Premium
Primary screening of siRNA to determine genes involved in vascular leakage (711.3)
Author(s) -
Hoke Allison,
Jibitu Meskerem,
Gautam Aarti,
Hammamieh Rasha,
Jett Marti
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.711.3
Subject(s) - gene knockdown , small interfering rna , microbiology and biotechnology , in vitro , vascular permeability , gene , cell , biology , chemistry , transfection , biochemistry , endocrinology
Endothelial cells lining the vessels of the vasculature and the cell‐cell junctions provide the primary barrier for the passage of fluids, immune cells, and macromolecules between the bloodstream and tissues. Appropriate and dynamic regulation of this barrier is required for normal physiological processes. To identify genes implicated in vascular leakage, we combined siRNA knockdown screens with an in vitro assay that models the human endothelium by using primary human microvascular endothelial cells (HMVEC‐L) plated on a 96‐transwell collagen‐coated filter unit. To investigate genes related to vascular leakage, a primary screen of ~500 siRNAs against drugable target genes was performed and the cells were plated for 48 hours for monolayer formation. The monolayer permeability was evaluated using dextran‐FITC to detect leakage through small gaps present in the early stages of permeability induction. The viability of the siRNA knockdown HMVEC‐Ls was also determined. We have identified genes from the primary screen that lead to increased permeability when knocked down. These results need to be validated. With the application of this in vitro assay, genes relating to vascular leakage can be more fully investigated and the siRNA knockdown results will help identify genes with a potential for therapeutic roles. Grant Funding Source : Supported by the Transformational Medical Technologies program contract TMTI.DRUG.02.10.WR.023.