Drive Gene Expression Research Articles

Transcriptional Regulation of AQP-8, a Caenorhabditis elegans Aquaporin Exclusively Expressed in the Excretory System, by the POU Homeobox Transcription Factor CEH-6

Due to the ever changing environmental conditions in soil, regulation of osmotic homeostasis in the soil-dwelling nematode Caenorhabditis elegans is critical. AQP-8 is a C. elegans aquaporin that is expressed in the excretory cell, a renal equivalent tissue, where the protein participates in maintaining water balance. To better understand the regulation of AQP-8, we undertook a promoter analysis to identify the aqp-8 cis-regulatory elements. Using progressive 5' deletions of upstream sequence, we have mapped an essential regulatory region to roughly 300 bp upstream of the translational start site of aqp-8. Analysis of this region revealed a sequence corresponding to a known DNA functional element (octamer motif), which interacts with POU homeobox transcription factors. Phylogenetic footprinting showed that this site is perfectly conserved in four nematode species. The octamer site's function was further confirmed by deletion analyses, mutagenesis, functional studies, and electrophoretic mobility shift assays. Of the three POU homeobox proteins encoded in the C. elegans genome, CEH-6 is the only member that is expressed in the excretory cell. We show that expression of AQP-8 is regulated by CEH-6 by performing RNA interference experiments. CEH-6's mammalian ortholog, Brn1, is expressed both in the kidney and the central nervous system and binds to the same octamer consensus binding site to drive gene expression. These parallels in transcriptional control between Brn1 and CEH-6 suggest that C. elegans may well be an appropriate model for determining gene-regulatory networks in the developing vertebrate kidney.

Promoter Choice for Retroviral Vectors: Transcriptional Strength Versus Trans-Activation Potential

Gene expression from retroviral vectors can be driven by either the retroviral long terminal repeat (LTR) promoter or by cellular or viral promoters located internally in an LTR-deleted self-inactivating vector design. Adverse events in a gene therapy clinical trial for X-linked severe combined immune deficiency have led to the realization that the enhancer/promoter elements contained within integrated vectors may also act outside the vector genome to trans-activate host genes. Ideally, the gene expression system chosen for a vector should possess a low probability of trans-activation while still being able to support adequate levels of transgene expression. However, the parameters that define these specific characteristics are unknown. To gain insight into the mechanism of trans-activation, we compared a panel of commonly used retroviral LTRs and cellular and viral promoters for their ability to drive gene expression and to trans-activate a nearby minimal promoter in three different cell lines. These studies identified two elements, the cytomegalovirus enhancer/chicken beta-actin (CAG) and elongation factor (EF)-1alpha promoters, as being of potential value for use in vectors targeting lymphoid cells, as these elements exhibited both high levels of reporter gene expression and relatively low levels of trans-activation in T cells.

Recent advances in barley transformation

Barley, an important member of the cereals, has been successfully transformed through various methods such as particle bombardment, Agrobacterium tumefaciens, DNA uptake, and electroporation. Initially, the transformation in barley concentrated on developing protocols using marker genes such as gus, bar, and hpt. Immature embryos and callus derived from immature embryos were targeted for transformation. Subsequently, genes of agronomic and malting importance have been deployed in barley. Particle bombardment appears to be the preferred choice for barley transformation in the majority of the reports, although Agrobacterium-mediated transformation is being used more often. The current review focuses on the challenges encountered in barley transformation such as somaclonal variation, development of transformation systems for commercial cultivars, gene expression, stability and inheritance, and gene flow. Newer markers such as the green fluorescent protein (gfp), firefly luciferase, and phosphomannose isomerase were found to be useful in the selection of transgenic plants. Tissue-specific promoters such as those for B1-hordein and D-hordein genes, and spike-specific promoters, are increasingly used to drive gene expression. The review also describes recent research on gene-tagging through transformation, insertion of disease resistance, and abiotic stress resistance genes, transformation with genes for improved malting quality, nutrient content, feed quality, and the production of feed enzymes and pharmaceutical compounds.

