Short Hairpin RNA Expression Research Articles

Targeting species D adenoviruses replication to counteract the epidemic keratoconjunctivitis.

Human adenoviruses are non-enveloped DNA viruses causing various infections; their pathogenicity varies dependent on virus species and type. Although acute infections can sometimes take severe courses, they are rarely fatal in immune-competent individuals. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are hyperacute and highly contagious infections of the eye caused by human adenovirus types within species D. Currently there is no causal treatment available to counteract these diseases effectively. The E2B region of the adenovirus genome encodes for the viral DNA polymerase, which is required for adenoviral DNA replication. Here we propose novel model systems to test this viral key factor, DNA polymerase, as a putative target for the development of efficient antiviral therapy based on RNA interference. Using our model cell lines we found that different small interfering RNAs mediate significant suppression (up to 90%) of expression levels of viral DNA polymerase upon transfection. Moreover, permanent expression of short hairpin RNA based on the most effective small interfering RNA led to a highly significant, more than tenfold reduction in replication for different human group D adenoviruses involved in ocular infections.

Alternative methods for the efficient construction of short hairpin RNA expression vectors

Short hairpin RNA (shRNA)-mediated RNA interference has become a basic technique in modern molecular biology and biochemistry for studying gene function and biological pathways. Here, we report two alternative and efficient methods to construct shRNA expression vectors based respectively on multiple-step sequential PCR and primer extension–homologous recombination (PE-HR). Neither method requires synthesizing long oligonucleotides containing hairpin sequences as used in traditional approaches. The hairpin sequences may produce mutations during oligo synthesis, pose problems in annealing, and lead to inefficient cloning. The PE-HR method further provides rapid and economical construction of shRNA expression vectors without needing the ligation procedure.

The impact of HIV-1 genetic diversity on the efficacy of a combinatorial RNAi-based gene therapy.

A hurdle for human immunodeficiency virus (HIV-1) therapy is the genomic diversity of circulating viruses and the possibility that drug-resistant virus variants are selected. Although RNA interference (RNAi) is a powerful tool to stably inhibit HIV-1 replication by the expression of antiviral short hairpin RNAs (shRNAs) in transduced T cells, this approach is also vulnerable to pre-existing genetic variation and the development of viral resistance through mutation. To prevent viral escape, we proposed to combine multiple shRNAs against important regions of the HIV-1 RNA genome, which should ideally be conserved in all HIV-1 subtypes. The vulnerability of RNAi therapy to viral escape has been studied for a single subtype B strain, but it is unclear whether the antiviral shRNAs can inhibit diverse virus isolates and subtypes, including drug-resistant variants that could be present in treated patients. To determine the breadth of the RNAi gene therapy approach, we studied the susceptibility of HIV-1 subtypes A-E and drug-resistant variants. In addition, we monitored the evolution of HIV-1 escape variants. We demonstrate that the combinatorial RNAi therapy is highly effective against most isolates, supporting the future testing of this gene therapy in appropriate in vivo models.

Construction of shRNA-Bmi-1 plasmid and its effect on proliferation of ECA109 cells.

To explore the role of Bmi-1 gene in the proliferation of squamous carcinoma cells and whether the silencing Bmi-1 can inhibit the growth of squamous cell carcinomas cells. The expressions of Bmi-1 in primary cultured Fibroblasts, karatinocyte cell line Hacat,squamous carcinoma cell line A431, and ECA109 were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Recombinant plasmid inserted with Bmi-1 gene short hairpin RNA (shRNA) expression vector PGPU6/GFP/Neo-shBmi-1 was constructed and transfected into ECA109 cells with control set. After transfection for 48 and 72 hours,the mRNA and protein levels of Bmi-1 were examined with RT-PCR and Western blot analysis, respectively. The proliferation of the ECA109 cells was evaluated by MTT method and flow cytometry. Bmi-1 was highly expressed in A431 and ECA109 cells than in Fibroblast cells and Hacat cells. The mRNA and protein expressions of Bmi-1 were significantly silenced in ECA109 cells after recombinant expression vector PGPU6/GFP/Neo-shBmi-1 transfection (P<0.05). Compared with the control groups,the proliferation of ECA109 transfected with PGPU6/GFP/Neo-shBmi-1 was significantly inhibited (P<0.05), and cells in G1 phase increased while in S phase decreased (P<0.05). Bmi-1 is involved in the proliferation of squamous carcinoma cells. After the silencing of Bmi-1 expression,the proliferation ECA109 cells is suppressed due to the altered cell cycle.

