H1 Promoter Research Articles

A simplified and versatile system for the simultaneous expression of multiple siRNAs in mammalian cells using Gibson DNA Assembly.

RNA interference (RNAi) denotes sequence-specific mRNA degradation induced by short interfering double-stranded RNA (siRNA) and has become a revolutionary tool for functional annotation of mammalian genes, as well as for development of novel therapeutics. The practical applications of RNAi are usually achieved by expressing short hairpin RNAs (shRNAs) or siRNAs in cells. However, a major technical challenge is to simultaneously express multiple siRNAs to silence one or more genes. We previously developed pSOS system, in which siRNA duplexes are made from oligo templates driven by opposing U6 and H1 promoters. While effective, it is not equipped to express multiple siRNAs in a single vector. Gibson DNA Assembly (GDA) is an in vitro recombination system that has the capacity to assemble multiple overlapping DNA molecules in a single isothermal step. Here, we developed a GDA-based pSOK assembly system for constructing single vectors that express multiple siRNA sites. The assembly fragments were generated by PCR amplifications from the U6-H1 template vector pB2B. GDA assembly specificity was conferred by the overlapping unique siRNA sequences of insert fragments. To prove the technical feasibility, we constructed pSOK vectors that contain four siRNA sites and three siRNA sites targeting human and mouse β-catenin, respectively. The assembly reactions were efficient, and candidate clones were readily identified by PCR screening. Multiple β-catenin siRNAs effectively silenced endogenous β-catenin expression, inhibited Wnt3A-induced β-catenin/Tcf4 reporter activity and expression of Wnt/β-catenin downstream genes. Silencing β-catenin in mesenchymal stem cells inhibited Wnt3A-induced early osteogenic differentiation and significantly diminished synergistic osteogenic activity between BMP9 and Wnt3A in vitro and in vivo. These findings demonstrate that the GDA-based pSOK system has been proven simplistic, effective and versatile for simultaneous expression of multiple siRNAs. Thus, the reported pSOK system should be a valuable tool for gene function studies and development of novel therapeutics.

Eating habits modulate short term memory and epigenetical regulation of brain derived neurotrophic factor in hippocampus of low- and high running capacity rats

Exercise capacity and dietary restriction (DR) are linked to improved quality of life, including enhanced brain function and neuro-protection. Brain derived neurotrophic factor (BDNF) is one of the key proteins involved in the beneficial effects of exercise on brain. Low capacity runner (LCR) and high capacity runner (HCR) rats were subjected to DR in order to investigate the regulation of BDNF. HCR-DR rats out-performed other groups in a passive avoidance test. BDNF content increased significantly in the hippocampus of HCR-DR groups compared to control groups (p<0.05). The acetylation of H3 increased significantly only in the LCR-DR group. However, chip-assay revealed that the specific binding between acetylated histone H3 and BNDF promoter was increased in both LCR-DR and HCR-DR groups. In spite of these increases in binding, at the transcriptional level only, the LCR-DR group showed an increase in BDNF mRNA content. Additionally, DR also induced the activity of cAMP response element-binding protein (CREB), while the content of SIRT1 was not altered. Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) was elevated in HCR-DR groups. But, based on the levels of nuclear respiratory factor-1 and cytocrome c oxidase, it appears that DR did not cause mitochondrial biogenesis. The data suggest that DR-mediated induction of BDNF levels includes chromatin remodeling. Moreover, DR does not induce mitochondrial biogenesis in the hippocampus of LCR/HCR rats. DR results in different responses to a passive avoidance test, and BDNF regulation in LCR and HCR rats.