Efficiency of the Elongation Factor-1α Promoter in Mammalian Embryonic Stem Cells Using Lentiviral Gene Delivery Systems

The establishment of new technology for genetic modification in human embryonic stem (ES) cell lines has raised great hopes for achieving new ground in basic and clinical research. Recently, lentiviral vector technology has been shown to be highly effective and therefore could emerge as a popular tool for human ES cell genetic modification. The objectives of this study were to evaluate the efficiency of promoters in lentiviral gene delivery systems in mammalian ES cells, including mouse, monkey, and human, and to construct efficient and optimized conditions for lentivirus-mediated transfection systems. Mammalian ES cells were transfected with self-inactivating (SIN) human immunodeficiency virus type-1 (HIV-1)-based lentiviral vectors containing the human polypeptide chain elongation factor-1alpha (EF-1alpha) promoter or cytomegalovirus (CMV) promoter and analyzed by fluorescence-activated cell sorting (FACS) analysis for the expression of the enhanced green fluorescent protein (eGFP) reporter gene. The efficiency of the EF-1alpha promoter was higher than that of the CMV promoter in all ES cells tested. The EF-1alpha promoter efficiently drove gene expression (14.74%) compared with CMV promoter (3.69%) in human ES cells. We generated a stable eGFP+ human ES cell line (CHA3-EGFP human ES cells) that continuously expressed high levels of EGFP ( approximately 95%) from the EF-1alpha promoter and was maintained for up to 60 weeks with undifferentiated proliferation. The established CHA3-EGFP human ES cell lines were characterized as being negative for nondifferentiation markers and teratoma formation. These results imply that genetic modification by lentiviral vectors with specific promoters in ES cells constitute a powerful tool for guided differentiation as well as gene therapy.

Specific expression of lacZ and cre recombinase in fetal thymic epithelial cells by multiplex gene targeting at the Foxn1 locus

BackgroundThymic epithelial cells (TECs) promote thymocyte maturation and are required for the early stages of thymocyte development and for positive selection. However, investigation of the mechanisms by which TECs perform these functions has been inhibited by the lack of genetic tools. Since the Foxn1 gene is expressed in all presumptive TECs from the early stages of thymus organogenesis and broadly in the adult thymus, it is an ideal locus for driving gene expression in differentiating and mature TECs.ResultsWe generated two knock-in alleles of Foxn1 by inserting IRES-Cre or IRES-lacZ cassettes into the 3' UTR of the Foxn1 locus. We simultaneously electroporated the two targeting vectors to generate the two independent alleles in the same experiment, demonstrating the feasibility of multiplex gene targeting at this locus. Our analysis shows that the knockin alleles drive expression of Cre or lacZ in all TECs in the fetal thymus. Furthermore, the knockin alleles express Cre or lacZ in a Foxn1-like pattern without disrupting Foxn1 function as determined by phenotype analysis of Foxn1 knockin/Foxn1 null compound heterozygotes.ConclusionThese data show that multiplex gene targeting into the 3' UTR of the Foxn1 locus is an efficient method to express any gene of interest in TECs from the earliest stage of thymus organogenesis. The resulting alleles will make possible new molecular and genetic studies of TEC differentiation and function. We also discuss evidence indicating that gene targeting into the 3' UTR is a technique that may be broadly applicable for the generation of genetically neutral driver strains.

