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 ...

Tumors exhibit numerous recurrent hemizygous focal deletions that contain no known tumor suppressors and are poorly understood. To investigate whether these regions contribute to tumorigenesis, we searched genetically for genes with cancer-relevant properties within these hemizygous deletions. We identified STOP and GO genes, which negatively and positively regulate proliferation, respectively. STOP genes include many known tumor suppressors, whereas GO genes are enriched for essential genes. Analysis of their chromosomal distribution revealed that recurring deletions preferentially over represent STOP genes and under represent GO genes. We propose a hypothesis called the cancer gene island model whereby gene islands encompassing high densities of STOP genes and low densities of GO genes are hemizygously deleted to maximize proliferative fitness through cumulative haploinsufficiencies. Because hundreds to thousands of genes are hemizygously deleted per tumor, this mechanism may help drive tumorigenesis across many cancer types.

A variety of tumor-suppressor mechanisms exist to promote genome integrity and organismal survival. One such mechanism is cellular senescence. In response to replicative aging, DNA damage, and oncogenic stimuli, the p53 and Rb pathways are activated to prevent the proliferation of damaged cells by inducing senescence or apoptosis. We have performed a loss-of-function genetic screen in primary human cells to identify components of the senescence machinery. Here we describe BRD7 and BAF180 as unique regulators of replicative senescence in human cells. Both regulate p53 transcriptional activity toward a subset of its target genes required for replicative and oncogenic stress senescence induction, and BRD7 physically interacts with p53. BRD7 is a deletion target in human cancer, suggesting that loss of BRD7 may provide an additional mechanism to antagonize p53 function in cancer cells.