The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo

TLDR

RNAi has revolutionized loss‑of‑function studies in mammals, yet challenges remain for inducible and uniform gene control needed for genetic screens and in vivo experiments. The authors developed the lentiviral pINDUCER series to provide inducible RNAi in vivo. pINDUCER uses a multicistronic design that tracks viral transduction and shRNA or cDNA induction, delivering uniform, temporal, dose‑dependent, reversible control via fluorescence‑based quantification of reverse Tet‑transactivator across heterogeneous cell populations. This system allows isolation of cell populations with potent, inducible target knockdown in vitro and in vivo, enabling their use in human xenotransplantation models to investigate cancer drug targets.

Abstract

The discovery of RNAi has revolutionized loss-of-function genetic studies in mammalian systems. However, significant challenges still remain to fully exploit RNAi for mammalian genetics. For instance, genetic screens and in vivo studies could be broadly improved by methods that allow inducible and uniform gene expression control. To achieve this, we built the lentiviral pINDUCER series of expression vehicles for inducible RNAi in vivo. Using a multicistronic design, pINDUCER vehicles enable tracking of viral transduction and shRNA or cDNA induction in a broad spectrum of mammalian cell types in vivo. They achieve this uniform temporal, dose-dependent, and reversible control of gene expression across heterogenous cell populations via fluorescence-based quantification of reverse tet-transactivator expression. This feature allows isolation of cell populations that exhibit a potent, inducible target knockdown in vitro and in vivo that can be used in human xenotransplantation models to examine cancer drug targets.