Transient receptor potential melastatin-3 (TRPM3)-induced activation of AP-1 requires Ca2+ ions and the transcription factors c-Jun, ATF2, and ternary complex factor.

The steroid pregnenolone sulfate activates the transcription factor activator protein-1 (AP-1) via stimulation of transient receptor potential melastatin-3 (TRPM3) channels. Here, we show that the signaling pathway requires an influx of Ca(2+) ions into the cells and a rise in the intracellular Ca(2+) levels. The upregulation of AP-1 was attenuated in cells that overexpressed mitogen activated protein kinase phosphatase-1, indicating that Ca(2+) ions prolong the signaling cascade via activation of mitogen activated protein kinases. On the transcriptional level, expression of a dominant-negative mutant of the basic region leucine zipper protein c-Jun, a major constituent of the AP-1 transcription factor complex, or expression of a c-Jun-specific short hairpin RNA attenuated pregnenolone sulfate-induced AP-1 activation. In addition, stimulation of TRPM3 channels increased the transcriptional activation potential of the basic region leucine zipper protein ATF2. Inhibition of ATF2 target gene expression via expression of a dominant-negative mutant of ATF2 or expression of an ATF2-specific short hairpin RNA interfered with TRPM3-mediated stimulation of AP-1. Moreover, we show that a dominant-negative mutant of the ternary complex factor (TCF) Elk-1 attenuated the upregulation of AP-1 following stimulation of TRPM3 channels. Thus, c-Jun, ATF2, and TCFs are required to connect the intracellular signaling cascade elicited by activation of TRPM3 channels with enhanced transcription of AP-1-regulated genes. We conclude that pregnenolone sulfate-induced TRPM3 channel activation changes the gene expression pattern of the cells by activating transcription of c-Jun-, ATF2-, and TCF-controlled genes.

Functional Characterization of Porcine (&amp;lt;i&amp;gt;Sus scrofa&amp;lt;/i&amp;gt;) BCL10

Human BCL10 (hBCL10) protein is a signal transduction molecule originally identified because of its direct involvement in a subset of mucosa-associated lymphoid tissue (MALT) lymphomas, and later recognized as a crucial factor in regulating activation of NF-kB transcription factor following antigen receptor stimulation on lymphocytes. In this study, we characterized the NF-kB inducing activity of porcine BCL10 (pBCL10). pBCL10 oligimerizes, binds to components of the CARMA/ BCL10/MALT1 complex and forms cytoplasmic filaments. Functionally, in human cells pBCL10 is more effective in activating NF-kB compared to hBCL10, possibly due to the lack of carboxy-terminal inhibitory serine residues present in the human protein. Also, depletion experiments carried out through expression of short hairpin RNAs targeting hBCL10 indicate that pBcl10 can functionally replace the human protein and retains its higher NF-kB-inducing property in the absence of hBCL10. Our results contribute useful information on BCL10 protein in pigs, and may help the development of strategies based on the control of the immune response in pigs.

Silencing effect of lentiviral vectors encod-ing shRNA of Herp on endoplasmic reticulum stress and inflammatory responses in RAW 264.7 macrophages.

Herp, a mammalian protein with a ubiquitin-like domain, can be strongly upregulated by endoplasmic reticulum (ER) stress during ER-associated protein degradation. However, the other cellular functions of Herp remain unclear. We explored the effect of Herp on ER stress and inflammatory responses in RAW 264.7 macrophages that had been exposed to tunicamycin or thapsigargin. We successfully constructed recombinant lentiviral vectors for Herp short-hairpin RNA (shRNA) expression to better understand the contribution made by Herp to other signaling pathways. Western blotting revealed that the recombinant Herp lentiviral shRNA vector significantly inhibited the expression of the Herp protein in the thapsigargin-treated RAW 264.7 macrophages. The reverse transcription quantitative polymerase chain reaction results showed that knockdown Herp inhibited the expression of ER stress-related genes during exposure to tunicamycin or thapsigargin. In RAW 264.7 macrophages, knockdown Herp markedly attenuated the expression of inflammatory cytokines when exposed to tunicamycin; however, it strongly enhanced the expression of inflammatory cytokines when exposed to thapsigargin. We concluded that Herp lentiviral shRNA vectors had been successfully constructed; knockdown Herp inhibited ER stress and had a different effect on inflammatory responses in RAW 264.7 macrophages depending on whether they were exposed to tunicamycin or thapsigargin.