A rational study for identification of highly effective siRNAs against hepatitis B virus

RNA interference (RNAi) is a powerful gene knockdown technique used for study gene function. It also potentially provides effective agents for inhibiting infectious and genetic diseases. Most of RNAi studies employ a single siRNA designing program and then require large-scale screening experiments to identify functional siRNAs. In this study, we demonstrate that an assembly of results generated from different siRNA designing programs could provide clusters of predicting sites that aided selection of potent siRNAs. Based on the clusters, three siRNA target sites were selected on a conserved RNA region of hepatitis B virus (HBV), known as HBV post-transcriptional regulatory element (HBV PRE) at nucleotide positions 1317–1337, 1357–1377 and 1644–1664. All three chosen siRNAs driven by H1 promoter were highly effective and could drastically decrease expression of HBV transcripts (core, surface and X) and surface protein without induction of interferon response and cell cytotoxicity in liver cancer cell line (HepG2). Based on prediction of secondary structures, the silencing effects of siRNAs were less effective against a loop sequence of the mRNA target with hairpin structure. In summary, we demonstrate an effectual approach for identification of functional siRNAs. Moreover, highly potent siRNAs identified here may serve as novel agents for development of nucleic acid-based HBV therapy.

Comparison of anthocyanin components, expression of anthocyanin biosynthetic structural genes, and TfF3′H1 sequences between Tulipa fosteriana ‘Albert heijn’ and its reddish sport

A reddish flower tulip sport Tulipa fosteriana ‘SN09’ was bred from a pink flower cultivar T. fosteriana ‘Albert heijn’ (AH). To investigate the molecular basis underlying this color difference, SN09 and AH were compared using biochemical and molecular approaches. Ultra performance liquid chromatography (UPLC) profiles revealed that major cyanidin 3-rutinoside and minor pelargonidin 3-rutinoside were accumulated in the petals of AH, whereas major pelargonidin anthocyanins (pelargonidin 3-acetylrutinoside and pelargonidin 3-rutinoside) and minor cyanidin 3-glucoside were detected in SN09. The total anthocyanin content in petals of AH and SN09 were not significantly different at Stage 4, and correlated with the expression patterns of dihydroflavonol 4-reductase (TfDFR1) and anthocyanidin synthase (TfANS1). In addition, more transcripts of chalcone synthase (TfCHS1), chalcone isomerase (TfCHI2), and flavanone 3-hydroxylase (TfF3H1) contributed to higher accumulation of flavone and flavonol in SN09 than in AH. The transcription of flavonoid 3′-hydroxylase (TfF3′H1) was significantly lower in SN09 than in AH throughout flower development. Accordingly, the nucleotide sequences of TfF3′H1 in AH and SN09 were compared. Results showed that the deduced amino acids of TfF3′H1 in AH and SN09 were not different, but an extra fragment (255bp) was found in the promoter of TfF3′H1 in SN09. Consequently, the promoters of TfF3′H1 in AH and SN09 were isolated, fused with the β-glucuronidase (GUS), and then transformed to tobacco and Arabidopsis thaliana. Results demonstrated that the GUS activity in Arabidopsis with TfF3′H1 promoter from SN09 was only 5.4% of that from AH, suggesting that the insert mutation in the TfF3′H1 promoter hampered the accumulation of cyanidin anthocyanins in SN09 through the reduction of TfF3′H1 transcription.

A MicroRNA Processing Defect in Smokers' Macrophages Is Linked to SUMOylation of the Endonuclease DICER

Despite the fact that alveolar macrophages play an important role in smoking-related disease, little is known about what regulates their pathophysiologic phenotype. Evaluating smoker macrophages, we found significant down-regulation of multiple microRNAs (miRNAs). This work investigates the hypothesis that cigarette smoke alters mature miRNA expression in lung macrophages by inhibiting processing of primary miRNA transcripts. Studies on smoker alveolar macrophages showed a defect in miRNA maturation. Studies on the miRNA biogenesis machinery led us to focus on the cytosolic RNA endonuclease, DICER. DICER cleaves the stem-loop structure from pre-miRNAs, allowing them to dissociate into their mature 20-22-nucleotide single-stranded form. DICER activity assays confirmed impaired DICER activity following cigarette smoke exposure. Further protein studies demonstrated a decreased expression of the native 217-kDa form of DICER and an accumulation of high molecular weight forms with cigarette smoke exposure. This molecular mass shift was shown to contain SUMO moieties and could be blocked by silencing RNA directed at the primary SUMOylating ligase, Ubc9. In determining the cigarette smoke components responsible for changes in DICER, we found that N-acetylcysteine, an antioxidant and anti-aldehyde, protected DICER protein and activity from cigarette smoke extract. This massive down-regulation of miRNAs (driven in part by alterations in DICER) may be an important regulator of the disease-promoting macrophage phenotype found in the lungs of smokers.

