaling represents a viable therapeutic vulnerability of KRAS-driven somatic brain arteriovenous malformations

Brain arteriovenous malformations (bAVMs) are direct connections between arteries and veins that remodel into a complex nidus susceptible to rupture and hemorrhage. Most sporadic bAVMs feature somatic activating mutations within KRAS, and endothelial-specific expression of the constitutively active variant KRASG12D models sporadic bAVM in mice. By leveraging 3D-based micro-CT imaging, we demonstrate that KRASG12D-driven bAVMs arise in stereotypical anatomical locations within the murine brain, which coincide with high endogenous Kras expression. We extend these analyses to show that a distinct variant, KRASG12C, also generates bAVMs in predictable locations. Analysis of 15,000 human patients revealed that, similar to murine models, bAVMs preferentially occur in distinct regions of the adult brain. Furthermore, bAVM location correlates with hemorrhagic frequency. Quantification of 3D imaging revealed that G12D and G12C alter vessel density, tortuosity, and diameter within the mouse brain. Notably, aged G12D mice feature increased lethality, as well as impaired cognition and motor function. Critically, we show that pharmacological blockade of the downstream kinase, MEK, after lesion formation ameliorates KRASG12D-driven changes in the murine cerebrovasculature and may also impede bAVM progression in human pediatric patients. Collectively, these data show that distinct KRAS variants drive bAVMs in similar patterns and suggest MEK inhibition represents a non-surgical alternative therapy for sporadic bAVM.

molecular biology↗

bioRxiv2021

Defining the Transcriptional and Epigenetic Basis of Organotypic Endothelial Diversity in the Developing and Adult Mouse

Significant phenotypic differences exist between the vascular endothelium of different organs, including cell-cell junctions, paracellular fluid transport, shape, and mural cell coverage. These organ-specific morphological features ultimately manifest as different functional capacities, as demonstrated by the dramatic differences in capillary permeability between the leaky vessels of the liver compared to the almost impermeable vasculature found in the brain. While these morphological and functional differences have been long appreciated, the molecular basis of endothelial organ specialization remains unclear. To determine the epigenetic and transcriptional mechanisms driving this functional heterogeneity, we profiled accessible chromatin, as well as gene expression, in six different organs, across three distinct time points, during murine development and in adulthood. After identifying both common, and organ-specific DNA motif usage and transcriptional signatures, we then focused our studies on the endothelium of the central nervous system. Using single cell RNA-seq, we identified key gene regulatory networks governing brain blood vessel maturation, including TCF/LEF and FOX transcription factors. Critically, these unique regulatory regions and gene expression signatures are evolutionarily conserved in humans. Collectively, this work provides a valuable resource for identifying the transcriptional regulators controlling organ-specific endothelial specialization and provides novel insight into the gene regulatory networks governing the maturation and maintenance of the cerebrovasculature.

developmental biology↗