Solid lipid nanoparticles as non-viral vector for the treatment of chronic hepatitis C by RNA interference

RNA interference (RNAi) is a promising strategy to treat the chronic infection by hepatitis C virus (HCV). The objective of this work was to develop a non-viral vector based on solid lipid nanoparticles (SLN) and RNAi to inhibit the internal ribosome entry site (IRES) mechanism of the HCV. The vectors were prepared with SLN, protamine, hylauronic acid (HA) or dextran (DX), and a short-hairpin RNA expression plasmid targeted to the stem loop II of the 5′ UTR (shRNA74). The particle size, surface charge, and capacity to bind, release and protect the shRNA74 against nucleases were evaluated. Cell uptake, silencing capacity and cell viability were evaluated in HepG2 cells. All the vectors presented particle size in the range of nanometers and positive surface charge, and they were able to protect the shRNA74 against DNase. An effective and rapid uptake into the cells was observed. Silencing capacity ranged from 3% to 67% depending on the presence of DX or HA in the vector, the shRNA74 to SLN ratio, and the shRNA74 dose. Vectors prepared with HA showed to be twice more effective than those prepared with DX. Differences in the intracellular trafficking may justify the higher efficacy of the HA-prepared vectors.

Role of calpastatin in the regulation of mRNA expression of calpain, caspase, and heat shock protein systems in bovine muscle satellite cells.

Calpastatin participates in apoptotic cell death and cell signaling, but its role in skeletal myoblast development and molecular involvements in cell growth still remains unknown. The current study aimed to investigate the role of calpastatin on the expression patterns of calpains, caspases, and heat shock proteins (HSPs). In addition, the cell viability during myoblast growth under calpastatin silence condition was also investigated. Three small interference RNA sequences (siRNAs) were used to silence calpastatin gene and ligated into pSilencer plasmid vector to construct short hairpin RNA (shRNA) expression. The all three siRNAs significantly silence the calpastatin gene. Moreover, suppression of calpastatin significantly reduced the viability of myoblasts during growth phase when compared to control cells. Additionally, knockdown of calpastatin significantly increased the mRNA expression of μ-calpain, caspase-3, caspase-7, and caspase-9, as well as HSP-27, -70, and -90. The present study results suggested that the suppression of calpastatin resulted in the increased expression of μ-calpain, caspases, and HSPs which in turn regulate the apoptotic cell death. The present study throws light on the central role of calpastatin in the control of calpain activity, cell proliferation, cell survival, and apoptotic pathways.

All-in-one system for the inducible expression of short-hairpin RNA

Objective To describe how to contribute all-in-one system for the inducible expression of short hairpin RNA (shRNA).Methods PLKO-Tet-On empty vector was digested by special site restriction enzymes.The shRNA oligos with Age Ⅰ and EcoR Ⅰ digesting sites were designed by using commercial software RNAi designer.T4 ligase was used for ligation of shRNA oligos and cut of vectors.Then Transformation of ligation product was performed into E.coli.After expansion and isolation,the plasmid was used for lenti-virus packing in order to proceed silencing the target gene.Results Inducible mouse sex determining region Y-box 2 (SOX2) shRNAs were successfully subcloned,and the efficiency was tested by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) assay and Western blotting.RT-qPCR showed that the knock-down efficiency was (83.5 ± 7.1) ％ for shSOX2#1,62.3 ± 10.2％ for shSOX2#2 and 77.4 ± 4.57％ for shSOX2#3,respectively.Western blotting revealed that protein level of knock-down groups was significantly down-regulated by doxycycline treatment.Conclusion The results indicted that inducible shRNA is an efficient way for silencing target genes. Key words: Inducible short hairpin RNA; Tet-On; Sex determining region Y-box 2

Bone Marrow-Derived MSCs Stimulated by IFN-γ Inhibited the Growth of ToxoplasmaGondii Via up-Regulation of GBP1

