To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China). KEYWORDS RNAi; Drosophila; screens; phenotypes; functional genomics A striking finding from the genomic revolution and wholegenome sequencing is the amount of information missing on gene function. Although Drosophila is arguably the bestunderstood multicellular organism and a proven model system for human diseases, mutations mapped to specific genes with readily detectable phenotypes have been isolated for 15% of the .13919 annotated fly coding genes (http:// flybase.org/; FlyBase R6.06). The lack of information on the majority of genes (the "phenotype gap") suggests that researchers have been unable to either assay their roles experimentally and/or resolve issues of functional redundancy. In addition, some phenotypes may be only detected on specific diets and environments. Further, our understanding of the function of many genes for which we have some information is limited by pleiotropy, whereby an earlier function of the gene prevents analysis of later functions.The availability of in vivo RNAi has revolutionized the ability of Drosophila researchers to disrupt the activity of single genes with spatial and temporal resolution (Dietzl et al. 2007; see review by Perrimon et al. 2010), and thus address the phenotype gap. Motivated by the power of the approach and the needs of the community, three large-scale efforts, the Vienna Drosophila RNAi Center (VDRC, http:// stockcenter.vdrc.at/control/main), the National Institute of Genetics (NIG, http://www.shigen.nig.ac.jp/fly/nigfly/index.jsp), and the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) (http://www.flyrnai.org/TRiP-HOME. html) have over the years generated large numbers of RNAi lines that aim to cover all Drosophila genes. These resources are proving invaluable to address a myriad of questions in various biological and biomedical fields including but not limite...

Infection by carcinogenic human papillomaviruses (HPV) results in precancers [cervical intraepithelial neoplasia (CIN)] and cancers near the ectoendocervical squamocolumnar (SC) junction of the cervix. However, the specific cells targeted by HPV have not been identified and the cellular origin of cervical cancer remains elusive. In this study, we uncovered a discrete population of SC junctional cells with unique morphology and gene-expression profile. We also demonstrated that the selected junctional biomarkers were expressed by a high percentage of high-grade CIN and cervical cancers associated with carcinogenic HPVs but rarely in ectocervical/transformation zone CINs or those associated with noncarcinogenic HPVs. That the original SC junction immunophenotype was not regenerated at new SC junctions following excision, not induced by expression of viral oncoproteins in foreskin keratinocytes, and not seen in HPV-related precursors of the vagina, vulva, and penis further support the notion that junctional cells are the source of cervical cancer. Taken together, our findings suggest that carcinogenic HPV-related CINs and cervical cancers are linked to a small, discrete cell population that localizes to the SC junction of the cervix, expresses a unique gene expression signature, and is not regenerated after excision. The findings in this study uncover a potential target for cervical cancer prevention, provide insight into the risk assessment of cervical lesions, and establish a model for elucidating the pathway to cervical cancer following carcinogenic HPV infection.

Mutations in the alanine-glyoxylate amino transferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. We generated a null mutant mouse by targeted mutagenesis of the homologous gene, Agxt, in embryonic stem cells. Mutant mice developed normally, and they exhibited hyperoxaluria and crystalluria. Approximately half of the male mice in mixed genetic background developed calcium oxalate urinary stones. Severe nephrocalcinosis and renal failure developed after enhancement of oxalate production by ethylene glycol administration. Hepatic expression of human AGT1, the protein encoded by AGXT, by adenoviral vector-mediated gene transfer in Agxt ؊/؊ mice normalized urinary oxalate excretion and prevented oxalate crystalluria. Subcellular fractionation and immunofluorescence studies revealed that, as in the human liver, the expressed wild-type human AGT1 was predominantly localized in mouse hepatocellular peroxisomes, whereas the most common mutant form of AGT1 (G170R) was localized predominantly in the mitochondria.gene therapy ͉ knockout mouse ͉ oxalate ͉ urolithiasis ͉ nephrocalcinosis

Contractile injection systems (CISs) are cell-puncturing nanodevices that share ancestry with contractile tail bacteriophages. Photorhabdus virulence cassette (PVC) represents one group of extracellular CISs that are present in both bacteria and archaea. Here, we report the cryo-EM structure of an intact PVC from P. asymbiotica. This over 10-MDa device resembles a simplified T4 phage tail, containing a hexagonal baseplate complex with six fibers and a capped 117-nanometer sheath-tube trunk. One distinct feature of the PVC is the presence of three variants for both tube and sheath proteins, indicating a functional specialization of them during evolution. The terminal hexameric cap docks onto the topmost layer of the inner tube and locks the outer sheath in pre-contraction state with six stretching arms. Our results on the PVC provide a framework for understanding the general mechanism of widespread CISs and pave the way for using them as delivery tools in biological or therapeutic applications.

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