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

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 tettransactivator 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.lentivirus | vector L oss-of-function studies represent a powerful means by which to gain insight into the molecular mechanisms behind complex biological processes. Until recently, these studies were practical for only a small set of genetically tractable model organisms (1, 2). The discovery of gene silencing through RNAi has made it possible to carry out loss-of-function studies in mammals. RNAi has transformed the way in which gene function can be investigated and has quickly become a versatile tool for a wide range of applications, including reverse genetics, highthroughput screens, and therapeutics (3). The application of RNAi technology both in vitro and in vivo has tremendous potential to further our knowledge of the molecular mechanisms that underpin both normal biology and human disease.To achieve efficient and long-term gene silencing, we have previously generated the retroviral-encoded microRNA-based shRNA PRIME (potent RNA interference using microRNA expression) vectors that are effective in suppressing expression of their intended gene targets from single proviral integrations (4). These vectors use the miR30 backbone and are expressed from RNA polymerase II promoters, which allows for exquisite regulation of shRNA expression. We have further generated genomewide shRNA libraries in these vectors that cover large numbers of mammalian transcripts for systematic study of gene function (5, 6). These shRNA libraries, as well as shRNA libraries generated by other laboratories (7,8), have been used to perform genetic screens in mammalian cell culture models for a variety of phenotypes, including cell transformation, synthetic lethal interactions, and resistance to chemotherapeutic treatments (9-15).Despite the success demonstrated by the above constitutive shRNA vectors, an inducible shRNA system would have obvious advantages in many experimental settings. First, the inducible shRNA system allows for the study of essential genes. Because constitutively expressed shRNAs targeting essential genes will be ...

Knockdown of HMGN5 Expression by RNA Interference Induces Cell Cycle Arrest in Human Lung Cancer Cells

“…Current lentiviral technology has facilitated a wide range of loss-and gain-of-function genetic studies in mammalian systems (Meerbrey et al, 2011). In the present study, we employed the lentivirus-mediated siRNA to knockdown HMGN5 expression and investigated the effects of HMGN5 silencing on the cell proliferation and cell cycle progression in human lung cancer cell lines A549 and H1299.…”

AIM2 inflammasome is activated by pharmacological disruption of nuclear envelope integrity

“…Lentiviral pINDUCER21 plasmid was obtained from Steve Elledge, Harvard Medical School, Boston (74). A human Flag-tagged AIM2 or TREX1 construct were cloned into the pENTR 1A dual selection vector (Invitrogen) and then cloned in the pINDUCER21, a GFP tet-inducible lentiviral vector plasmid.…”

Common nonmutationalNOTCH1activation in chronic lymphocytic leukemia

“…S3A), we sought to investigate NOTCH1-dependent programs using an inducible lentiviral system expressing either an HA-tagged constitutively active form of NOTCH1 (ICN1-HA) or control eGFP upon doxycycline addition (Fig. S3B) (30). Induction of NOTCH1 signaling was demonstrated by HA immunoblot and quantitative RT-PCR (qRT-PCR) for DTX1, a well-established NOTCH1-direct target gene (11) (Fig.…”