Background Mesenchymal stromal cells (MSCs) are a heterogeneous cell population endowed with multi-lineage differentiation potential and extensive immunomodulatory properties. MSCs have been successfully used for prevention and treatment of immune disorders such as graft-versus-host disease. Emerging preclinical studies suggest that MSCs might also protect against infectious challenge.Aims This study aimed to rule out the potential mechanism of human MSCs against Toxoplasma gondii (T. gondii).Methods Human bone marrow-derived MSCs (hMSCs) were pretreated for 24h with a series of concentrations of IFN-γ and then infected with T. gondii strains of variant virulences (virulent RH and avirulent ME49). RNA-seq and westernblots were used to analyze gene and protein expression patterns of hMSCs in IFN-γ-stimulated and unstimulated conditions. The intracellular parasites (with fluorescence labeled) were counted microscopically at multiple time points postinfection. The short hairpin RNA (shRNA) expression was used to generate RNAi of GBP-1, GBP-2 and GBP-5.Results Human MSCs stimulated with IFN-γ were capable to inhibit the growth of T. gondii (eg: at IFN-γ 10ng/ml, the inhibition rates are 26.5% (RH) and 37.5% (ME49) 12hr postinfection) in a dose-dependent manner. Compared with the unstimulated MSCs (controls), IFN-γ treatment at 5, 10, 20ng/ml inhibited T. gondii (ME49) growth by percent of 27.1±7.9, 37.5±6.2, 47.0±7.6 (mean±SD, n=4) 12 hr postinfection and the inhibition rates are 54.5±2.1%, 62.5±4.9% and 78.5±2.1 at 24 hr postinfection, respectively. After 48 hr postinfection, the ratio between parasites per parasitophorous vacuole (PV) containing rosettes and single paraites in IFN-γ-stimulated MSCs was significantly reduced compared with that in the unstimulated MSCs (p<0.01, p<0.01, p<0.001 for ME49 at IFN-γ 5, 10, 20ng/ml, respectively). Furthermore, There was no significant effect of conditioned medium (CM) from IFN-γ-stimulated MSCs on T. gondii growth in comparison with CM from unstimulated MSCs (p=0.74 for RH and p=0.69 for ME49). We observed that the resistance in hMSCs does not depend on IDO (p=0.85 for RH and p=0.79 for ME49). RNA-seq data showed that IFN-γ-inducible p65 guanylate-binding proteins (GBPs) might play pivotal roles in the inhibition of T. gondii growth. Reads per kilobase-pairs per million (RPKM) mean values of GBP1, 2, 5 in IFN-γ-stimulated MSCs are 1093.3, 443.3, 348.2, respectively. By RNAi knockdown, the results showed that silencing of GBP1 (but not GBP2, GBP5) in hMSCs resulted in recovery of T. gondii growth inhibition at 12 hr and 24 hr postinfection (p<0.05 and p<0.001 for ME49).Conclusion: Human MSCs pre-stimulated with IFN-γ inhibited the growth of T. gondii in a dose-dependent manner via up-regulation of GBP-1 expression. DisclosuresLiu:the project of the Zhujiang Science & Technology Star of Guangzhou city (2013027): Research Funding; the Technology Plan of Guangdong Province of China (2012B031800403): Research Funding; the project of health collaborative innovation of Guangzhou city (201400000003-4, 201400000003-1): Research Funding; Natural Science Foundation of Guangdong Province (S2012010009299): Research Funding; National Public Health Grand Research Foundation (201202017): Research Funding; National High Technology Research and Development Program of China (863 Program) (2011AA020105): Research Funding; National Natural Science Foundation of China (81270647, 81300445, 81200388): Research Funding.

Restitution of gene expression and histone acetylation signatures altered by hepatitis B virus through antiviral microRNA-like molecules in nontransformed murine hepatocytes.

