PT  - JOURNAL ARTICLE
AU  - Claire Simonneau
AU  - Junning Yang
AU  - Xianguo Kong
AU  - Robert Kilker
AU  - Leonard Edelstein
AU  - Paolo Fortina
AU  - Eric Londin
AU  - Arie Horowitz
TI  - Validation of a Miniaturized Permeability Assay Compatible with CRISPR-Mediated Genome-Wide Screen
AID  - 10.1101/471854
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 471854
4099  - http://biorxiv.org/content/early/2019/07/18/471854.short
4100  - http://biorxiv.org/content/early/2019/07/18/471854.full
AB  - 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’s 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 experimentation and for genome-wide screens for genes that mediate the disruptive effect of thrombin on endothelial cell junctions.