A Cell Surface Biotinylation Assay to Reveal Membrane-associated Neuronal Cues: Negr1 Regulates Dendritic Arborization

A complex and still not comprehensively resolved panel of transmembrane proteins regulates the outgrowth and the subsequent morphological and functional development of neuronal processes. In order to gain a more detailed description of these events at the molecular level, we have developed a cell surface biotinylation assay to isolate, detect, and quantify neuronal membrane proteins. When we applied our assay to investigate neuron maturation in vitro, we identified 439 differentially expressed proteins, including 20 members of the immunoglobulin superfamily. Among these candidates, we focused on Negr1, a poorly described cell adhesion molecule. We demonstrated that Negr1 controls the development of neurite arborization in vitro and in vivo. Given the tight correlation existing among synaptic cell adhesion molecules, neuron maturation, and a number of neurological disorders, our assay results are a useful tool that can be used to support the understanding of the molecular bases of physiological and pathological brain function.

Antitumor activity and immunogenicity of recombinant vaccinia virus expressing HPV 16 E7 protein SigE7LAMP is enhanced by high-level coexpression of IGFBP-3

We constructed recombinant vaccinia viruses (VACVs) coexpressing the insulin-like growth factor-binding protein-3 (IGFBP-3) gene and the fusion gene encoding the SigE7Lamp antigen. The expression of the IGFBP-3 transgene was regulated either by the early H5 promoter or by the synthetic early/late (E/L) promoter. We have shown that IGFBP-3 expression regulated by the H5 promoter yielded higher amount of IGFBP-3 protein when compared with the E/L promoter. The immunization with P13-SigE7Lamp-H5-IGFBP-3 virus was more effective in inhibiting the growth of TC-1 tumors in mice and elicited higher T-cell response against VACV-encoded antigen than the P13-SigE7Lamp-TK(-) control virus. We found that high-level production of IGFBP-3 enhanced virus replication both in vitro and in vivo, resulting in more profound antigen stimulation. Production of IGFBP-3 was associated with a higher adsorption rate of P13-SigE7Lamp-H5-IGFBP-3 to CV-1 cells when compared with P13-SigE7Lamp-TK(-). Intracellular mature virions (IMVs) of the IGFBP-3-expressing virus P13-SigE7Lamp-H5-IGFBP-3 have two structural differences: they incorporate the IGFBP-3 protein and they have elevated phosphatidylserine (PS) exposure on outer membrane that could result in increased uptake of IMVs by macropinocytosis. The IMV PS content was measured by flow cytometry using microbeads covered with immobilized purified VACV virions.

RNAi-Mediated Knockdown of IKK1 in Transgenic Mice Using a Transgenic Construct Containing the Human H1 Promoter

Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models.

The Compass-like Locus, Exclusive to the Ambulacrarians, Encodes a Chromatin Insulator Binding Protein in the Sea Urchin Embryo