BackgroundVirus-host interactions result in altered gene expression profiles in host cell nuclei and enable virus particle production, thus obligatorily involving changes in their epigenomes. Neither such epigenome changes nor their response to antiviral treatment have been extensively studied to date, although viral infections are known to contribute to the long-term development of severe secondary diseases, for example, hepatocellular carcinoma. This may be causally linked to virus-induced persistent plastic chromatin deformations.ResultsWe studied whether impaired hepatitis B virus (HBV) replication can lead to the restitution of epigenome signatures hypothesizing that hepatocytes alternatively could adopt a ‘memory’ state of the infection; that is, the chromatin could persist in a HBV-induced configuration potentially inheritable between dividing hepatocytes. We therefore determined epigenomic signatures and gene expression changes altered by HBV and the effects of suppressed HBV replication in nontransformed hepatocytes of newborn mice. Further we investigated differential histone acetyltransferase and histone deacetylase activities in HBV-negative and HBVpositive hepatocytes, as well as the effects of HBV suppression on gene expression and the chromatin landscape. We show that the expression of several genes and the chromatin landscape become altered upon HBV infection, including global hypoacetylation of H2A.Z and H3K9. Reporter assays monitoring the activities of histone acetyltransferases or histone deacetylases, respectively, suggest that hypoacetylation most probably depends on elevated sirtuin deacetylase activity, but not on class I/II histone deacetylases. Using Micrococcus nuclease to study the chromatin accessibility in met murine-D3 and hepatitis B virus met murine hepatocytes, we demonstrate that the observed differences in H2A.Z/H3K9 acetylation lead to global chromatin structure changes. At all selected sites examined by chromatin immunoprecipitation and quantitative real-time PCR, these effects can be partly restituted via the nucleoside analog reverse transcriptase inhibitor 3TC or using anti-HBV microRNA-like molecules.ConclusionsIncreased sirtuin activity might lead to global histone hypoacetylation signatures, which could contribute to the HBV-induced pathomechanism in nontransformed hepatocytes. Using several techniques to suppress HBV replication, we observed restituted gene expression and chromatin signature patterns reminiscent of noninfected hepatocytes. Importantly, ectopic expression of antiviral short-hairpin RNA, but not microRNA-like molecules, provoked intolerable off-target effects on the gene expression level.Electronic supplementary materialThe online version of this article (doi:10.1186/1868-7083-6-26) contains supplementary material, which is available to authorized users.

Construction and expression of lentiviral vectors encoding recombinant mouse CREBZF in NIH 3T3 cells

CREBZF, also known as Zhangfei or SMILE, is a member of the CREB/ATF protein family. CREBZF has mainly been considered as a basic region-leucine zipper transcription factor that functions in coordination with other transcription factors and plays a role in latent HSV-1 infection, apoptosis and the mammalian endoplasmic reticulum stress and unfolded protein response. In this study, we constructed recombinant lentiviral vectors for CREBZF short hairpin RNA (shRNA) expression and over-expression to improve understanding of the mechanisms regulating CREBZF. The CREBZF ORF sequence was cloned into the lentiviral shuttle plasmid pCD513B-1, and various shRNA oligonucleotides and one negative control (shN) were cloned into the pCD513B-U6 expression vector. The recombinant lentivirus was packaged and transduced into NIH 3T3 cells. CREBZF mRNA and protein expression were examined using real-time reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting, respectively. The over-expression vector and the most effective shRNA vector significantly affected the expression of CREBZF mRNA and protein. Both of the CREBZF recombinant lentiviral vectors were successfully constructed. The over-expression vector significantly increased the expression of exogenous CREBZF and inhibited the growth of NIH 3T3 cells compared to controls. The most effective shRNA lentiviral vector, pCD513B-U6-CREBZF-shRNA-3, was transformed, leading to significant knockdown of the CREBZF gene. We conclude that CREBZF the recombinant lentiviral vectors are promising tools for regulating the expression of CREBZF in NIH 3T3 cells.

Breast Cancer: Coordinated Regulation of CCL2 Secretion by Intracellular Glycosaminoglycans and Chemokine Motifs

The chemokine CCL2 (MCP-1) has been identified as a prominent tumor-promoting factor in breast cancer. The major source for CCL2 is in the tumor cells; thus, identifying the mechanisms regulating CCL2 release by these cells may enable the future design of modalities inhibiting CCL2 secretion and consequently reduce tumorigenicity. Using cells deficient in expression of glycosaminoglycans (GAGs) and short hairpin RNAs reducing heparan sulfate (HS) and chondroitin sulfate (CS) expression, we found that intracellular HS and CS (=GAGs) partly controlled the trafficking of CCL2 from the Golgi toward secretion. Next, we determined the secretion levels of GFP-CCL2-WT and GFP-CCL2-variants mutated in GAG-binding domains and/or in the 40s loop of CCL2 (45TIVA48). We have identified partial roles for R18+K19, H66, and the 45TIVA48 motif in regulating CCL2 secretion. We have also demonstrated that in the absence of R24 or R18+K19+45TIVA48, the secretion of CCL2 by breast tumor cells was almost abolished. Analyses of the intracellular localization of GFP-CCL2-mutants in the Golgi or the endoplasmic reticulum revealed particular intracellular processes in which these CCL2 sequences controlled its intracellular trafficking and secretion. The R24, 45TIVA48 and R18+K19+45TIVA48 domains controlled CCL2 secretion also in other cell types. We propose that targeting these chemokine regions may lead to reduced secretion of CCL2 by breast cancer cells (and potentially also by other malignant cells). Such a modality may limit tumor growth and metastasis, presumably without affecting general immune activities (as discussed below).

