DOI: 10.1101/471854Nov 16, 2018Paper

Validation of a Miniaturized Permeability Assay Compatible with CRISPR-Mediated Genome-Wide Screen

BioRxiv : the Preprint Server for Biology
Claire SimonneauArie Horowitz

Abstract

The impermeability of the luminal endothelial cell monolayer is crucial for the normal performance of the vascular and lymphatic systems. A key to this function is the integrity of the monolayer intercellular junctions. The known repertoire of junction-regulating genes is incomplete. Current permeability assays are incompatible with high-throughput genome-wide screens that could identify these genes. To overcome these limitations, we designed a new permeability assay that consists of cell monolayers grown on ~150 μm microcarriers. Each microcarrier functions as a miniature individual assay of permeability (MAP). We demonstrate that false-positive results can be minimized, and that MAP sensitivity to thrombin-induced increase in monolayer permeability is similar to the sensitivity of the measurement of impedance. We validate the assay by showing that the expression of single guide RNAs (sgRNAs) that target genes encoding known thrombin signaling proteins blocks effectively thrombin-induced junction disassembly, and that MAPs carrying such cells can be separated effectively by a fluorescent probe from those that carry cells expressing non-targeting sgRNAs. These results indicate that MAPs are suitable for high-throughput experime...Continue Reading

Related Concepts

Blood Vessel
Fluorescent Probes
Genes
Genome
Microtubule-Associated Proteins
RNA
Thrombin
Electric Impedance
Endothelial Cells
Thrombin Activity

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