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Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins.

The controlled overexpression or knockdown of gene expression in primary organoid cultures of mouse endodermal epithelia is described. This should enable ex vivo studies of mammalian gene function. The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive and time-consuming. We present here a method based on Cre recombinase–inducible retrovirus vectors that allows the conditional manipulation of gene expression in primary mouse organoid culture systems.

Conditional expression of hairpin constructs in Drosophila is a powerful method to disrupt the activity of single genes with a spatial and temporal resolution that is impossible, or exceedingly difficult, using classical genetic methods. We previously described a method (Ni  et al. 2008) whereby RNAi constructs are targeted into the genome by the phiC31-mediated integration approach using Vermilion-AttB-Loxp-Intron-UAS-MCS (VALIUM), a vector that contains vermilion as a selectable marker, an attB sequence to allow for phiC31-targeted integration at genomic attP landing sites, two pentamers of UAS, the hsp70 core promoter, a multiple cloning site, and two introns. As the level of gene activity knockdown associated with transgenic RNAi depends on the level of expression of the hairpin constructs, we generated a number of derivatives of our initial vector, called the “VALIUM” series, to improve the efficiency of the method. Here, we report the results from the systematic analysis of these derivatives and characterize VALIUM10 as the most optimal vector of this series. A critical feature of VALIUM10 is the presence of gypsy insulator sequences that boost dramatically the level of knockdown. We document the efficacy of VALIUM as a vector to analyze the phenotype of genes expressed in the nervous system and have generated a library of 2282 constructs targeting 2043 genes that will be particularly useful for studies of the nervous system as they target, in particular, transcription factors, ion channels, and transporters.

Here we describe “Supernova” series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood. Sparseness levels are adjustable. Labeled cell-specific gene knockout was accomplished by introducing Cre/loxP-based Supernova vectors into floxed mice. Furthermore, by combining with RNAi, TALEN, and CRISPR/Cas9 technologies, IUE-based Supernova achieved labeled cell-specific gene knockdown and editing/knockout without requiring genetically altered mice. Thus, Supernova system is highly extensible and widely applicable for single-cell analyses in complex organs, such as the mammalian brain.

MicroRNAs (miRNAs) are small silencing RNAs with regulatory roles in gene expression. miRNAs interact with Argonaute (AGO) proteins to form effector complexes that cleave target mRNAs or repress translation. Rice (Oryza sativa) encodes four AGO1 homologs (AGO1a, AGO1b, AGO1c, and AGO1d). We used RNA interference (RNAi) to knock down the four AGO1s. The RNAi lines displayed pleiotropic developmental phenotypes and had increased accumulation of miRNA targets. AGO1a, AGO1b, and AGO1c complexes were purified and further characterized. The three AGO1s all have a strong preference for binding small RNAs (sRNAs) with 5' U and have Slicer activity. We cataloged the sRNAs in each AGO1 complex by deep sequencing and found that all three AGO1s predominantly bound known miRNAs. Most of the miRNAs were evenly distributed in the three AGO1 complexes, suggesting a redundant role for the AGO1s. Intriguingly, a subset of miRNAs were specifically incorporated into or excluded from one of the AGO1s, suggesting functional specialization among the AGO1s. Furthermore, we identified rice miRNA targets at a global level. The validated targets include transcription factors that control major stages of development and also genes involved in a variety of physiological processes, indicating a broad regulatory role for miRNAs in rice.

Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 ( eEF-2 ) gene] using the C lustered R egularly I nterspaced S hort P alindromic R epeats/ C rispr as sociated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [ orthodenticle homeobox 2 ( Otx2 )] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.