Chromatin insulators are eukaryotic genome elements that upon binding of specific proteins display barrier and/or enhancer-blocking activity. Although several insulators have been described throughout various metazoans, much less is known about proteins that mediate their functions. This article deals with the identification and functional characterization in Paracentrotus lividus of COMPASS-like (CMPl), a novel echinoderm insulator binding protein. Phylogenetic analysis shows that the CMPl factor, encoded by the alternative spliced Cmp/Cmpl transcript, is the founder of a novel ambulacrarian-specific family of Homeodomain proteins containing the Compass domain. Specific association of CMPl with the boxB cis-element of the sns5 chromatin insulator is demonstrated by using a yeast one-hybrid system, and further corroborated by ChIP-qPCR and trans-activation assays in developing sea urchin embryos. The sns5 insulator lies within the early histone gene cluster, basically between the H2A enhancer and H1 promoter. To assess the functional role of CMPl within this locus, we challenged the activity of CMPl by two distinct experimental strategies. First we expressed in the developing embryo a chimeric protein, containing the DNA-binding domain of CMPl, which efficiently compete with the endogenous CMPl for the binding to the boxB sequence. Second, to titrate the embryonic CMPl protein, we microinjected an affinity-purified CMPl antibody. In both the experimental assays we congruently observed the loss of the enhancer-blocking function of sns5, as indicated by the specific increase of the H1 expression level. Furthermore, microinjection of the CMPl antiserum in combination with a synthetic mRNA encoding a forced repressor of the H2A enhancer-bound MBF1 factor restores the normal H1 mRNA abundance. Altogether, these results strongly support the conclusion that the recruitment of CMPl on sns5 is required for buffering the H1 promoter from the H2A enhancer activity, and this, in turn, accounts for the different level of accumulation of early linker and nucleosomal transcripts.

Studies of Efficacy and Liver Toxicity Related to Adeno-Associated Virus–Mediated RNA Interference

Adeno-associated virus (AAV)-mediated RNA interference shows promise as a therapy for chronic hepatitis B virus (HBV) infection, but its low efficacy and hepatotoxicity pose major challenges. We have generated AAV vectors containing different promoters and a panel of HBV-specific short hairpin RNAs (shRNAs) to investigate factors that contribute to the efficacy and pathogenesis of AAV-mediated RNA interference. HBV transgenic mice injected with high doses of AAV vectors containing the U6 promoter produced abundant shRNAs, transiently inhibited HBV, but induced severe hepatotoxicity. Sustained HBV suppression without liver toxicity can be achieved by lowering the dose of AAV-U6 vectors. AAVs containing the weaker H1 promoter did not cause liver injury, but their therapeutic efficacy was highly dependent on the sequence of the shRNA. Mice treated with the toxic U6-promoter-driven shRNA showed little change in hepatic microRNA levels, but a dramatic increase in hepatic leukocytes and inflammatory cytokines and chemokines. Hepatotoxicity was completely absent in immunodeficient mice and significantly alleviated in wild-type mice depleted of macrophages and granulocytes, suggesting that host inflammatory responses are the major cause of liver injury induced by the overexpressed shRNAs from AAV-U6 vectors. Our results demonstrate that selection of a highly potent shRNA and control its expression level is critical to achieve sustained HBV suppression without inducing inflammatory side effects.

An HDAC inhibitor enhances cancer therapeutic efficiency of RNA polymerase III promoter-driven IDO shRNA

Histone deacetylase (HDAC) inhibitors are used in treating certain human malignancies. Our laboratories demonstrated their capability in enhancing antitumor effect of DNA vaccine driven by an RNA polymerase II (RNA pol II) promoter. However, it is unknown whether HDAC inhibitors enhance the therapeutic short hairpin RNA (shRNA) expressed by an RNA polymerase III (RNA pol III) promoter. We investigated whether HDAC inhibitors augmented antitumor effect of indoleamine 2,3 dioxygenase (IDO) shRNA. HDAC inhibitor OSU-HDAC42 and suberoylanilide hydroxamic acid enhanced RNA pol III-driven U6 and H1 promoter activity in three different cell types in vitro: 293, NIH3T3 and dendritic cell line DC2.4. Subcutaneous injection of OSU-HDAC42 enhanced U6 and H1 promoter activity on abdominal skin of mice in vivo. Combination of IDO shRNA and OSU-HDAC42 increased antitumor effect of IDO shRNA in MBT-2 murine bladder tumor model. IDO shRNA induced tumor-infiltrating CD8⁺ and CD4⁺ T cells, whereas OSU-HDAC42 treatment induced tumor-infiltrating CD4⁺ T cells. Combination of OSU-HDAC42 and IDO shRNA further induced tumor-infiltrating natural killer cells and enhanced interferon-γ in lymphocytes, but suppressed interleukin (IL)-4 expression of lymphocytes. In addition, OSU-HDAC42 treatment did not alter mRNA expression of IL-12 and tumor necrosis factor-α. In conclusion, HDAC inhibitor OSU-HDAC42 may serve as adjuvant of the therapeutic shRNA expressed by an RNA pol III promoter.