Exclusive developmental functions of gatae cis-regulatory modules in the Strongylocentrorus purpuratus embryo

The gatae gene of Strongylocentrotus purpuratus is orthologous to vertebrate gata-4,5,6 genes. This gene is expressed in the endomesoderm in the blastula and later the gut of the embryo, and is required for normal development. A gatae BAC containing a GFP reporter knocked into exon one of the gene was able to reproduce all aspects of endogenous gatae expression in the embryo. To identify putative gatae cis-regulatory modules we carried out an interspecific sequence conservation analysis with respect to a Lytechinus variegatus gatae BAC, which revealed 25 conserved non-coding sequence patches. These were individually tested in gene transfer experiments, and two modules capable of driving localized reporter expression in the embryo were identified. Module 10 produces early expression in mesoderm and endoderm cells up to the early gastrula stage, while module 24 generates late endodermal expression at gastrula and pluteus stages. Module 10 was then deleted from the gatae BAC by reciprocal recombination, resulting in total loss of reporter expression in the time frame in which it is normally active. Similar deletion of module 24 led to ubiquitous GFP expression in the gastrula and pluteus. These results show that Module 10 is uniquely necessary and sufficient to account for the early phase of gatae expression during endomesoderm specification. In addition, they imply a functional cis-regulatory module exclusion, whereby only a single module can associate with the basal promoter and drive gene expression at any given time.

Coupling the GAL4 UAS system with alcR for versatile cell type‐specific chemically inducible gene expression in Arabidopsis

The Aspergillus alc regulon encodes a transcription factor, ALCR, which regulates transcription from cognate promoters such as alcA(p). In the presence of suitable chemical inducers, ALCR activates gene expression from alcA(p). The alc regulon can be transferred to other species and can be used to control the expression of reporter, metabolic and developmental genes in response to low-level ethanol exposure. In this paper, we describe a versatile system for targeting the alc regulon to specific cell types in Arabidopsis by driving ALCR expression from the GAL4 upstream activator sequence (UAS). Large numbers of Arabidopsis lines are available in which GAL4 is expressed in a variety of spatial patterns and, in turn, drives the expression of any gene cloned downstream of the UAS. We have used a previously characterized line that directs gene expression to the endosperm to demonstrate spatially restricted ethanol-inducible gene expression. We also show that the domain of inducible gene expression can easily be altered by crossing the UAS::ALCR cassette into different driver lines. We conclude that this gene switch can be used to drive gene expression in a highly responsive, but spatially restricted, manner.

Functional characterization of putative promoter elements from infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp and in insect and fish cell lines

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) of shrimp contains a linear single-stranded DNA genome of approximately 4.1 kb with three putative open reading frames (ORFs) on the same DNA strand designated, the Left, Middle, and Right ORFs. The Left ORF codes for non-structural protein and the Right ORF codes for capsid protein, whereas the role of the Middle ORF is still unknown. Two putative promoters, designated P2 and P61, were detected upstream of the Left ORF and Right ORF, respectively. We evaluated the activities of these two promoters with or without a transcriptional enhancer element via the use of firefly luciferase reporter constructs in insect and fish cells, and in shrimp tail muscle. In insect and fish cells, the P2 promoter was stronger than the P61 promoter. The presence of the SV40 enhancer element negatively affected P2 but not P61 promoter activity in insect cells. However, in fish cells, the SV40 enhancer element dramatically increased the activities of both promoters. In shrimp, there was no significant difference in luciferase expression driven by these two promoters. In shrimp tail muscle, the presence of SV40 enhancer element in the construct had no significant effect on the P2 promoter and a negative effect on the P61 promoter. The IHHNV P2 and P61 promoters were found to be constitutive promoters that can drive gene expression in both invertebrate and vertebrate hosts.

