RNA interference (RNAi) was first recognized in Caenorhabditis elegans as a biological response to exogenous double-stranded RNA (dsRNA), which induces sequence-specific gene silencing. RNAi represents a conserved regulatory motif, which is present in a wide range of eukaryotic organisms. Recently, we and others have shown that endogenously encoded triggers of gene silencing act through elements of the RNAi machinery to regulate the expression of protein-coding genes. These small temporal RNAs (stRNAs) are transcribed as short hairpin precursors (∼70 nt), processed into active, 21-nt RNAs by Dicer, and recognize target mRNAs via base-pairing interactions. Here, we show that short hairpin RNAs (shRNAs) can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or can be transcribed from RNA polymerase III promoters in vivo, thus permitting the construction of continuous cell lines or transgenic animals in which RNAi enforces stable and heritable gene silencing.

Loss-of-function phenotypes often hold the key to understanding the connections and biological functions of biochemical pathways. We and others previously constructed libraries of short hairpin RNAs that allow systematic analysis of RNA interference-induced phenotypes in mammalian cells. Here we report the construction and validation of second-generation short hairpin RNA expression libraries designed using an increased knowledge of RNA interference biochemistry. These constructs include silencing triggers designed to mimic a natural microRNA primary transcript, and each target sequence was selected on the basis of thermodynamic criteria for optimal small RNA performance. Biochemical and phenotypic assays indicate that the new libraries are substantially improved over first-generation reagents. We generated large-scale-arrayed, sequence-verified libraries comprising more than 140,000 second-generation short hairpin RNA expression plasmids, covering a substantial fraction of all predicted genes in the human and mouse genomes. These libraries are available to the scientific community.

The establishment of the primitive streak and its derivative germ layers, mesoderm and endoderm, are prerequisite steps in the formation of many tissues. To model these developmental stages in vitro, an ES cell line was established that expresses CD4 from the foxa2 locus in addition to GFP from the brachyury locus. A GFP-Bry ؉ population expressing variable levels of CD4-Foxa2 developed upon differentiation of this ES cell line. Analysis of gene-expression patterns and developmental potential revealed that the CD4-Foxa2 hi GFP-Bry ؉ population displays characteristics of the anterior primitive streak, whereas the CD4-Foxa2 lo GFP-Bry ؉ cells resemble the posterior streak. Using this model, we were able to demonstrate that Wnt and TGF-␤͞nodal͞activin signaling simultaneously were required for the generation of the CD4-Foxa2 ؉ GFP-Bry ؉ population. Wnt or low levels of activin-induced a posterior primitive streak population, whereas high levels of activin resulted in an anterior streak fate. Finally, sustained activin signaling was found to stimulate endoderm commitment from the CD4-Foxa2 ؉ GFP-Bry ؉ ES cell population. These findings demonstrate that the early developmental events involved in germ-layer induction in the embryo are recapitulated in the ES cell model and uncover insights into the signaling pathways involved in the establishment of mesoderm and endoderm.gastrulation ͉ mesoderm ͉ endoderm T o be able to fully exploit the potential of embryonic stem (ES) cells in basic biology and regenerative medicine, it is essential to recapitulate the critical lineage induction events of the early embryo in this model system. One of the earliest commitment steps in embryogenesis is the formation of the primary germ layers, mesoderm, endoderm, and ectoderm, the founder populations of all somatic cell types in the body (1). Mesoderm and endoderm are formed during gastrulation, a process that involves the movement of undifferentiated epiblast cells through a structure called the primitive streak (PS). The PS can be subdivided into distinct regions, posterior, middle, and anterior, based on lineage development and gene-expression patterns. With respect to developmental potential, distinct subpopulations of mesoderm are induced in each of the different regions, whereas definitive endoderm forms from the anterior PS of the early-PS and mid-PS stages (2-5). These observations suggest that the different regions of the PS constitute different signaling environments that are responsible for the induction of specific lineages.To address questions regarding early commitment by using the ES cell model, it is essential to be able to track and isolate germ-layer populations from the differentiation cultures. By using an ES cell line with the green fluorescence protein (GFP) targeted to the PS and early mesodermal-specific gene brachyury (6) (GFPBry ES cells), it has been possible to quantify mesoderm induction and isolate and characterize different mesodermal populations (7,8). In addition, brachyury-expressing cells were found ...

Gene silencing by RNA interference (RNAi) in mammalian cells using small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) has become a valuable genetic tool. Here, we report the construction and application of a shRNA expression library targeting 9,610 human and 5,563 mouse genes. This library is presently composed of about 28,000 sequence-verified shRNA expression cassettes contained within multi-functional vectors, which permit shRNA cassettes to be packaged in retroviruses, tracked in mixed cell populations by means of DNA 'bar codes', and shuttled to customized vectors by bacterial mating. In order to validate the library, we used a genetic screen designed to report defects in human proteasome function. Our results suggest that our large-scale RNAi library can be used in specific, genetic applications in mammals, and will become a valuable resource for gene analysis and discovery.

SummaryFactors that sustain self-renewal of mouse embryonic stem cells (ESCs) are well described. In contrast, the machinery regulating exit from pluripotency is ill defined. In a large-scale small interfering RNA (siRNA) screen, we found that knockdown of the tumor suppressors Folliculin (Flcn) and Tsc2 prevent ESC commitment. Tsc2 lies upstream of mammalian target of rapamycin (mTOR), whereas Flcn acts downstream and in parallel. Flcn with its interaction partners Fnip1 and Fnip2 drives differentiation by restricting nuclear localization and activity of the bHLH transcription factor Tfe3. Conversely, enforced nuclear Tfe3 enables ESCs to withstand differentiation conditions. Genome-wide location and functional analyses showed that Tfe3 directly integrates into the pluripotency circuitry through transcriptional regulation of Esrrb. These findings identify a cell-intrinsic rheostat for destabilizing ground-state pluripotency to allow lineage commitment. Congruently, stage-specific subcellular relocalization of Tfe3 suggests that Flcn-Fnip1/2 contributes to developmental progression of the pluripotent epiblast in vivo.