Lentivirus Mediated Gene Manipulation in Trophectoderm of Porcine Embryos

Development of tools that can manipulate gene expression specifically and efficiently in the trophectoderm (TE) lineage would greatly aid understanding the roles of different genetic pathways in TE versus embryonic lineages. Here, we showed first time that short-term lentivirus infection of porcine blastocysts could lead to rapid expression of transgene specifically in TE cells. Efficient TE-specific gene knockdown could also be achieved by lentivirus-mediated pol III-driven short hairpin RNA (shRNA) and TE-specific gene expression could be temporal controlled efficiently by combining this system with Tet-On system. This lentivirus lineage-specific infection system would facilitate gene function studies in porcine pre-implatation embryos by specifically knockdown or overexpression of these genes in TE.

Modulation of hepatitis C virus genome replication by glycosphingolipids and four-phosphate adaptor protein 2.

Hepatitis C virus (HCV) assembles its replication complex on cytosolic membrane vesicles often clustered in a membranous web (MW). During infection, HCV NS5A protein activates PI4KIIIα enzyme, causing massive production and redistribution of phosphatidylinositol 4-phosphate (PI4P) lipid to the replication complex. However, the role of PI4P in the HCV life cycle is not well understood. We postulated that PI4P recruits host effectors to modulate HCV genome replication or virus particle production. To test this hypothesis, we generated cell lines for doxycycline-inducible expression of short hairpin RNAs (shRNAs) targeting the PI4P effector, four-phosphate adaptor protein 2 (FAPP2). FAPP2 depletion attenuated HCV infectivity and impeded HCV RNA synthesis. Indeed, FAPP2 has two functional lipid-binding domains specific for PI4P and glycosphingolipids. While expression of the PI4P-binding mutant protein was expected to inhibit HCV replication, a marked drop in replication efficiency was observed unexpectedly with the glycosphingolipid-binding mutant protein. These data suggest that both domains are crucial for the role of FAPP2 in HCV genome replication. We also found that HCV significantly increases the level of some glycosphingolipids, whereas adding these lipids to FAPP2-depleted cells partially rescued replication, further arguing for the importance of glycosphingolipids in HCV RNA synthesis. Interestingly, FAPP2 is redistributed to the replication complex (RC) characterized by HCV NS5A, NS4B, or double-stranded RNA (dsRNA) foci. Additionally, FAPP2 depletion disrupts the RC and alters the colocalization of HCV replicase proteins. Altogether, our study implies that HCV coopts FAPP2 for virus genome replication via PI4P binding and glycosphingolipid transport to the HCV RC. Like most viruses with a positive-sense RNA genome, HCV replicates its RNA on remodeled host membranes composed of lipids hijacked from various internal membrane compartments. During infection, HCV induces massive production and retargeting of the PI4P lipid to its replication complex. However, the role of PI4P in HCV replication is not well understood. In this study, we have shown that FAPP2, a PI4P effector and glycosphingolipid-binding protein, is recruited to the HCV replication complex and is required for HCV genome replication and replication complex formation. More importantly, this study demonstrates, for the first time, the crucial role of glycosphingolipids in the HCV life cycle and suggests a link between PI4P and glycosphingolipids in HCV genome replication.

Efficient Transient Transfection of Human Multiple Myeloma Cells by Electroporation – An Appraisal

Cell lines represent the everyday workhorses for in vitro research on multiple myeloma (MM) and are regularly employed in all aspects of molecular and pharmacological investigations. Although loss-of-function studies using RNA interference in MM cell lines depend on successful knockdown, no well-established and widely applied protocol for efficient transient transfection has so far emerged. Here, we provide an appraisal of electroporation as a means to introduce either short-hairpin RNA expression vectors or synthesised siRNAs into MM cells. We found that electroporation using siRNAs was much more efficient than previously anticipated on the basis of transfection efficiencies deduced from EGFP-expression off protein expression vectors. Such knowledge can even confidently be exploited in “hard-to-transfect” MM cell lines to generate large numbers of transient knockdown phenotype MM cells. In addition, special attention was given to developing a protocol that provides easy implementation, good reproducibility and manageable experimental costs.