Induction of innate inflammatory responses is the major cause of liver toxicity associated with AAV-mediated RNA interference (P4007)

Abstract Adeno-associated virus (AAV)-mediated RNA interference shows promise as a therapy for chronic hepatitis B virus (HBV) infection, but its low efficacy and hepatotoxicity pose major challenges. We have generated AAV vectors containing different promoters and a panel of HBV-specific short hairpin RNAs (shRNAs) to investigate factors that contribute to the efficacy and pathogenesis of AAV-mediated RNA interference. HBV transgenic mice injected with AAV vectors containing the U6 promoter produced abundant shRNAs, resulting in significant, but transient, HBV suppression and severe liver injury. AAVs containing the weaker H1 promoter did not cause liver injury, but their therapeutic efficacy was highly dependent on the sequence of the shRNA. Mice treated with the toxic U6 promoter-driven shRNA showed little change in hepatic microRNA levels, but a dramatic increase in hepatic leukocytes and inflammatory cytokines and chemokines. Hepatotoxicity was completely absent in immunodeficient mice and significantly alleviated in wild-type mice depleted of macrophages and granulocytes, suggesting that host inflammatory responses are the major cause of liver injury induced by U6 promoter-driven shRNA. Our results demonstrate that a combination of the H1 promoter and a highly potent shRNA can achieve sustained HBV suppression without inducing inflammatory side-effects.

Essential Roles of the Kar2/BiP Molecular Chaperone Downstream of the UPR Pathway in Cryptococcus neoformans

The endoplasmic reticulum (ER) is a central hub where secreted or membrane-bound proteins are maturated and folded properly in eukaryotes. Maintenance of ER homeostasis is particularly important for human fungal pathogens, such as Cryptococcus neoformans, which encounter a plethora of host-mediated stresses during infection. Our previous study demonstrated that the unfolded protein response (UPR) pathway, composed of the evolutionarily conserved Ire1 kinase and the unique Hxl1 transcription factor, has pleiotropic roles in ER stress response, thermotolerance, antifungal drug resistance, and virulence in C. neoformans. Here, we functionally characterized an ER-resident molecular chaperone, Kar2/BiP, in C. neoformans. Conditional expression of KAR2 by the copper-regulated promoter revealed that Kar2 is essential for the viability of C. neoformans. Constitutive expression of KAR2 by the strong histone H3 promoter partially restores resistance to ER stress, cell wall stress, thermotolerance, and genotoxic stress in ire1Δ and hxl1Δ mutants, suggesting that Kar2 mainly functions downstream of the UPR pathway. Furthermore, Kar2 appears to control azole resistance in C. neoformans downstream of the UPR pathway without regulation of ERG11 or ERG3. Interestingly, we discovered that azole treatment is sensed as ER-stress and subsequently activates the Ire1-dependent Hxl1 splicing event and induction of KAR2 by the UPR pathway. In contrast, the constitutive expression of Kar2 is not sufficient to restore the Ire1-mediated regulation of capsule production in C. neoformans UPR mutants. In conclusion, this study demonstrates that Kar2 is not only essential for vegetative growth but also required for response and adaptation to the environmental stresses and antifungal drugs downstream of the UPR pathway in C. neoformans.

