Efficient gene delivery to primary alveolar epithelial cells by nucleofection

Abstract

Primary alveolar epithelial cells play a pivotal role in lung research, particularly when focusing on gas exchange, barrier function, and transepithelial transport processes. However, efficient transfection of primary alveolar epithelial cells continues to be a major challenge. In the present study, we applied nucleofection, a novel method of gene and oligonucleotide delivery to the nucleus of cells by electroporation, to achieve highly efficient transfection of primary alveolar epithelial type II (ATII) cells. To quantify the amount of ATII cells effectively transfected, we applied a plasmid expressing GFP and assessed the amount of GFP-expressing cells by flow cytometry. Analysis of the nucleofected ATII cells revealed a concentration-dependent transfection efficiency of up to 50% when using 3-8 μg plasmid DNA without affecting cell viability. Nucleofection of cultured A549 and H441 cells yielded similar transfection rates. Importantly, nucleofection of ATII cells did not interfere with the integrity of ATII monolayers even with use of relatively high concentrations of plasmid DNA. In subsequent studies, we also efficiently delivered small interfering RNAs to ATII cells by nucleofection, thereby silencing Akt and the multiligand receptor megalin, which has been recently shown to play a key role in removal of excess protein from the alveolar space, and effectively inhibited megalin-driven uptake and transcellular transport of albumin in ATII cells. Thus we report successful transfection of primary rat alveolar epithelial cells with both plasmids and oligonucleotides via nucleofection with high viability and consistently good transfection rates without impairing key physiological properties of the cells.