Development of a multipurpose GATEWAY-based lentiviral tetracycline-regulated conditional RNAi system (GLTR).

RNA interference (RNAi) has become an essential technology for functional gene analysis. Its success, however, depends on the effective expression of RNAi-inducing small double-stranded interfering RNA molecules (siRNAs) in target cells. In many cell types, RNAi can be achieved by transfection of chemically synthesised siRNAs, which results in transient knockdown of protein expression. Expression of double-stranded short hairpin RNA (shRNA) provides another means to induce RNAi in cells that are hard to transfect. To facilitate the generation of stable, conditional RNAi cell lines, we have developed novel one- and two-component vector GATEWAY-compatible lentiviral tetracycline-regulated RNAi (GLTR) systems. The combination of a modified RNA-polymerase-III-dependent H1 RNA promoter (designated ‘THT’) for conditional shRNA expression with different lentiviral delivery vectors allows (1) the use of fluorescent proteins for colour-coded combinatorial RNAi or for monitoring RNAi induction (pGLTR-FP), (2) selection of transduced cells (pGLTR-S), and (3) the generation of conditional cell lines using a one vector system (pGLTR-X). All three systems were found to be suitable for the analysis of essential genes, such as CDC27, a component of the mitotic ubiquitin ligase APC/C, in cell lines and primary human cells.

Pseudomonas aeruginosa–Induced Apoptosis in Airway Epithelial Cells Is Mediated by Gap Junctional Communication in a JNK-Dependent Manner

Chronic infection and inflammation of the airways is a hallmark of cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The response of the CF airway epithelium to the opportunistic pathogen Pseudomonas aeruginosa is characterized by altered inflammation and apoptosis. In this study, we examined innate immune recognition and epithelial responses at the level of the gap junction protein connexin43 (Cx43) in polarized human airway epithelial cells upon infection by PAO1. We report that PAO1 activates cell surface receptors to elicit an intracellular signaling cascade leading to enhancement of gap junctional communication. Expression of Cx43 involved an opposite regulation exerted by JNK and p38 MAPKs. PAO1-induced apoptosis was increased in the presence of a JNK inhibitor, but latter effect was prevented by lentiviral expression of a Cx43-specific short hairpin RNA. Moreover, we found that JNK activity was upregulated by pharmacological inhibition of CFTR in Calu-3 cells, whereas correction of a CF airway cell line (CF15 cells) by adenoviral expression of CFTR reduced the activation of this MAPK. Interestingly, CFTR inhibition in Calu-3 cells was associated with decreased Cx43 expression and reduced apoptosis. These results indicate that Cx43 expression is a component of the response of airway epithelial cells to innate immune activation by regulating the survival/apoptosis balance. Defective CFTR could alter this equilibrium with deleterious consequences on the CF epithelial response to P. aeruginosa.

Enhanced flexibility of place discrimination learning by targeting striatal cholinergic interneurons.

Behavioural flexibility is mediated through the neural circuitry linking the prefrontal cortex and basal ganglia. Here we conduct selective elimination of striatal cholinergic interneurons in transgenic rats by immunotoxin-mediated cell targeting. Elimination of cholinergic interneurons from the dorsomedial striatum (DMS), but not from the dorsolateral striatum, results in enhanced reversal and extinction learning, sparing the acquisition of place discrimination. This enhancement is prevented by infusion of a non-selective muscarinic acetylcholine receptor agonist into the DMS either in the acquisition, reversal or extinction phase. In addition, gene-specific silencing of M4 muscarinic receptor by lentiviral expression of short hairpin RNA (shRNA) mimics the place reversal learning promoted by cholinergic elimination, whereas shRNA-mediated gene silencing of M1 muscarinic receptor shows the normal performance of reversal learning. Our data indicate that DMS cholinergic interneurons inhibit behavioural flexibility, mainly through the M4 muscarinic receptor, suggesting that this role is engaged to the stabilization of acquired reward contingency and the suppression of response switch to changed contingency.