Imbalance in expression of hop (Humulus lupulus) chalcone synthase H1 and its regulators during hop stunt viroid pathogenesis

Viroid-derived small RNAs generated during hop stunt viroid (HSVd) pathogenesis may induce the symptoms found in the hop cultivar “Admiral”, including observed shifts in phenylpropanoid metabolites and changes in petiole coloration. Using quantitative RT-PCR, we examined hop lupulin gland-specific genes that have been shown to be involved in phenylpropanoid metabolism, for altered expression in response to infection with two HSVd isolates, HSVd-g and CPFVd. Most notably, the expression of a gene encoding a key enzyme for phenylpropanoid synthesis, naringenin-chalcone synthase H1 (chs_H1), decreased up to 40-fold in infected samples. In addition, a marked decrease in the expression of HlbHLH2 and an increase in the expression of HlMyb3 were observed. These two genes encode transcription factors that form a ternary complex with HlWDR1 for chs_H1 promoter activation. In a transient expression assay, a decrease in the ternary complex potential to activate the chs_H1 promoter was observed upon the decrease of HlbHLH2 expression. In addition, targeting of the chs_H1 transcript by vd-sRNAs may contribute to these expression changes. Our data show that HSVd infection causes a significant imbalance in the expression of phenylpropanoid metabolite-affecting genes via a complex mechanism, possibly involving regulatory disorders and direct targeting by vd-sRNA.

High-level expression of IGFBP-3 by recombinant vaccinia virus leads to its incorporation into IMV and affects virus replication and immunogenicity

Event Abstract Back to Event High-level expression of IGFBP-3 by recombinant vaccinia virus leads to its incorporation into IMV and affects virus replication and immunogenicity Jan Musil1*, Luda Kutinova1, Kamila Zurkova1, Petr Hainz1, Katarina Babiarova1, Jitka Krystofova1 and Sarka Nemeckova1* 1 Institute of Hematology and Blood Transfusion, Czechia The aim of this study was to investigate, whether the tumor- suppressive properties of Insulin-like growth factor-binding protein-3 (IGFBP-3) can be used to enhance the efficiency of therapeutic immunization against the HPV16 E7 oncogene associated with cervical cancer. We constructed recombinant vaccinia viruses (rVACV) co-expressing the immunogen SigE7Lamp and IGFBP-3. The expression of IGFBP-3 was controlled either by the early promoter H5 or by the synthetic early-late (E/L) promoter. Our results show that IGFBP-3 expression under the H5 promoter (P13-SigE7Lamp-H5-IGFBP-3) is significantly higher than under E/L promoter (P13-SigE7Lamp-E/L-IGFBP-3) (***p<0.001). The mice bearing TC-1 tumors treated with P13-SigE7Lamp-H5-IGFBP-3 had significantly smaller tumors than PBS treated mice by day 11 (*p<0.05), whereas mice treated with P13-SigE7Lamp-E/L-IGFBP-3 or the control virus P13-SigE7Lamp-TK- had significantly smaller tumors by day 18 (***p<0.001). Co-expression of IGFBP-3 increased T cell response against VACV antigen but not against HPV16 E7 antigen as determined by ELISPOT IFN-γ. In vitro analysis of virus multiplication has shown that P13-SigE7Lamp-H5-IGFBP-3 has a significantly higher replication rate than P13-SigE7Lamp-TK- or P13-SigE7Lamp-E/L-IGFBP-3 (***p<0.001) and that adding an IGFBP-3 neutralizing antibody significantly decreased yields of P13-SigE7Lamp-H5-IGFBP-3 (**p<0.01). Furthermore, using western-blot analysis we detected insertion of IGFBP-3 into virions of P13-SigE7Lamp-H5-IGFBP-3. We therefore hypothesize that incorporation of IGFBP-3 into intracellular mature virus helps virus replication. Indeed, our results show that cell absorption rate of P13-SigE7Lamp-H5-IGFBP-3 is significantly higher than P13-SigE7Lamp-TK- (*p<0.05). Summarized, higher cell absorption leads to overall better replication and thus resulting in prolonged and enhanced antigen stimulation during immunization. Keywords: IGFBP-3, Vaccinia virus, T-cell response, Immunotherapy, Cancer Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Musil J, Kutinova L, Zurkova K, Hainz P, Babiarova K, Krystofova J and Nemeckova S (2013). High-level expression of IGFBP-3 by recombinant vaccinia virus leads to its incorporation into IMV and affects virus replication and immunogenicity. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00387 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Jan Musil, Institute of Hematology and Blood Transfusion, Prague, Czechia, musil@uhkt.cz Dr. Sarka Nemeckova, Institute of Hematology and Blood Transfusion, Prague, Czechia, Sarka.Nemeckova@uhkt.cz Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jan Musil Luda Kutinova Kamila Zurkova Petr Hainz Katarina Babiarova Jitka Krystofova Sarka Nemeckova Google Jan Musil Luda Kutinova Kamila Zurkova Petr Hainz Katarina Babiarova Jitka Krystofova Sarka Nemeckova Google Scholar Jan Musil Luda Kutinova Kamila Zurkova Petr Hainz Katarina Babiarova Jitka Krystofova Sarka Nemeckova PubMed Jan Musil Luda Kutinova Kamila Zurkova Petr Hainz Katarina Babiarova Jitka Krystofova Sarka Nemeckova Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Abstract 19036: Transgenic Mouse with Overexpression Hypoxamir-210: Implications on Infarct Size Attenuation and Preservation of Heart Function

