siRNA-mediated protein knockdown in precision-cut lung slices

Abstract

Small interfering RNA (siRNA) can induce RNA interference, which leads to the knockdown of messenger RNA (mRNA) and protein. As a result, siRNA is often used in vitro and in vivo to unravel the function of genes and as a therapeutic agent to disrupt excessive expression of disease-related genes. However, there is a large gap between in vitro and in vivo models in terms of simplicity, flexibility, throughput, and translatability. This gap could be bridged by using precision-cut tissue slices, which represent viable explants prepared from animal or human tissue that can be cultured ex vivo. Previously, we demonstrated that self-deliverable siRNA (Accell siRNA) induced significant mRNA knockdown in lung slices. The goal of this study, however, was to investigate whether Accell siRNA also induced protein knockdown in murine lung slices. Slices were incubated for up to 96 h with no siRNA (untransfected), non-targeting siRNA (control), or gene-targeting siRNA (Gapdh, Ppib, Serpinh1, and Bcl2l1). Overall, untransfected and transfected slices remained viable during an incubation of 96 h. In addition, gene-targeting siRNAs induced not only significant and specific mRNA knockdown but also protein knockdown. Finally, protein knockdown of fibrogenesis-related targets (Ppib, Serpinh1, and Bcl2l1) was shown to influence fibrogenesis on mRNA level, thereby demonstrating this model its utility in functional genomics and translational research.

Keywords: Explant culture; Gene silencing; Organotypic culture; Precision-cut tissue slices; Short interfering RNA; Transfection.