Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro

Abstract

Ramachandran S, Krishnamurthy S, Jacobi AM, WohlfordLenane C, Behlke MA, Davidson BL, McCray PB, Jr. Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro. Am J Physiol Lung Cell Mol Physiol 305: L23-L32, 2013. First published April 26, 2013 doi:10.1152/ajplung.00426.2012.-Polarized and pseudostratified primary airway epithelia present barriers that significantly reduce their transfection efficiency and the efficacy of RNA interference oligonucleotides. This creates an impediment in studies of the airway epithelium, diminishing the utility of loss-of-function as a research tool. Here we outline methods to introduce RNAi oligonucleotides into primary human and porcine airway epithelia grown at an air-liquid interface and difficult-totransfect transformed epithelial cell lines grown on plastic. At the time of plating, we reverse transfect small-interfering RNA (siRNA), Dicer-substrate siRNA, or microRNA oligonucleotides into cells by use of lipid or peptide transfection reagents. Using this approach we achieve significant knockdown in vitro of hypoxanthine-guanine phosphoribosyltransferase, IL-8, and CFTR expression at the mRNA and protein levels in 1-3 days. We also attain significant reduction of secreted IL-8 in polarized primary pig airway epithelia 3 days posttransfection and inhibition of CFTR-mediated Cl Ϫ conductance in polarized air-liquid interface cultures of human airway epithelia 2 wk posttransfection. These results highlight an efficient means to deliver RNA interference reagents to airway epithelial cells and achieve significant knockdown of target gene expression and function. The ability to reliably conduct loss-of-function assays in polarized primary airway epithelia offers benefits to research in studies of epithelial cell homeostasis, candidate gene function, gene-based therapeutics, microRNA biology, and targeting the replication of respiratory viruses. primary airway epithelium; transfection; gene silencing; RNA interference; siRNA; air-liquid interface; porcine airway epithelial cells SMALL INTERFERING RNAs (siRNA) mediate posttranscriptional inhibition of targeted genes, offering a novel approach to achieve specific silencing in the airway epithelium. These approaches have broad applications for studies of cell biology, disease pathogenesis, and therapeutics. Primary cultures of airway epithelia have emerged as a powerful model to study epithelial cell biology and the impact of diseases on tissue function (18,22,39). Cells grown at the air-liquid interface (ALI) form a polarized, pseudostratified columnar epithelium that closely resembles the morphology of the in vivo surface epithelium of the conducting airways (2,18,24,33,63,64), providing an opportunity to study cell biology, disease pathogenesis, and treatments (66).Histological studies have shown that ALI cultures of primary airways grown for 4 wk or more form a well-differentiated epithelium resembling the in vivo architecture (12, 56). However, another study has observed that airway epithelia...

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