Background. Hypoxamir-210 (miR-210) is a universal responder to activation of HIF-1 signaling and plays a significant role in cell survival under ischemic stress. To assess the impact of constitutive over expression of hypoxamir-210 on infarct size expansion and preservation of cardiac function, we report the development of first transgenic (TG) mouse model with miR-210 overexpression ( 210 TG). Methods and Results. miR-210 transgenic mouse was generated at the University of Cincinnati transgenic core using the C57BL/6 mouse strain. Briefly precursor miR-210 along with H1 promoter (HmiR0167-MR01 vector from Genocopiea) was amplified and cloned into pShuttle vector (Stratagene) using restriction enzymes Not1 and Sal1. Amplified vector was digested, purified and used for microinjection into the pronuclei of single cell embryos derived from superovulated C57BL/6 females. The surviving embryos were implanted into foster female mice. TG mice were identified genotyping of the tail genomic DNA by PCR. As compared to control littermates (NTG), 210 TG had significantly higher miR-210 expression in different tissues and organs including the heart and the bone marrow. Molecular studies showed altered expression of miR-210 putative traget Dffb in 210 TG animals. Age and sex-matched mouse model of permanent LAD coronary artery ligation was developed in 210 TG(n=12 animals/group) using NTGas controls. Animals were harvested on day 7 (short term) and 28 (long term) after LAD ligation. Echocardiography performed prior to euthanasia showed that cardiac function was significantly preserved in 210 TG as compared to NTG as indicated by LVEF (45.2±1.06 vs 38.9±1 p &lt;0.001) and LVFS (18.3±1 vs 15±1), Histochemical studies showed preserved myocardial architecture and attenuated infarct size in 210 TG ( p &lt;0.05 vs NTG). TUNEL combined with fluorescence immunostaining for cardiac troponin-1, smooth muscle actin and CD31 showed significantly reduced TUNEL positivity of cardiomyocytes, smooth muscle and endothelial cells in 210 TG heart at day 7 ( p &lt;0.05 vs NTG hearts). Conclusions. Constitutive overexpression of hypoxamir-210 rescued global cardiac function after myocardial infarction by inhibition of cardiomyocyte apoptosis to attenuate infarct size in the heart.