Triple negative breast cancer (TNBC) is responsible for significant number of breast cancer-associated deaths because of lacking of successful molecular-targeted therapy. To explore a therapeutic target for TNBC, we performed a siRNA-mediated knockdown screening and identified Polo-like kinase 1 (PLK1) as a potential therapeutic target for TNBC. Knockdown of PLK1 as well as a small compound inhibitor for PLK1, BI-2536, induced G2/M arrest and created polyploid cell population, shown by increased DNA content and nuclear size. Inhibition of PLK1 eventually triggered apoptosis in multiple TNBC cell lines. In addition, we confirmed that PLK1 was significantly overexpressed in the tissues from TNBC patients compared with the tissues of normal mammary glands and benign breast tumors. Our data indicated that PLK1 plays a pivotal role in the regulation of mitosis of TNBC cells. Although future in vivo studies are warranted, targeting PLK1 by a selective inhibitor such as BI-2536 can be an attractive molecular-targeted therapy for TNBC.

ZO-1, ZO-2 and ZO-3 are tight junction-associated scaffold proteins that bind to transmembrane proteins of tight junctions and the underlying cytoskeleton. ZO-1 is involved in the regulation of cytoskeletal organization, but its detailed molecular mechanism is less well understood. Gene knockout is an ideal method to investigate the functions of proteins that might have redundant functions such as ZO proteins, when compared with methods such as RNA interference-mediated suppression of gene expression. In this study we applied transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout, and established ZO-1 knockout clones in Madin-Darby canine kidney (MDCK) cells. ZO-1 knockout induced striking changes in myosin organization at cell-cell contacts and disrupted the localization of tight junction proteins; these findings were previously unseen in studies of ZO-1 knockdown by RNA interference. Rescue experiments revealed that trace ZO-1 expression reversed these changes while excessive ZO-1 expression induced an intensive zigzag shape of cell-cell junctions. These results suggest a role for ZO-1 in the regulation of cytoskeleton and shape of cell-cell junctions in MDCK cells and indicate the advantage of knockout analysis in cultured cells.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by inattention, hyperactivity, increased impulsivity and emotion dysregulation. Linkage analysis followed by fine-mapping identified variation in the gene coding for Latrophilin 3 (LPHN3), a putative adhesion-G protein-coupled receptor, as a risk factor for ADHD. In order to validate the link between LPHN3 and ADHD, and to understand the function of LPHN3 in the etiology of the disease, we examined its ortholog lphn3.1 during zebrafish development. Loss of lphn3.1 function causes a reduction and misplacement of dopamine-positive neurons in the ventral diencephalon and a hyperactive/impulsive motor phenotype. The behavioral phenotype can be rescued by the ADHD treatment drugs methylphenidate and atomoxetine. Together, our results implicate decreased Lphn3 activity in eliciting ADHD-like behavior, and demonstrate its correlated contribution to the development of the brain dopaminergic circuitry.

BackgroundRecent evidence suggests that the targeting of membrane proteins specifically activated in cancer stem cells (CSCs) is an important strategy for cancer therapy. The objectives of the present study were to investigate the expression and activity of ion-transport-related molecules in the CSCs of esophageal squamous cell carcinoma.MethodsCells exhibiting strong aldehyde dehydrogenase 1 family member A1 (ALDH1A1) activity were isolated from TE8 cells by fluorescence-activated cell sorting, and CSCs were then generated with the sphere formation assay. The gene expression profiles of CSCs were examined by microarray analysis.ResultsAmong TE8 cells, ALDH1A1 messenger RNA and protein levels were higher in CSCs than in non-CSCs. The CSCs obtained were resistant to cisplatin and had the ability to redifferentiate. The results of the microarray analysis revealed that the expression of 50 genes encoding plasma membrane proteins was altered in CSCs, whereas that of several genes related to ion channels, including transient receptor potential vanilloid 2 (TRPV2), was upregulated. The TRPV2 inhibitor tranilast was more cytotoxic at a lower concentration in CSCs than in non-CSCs, and effectively decreased the number of tumorspheres. Furthermore, tranilast significantly decreased the cell population that strongly expressed ALDH1A1 among TE8 cells.ConclusionsThe results of the present study suggest that TRPV2 is involved in the maintenance of CSCs, and that its specific inhibitor, tranilast, has potential as a targeted therapeutic agent against esophageal squamous cell carcinoma.