Optimization and comparison of knockdown efficacy between polymerase II expressed shRNA and artificial miRNA targeting luciferase and Apolipoprotein B100

BackgroundControlling and limiting the expression of short hairpin RNA (shRNA) by using constitutive or tissue-specific polymerase II (pol II) expression can be a promising strategy to avoid RNAi toxicity. However, to date detailed studies on requirements for effective pol II shRNA expression and processing are not available. We investigated the optimal structural configuration of shRNA molecules, namely: hairpin location, stem length and termination signal required for effective pol II expression and compared it with an alternative strategy of avoiding toxicity by using artificial microRNA (miRNA) scaffolds.ResultsHighly effective shRNAs targeting luciferase (shLuc) or Apolipoprotein B100 (shApoB1 and shApoB2) were placed under the control of the pol II CMV promoter and expressed at +5 or +6 nucleotides (nt) with reference to the transcription start site (TSS). Different transcription termination signals (TTS), namely minimal polyadenylation (pA), poly T (T5) and U1 were also used. All pol II- expressed shRNA variants induced mild inhibition of Luciferase reporters carrying specific targets and none of them showed comparable efficacy to their polymerase III-expressed H1-shRNA controls, regardless of hairpin position and termination signal used. Extending hairpin stem length from 20 basepairs (bp) to 21, 25 or 29 bp yielded only slight improvement in the overall efficacy. When shLuc, shApoB1 and shApoB2 were placed in an artificial miRNA scaffold, two out of three were as potent as the H1-shRNA controls. Quantification of small interfering RNA (siRNA) molecules showed that the artificial miRNA constructs expressed less molecules than H1-shRNAs and that CMV-shRNA expressed the lowest amount of siRNA molecules suggesting that RNAi processing in this case is least effective. Furthermore, CMV-miApoB1 and CMV-miApoB2 were as effective as the corresponding H1-shApoB1 and H1-shApoB2 in inhibiting endogenous ApoB mRNA.ConclusionOur results demonstrate that artificial miRNA have a better efficacy profile than shRNA expressed either from H1 or CMV promoter and will be used in the future for RNAi therapeutic development.

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

Sheep 7SK promoter for short hairpin RNA expression

Background: Gene silencing mediated by small interfering RNA (siRNA) has become a powerful biological tool for the regulation of gene expression. For the synthesis of siRNA by vector-based expression systems, several mammalian small nuclear RNA (snRNA) promoters have been cloned and shown different transcriptional efficiencies. Results: In this study, we identified a sheep 7SK snRNA (s7SK) promoter based on the highly conserved polymerase III promoter elements. Promoter activity was measured by promoter-driven shRNA expression to suppress expression of an exogenous reporter gene and endogenous sheep gene. Conclusions: The knock down assay demonstrated that the s7SK induced more stronger inhibition effect than human U6 and H1 promoters. The use of this native sheep 7SK promoter for shRNA expressionis an important component for development of RNAi-based gene therapy and production of transgenic animals in sheep species.

Lipid Polarity Is Maintained in Absence of Tight Junctions

The role of tight junctions (TJs) in the establishment and maintenance of lipid polarity in epithelial cells has long been a subject of controversy. We have addressed this issue using lysenin, a toxin derived from earthworms, and an influenza virus labeled with a fluorescent lipid, octadecylrhodamine B (R18). When epithelial cells are stained with lysenin, lysenin selectively binds to their apical membranes. Using an artificial liposome, we demonstrated that lysenin recognizes the membrane domains where sphingomyelins are clustered. Interestingly, lysenin selectively stained the apical membranes of epithelial cells depleted of zonula occludens proteins (ZO-deficient cells), which completely lack TJs. Furthermore, the fluorescent lipid inserted into the apical membrane by fusion with the influenza virus did not diffuse to the lateral membrane in ZO-deficient epithelial cells. This study revealed that sphingomyelin-cluster formation occurs only in the apical membrane and that lipid polarity is maintained even in the absence of TJs.