C-Jun Homodimers Can Function as a Context-Specific Coactivator

Transcription factors can function as DNA-binding-specific activators or as coactivators. c-Jun drives gene expression via binding to AP-1 sequences or as a cofactor for PU.1 in macrophages. c-Jun heterodimers bind AP-1 sequences with higher affinity than homodimers, but how c-Jun works as a coactivator is unknown. Here, we provide in vitro and in vivo evidence that c-Jun homodimers are recruited to the interleukin-1beta (IL-1beta) promoter in the absence of direct DNA binding via protein-protein interactions with DNA-anchored PU.1 and CCAAT/enhancer-binding protein beta (C/EBPbeta). Unexpectedly, the interaction interface with PU.1 and C/EBPbeta involves four of the residues within the basic domain of c-Jun that contact DNA, indicating that the capacities of c-Jun to function as a coactivator or as a DNA-bound transcription factor are mutually exclusive. Our observations indicate that the IL-1beta locus is occupied by PU.1 and C/EBPbeta and poised for expression and that c-Jun enhances transcription by facilitating a rate-limiting step, the assembly of the RNA polymerase II preinitiation complex, with minimal effect on the local chromatin status. We propose that the basic domain of other transcription factors may also be redirected from a DNA interaction mode to a protein-protein interaction mode and that this switch represents a novel mechanism regulating gene expression profiles.

The pallial basal ganglia pathway modulates the behaviorally driven gene expression of the motor pathway

The discrete neural network for songbird vocal communication provides an effective system to study neural mechanisms of learned motor behaviors in vertebrates. This system consists of two pathways--a vocal motor pathway used to produce learned vocalizations and a vocal pallial basal ganglia loop used to learn and modify the vocalizations. However, it is not clear how the loop exerts control over the motor pathway. To study the mechanism, we used expression of the neural activity-induced gene ZENK (or egr-1), which shows singing-regulated expression in a social context-dependent manner: high levels in both pathways when singing undirected and low levels in the lateral part of the loop and in the robust nucleus of the arcopallium (RA) of the motor pathway when singing directed to another animal. Here, we show that there are two parallel interactive parts within the pallial basal ganglia loop, lateral and medial, which modulate singing-driven ZENK expression of the motor pathway nuclei RA and HVC, respectively. Within the loop, the striatal and pallial nuclei appear to have opposing roles; the striatal vocal nucleus lateral AreaX is required for high ZENK expression in its downstream nuclei, particularly during undirected singing, while the pallial vocal lateral magnocellular nucleus of the anterior nidopallium is required for lower expression, particularly during directed singing. These results suggest a dynamic molecular interaction between the basal ganglia pathway and the motor pathway during production of a learned motor behavior.

Evaluation of promoters for driving efficient transgene expression in neonatal porcine islets

There is considerable interest in the viral modification of insulin-producing islets, including porcine islets, in the context of islet xenotransplantation to treat type 1 diabetes. Adenovirus (Adv) gene delivery offers the potential to modify pre-transplant islets for enhanced survival. Modifications include transfer of cytoprotective molecules to ensure islet survival immediately post-transplant, and molecules to dampen the immune system and prevent chronic islet graft rejection. In this study, we compared different promoters (three promiscuous and two tissue-specific promoters) for their efficiency in driving gene expression in neonatal pig islet tissue after Adv delivery. We also compared the efficiency of these promoters in adult islets from mouse and human pancreata. We observed that the promiscuous cytomegalovirus promoter was the most potent, eliciting high luciferase expression in neonatal pig islets, as well as in human and mouse islets. In contrast, the mammalian EF1-alpha promoter educed comparatively intermediate gene expression. The mouse major histocompatibility complex class I promoter H-2K(b) and the pancreatic-specific promoters insulin and human pdx-1 (area II) performed poorly in islets from all three species. This has important implications for the generation of modified neonatal pig islets for transplantation into humans.

Generation of Inhibitory NFκB Complexes and Phosphorylated cAMP Response Element-binding Protein Correlates with the Anti-inflammatory Activity of Complement Protein C1q in Human Monocytes

The interaction of C1q with specific cells of the immune system induces activities, such as enhancement of phagocytosis in monocytes and stimulation of superoxide production in neutrophils. In contrast to some other monocyte activators, C1q itself does not induce pro-inflammatory cytokine production, but rather inhibits the lipopolysaccharide (LPS)-stimulated induction of certain pro-inflammatory cytokines and induces expression of interleukin-10. To investigate the molecular mechanism by which C1q exerts this effect on gene expression, the influence of C1q on the activation of transcription factors of the NFkappaB family and cAMP response element-binding protein (CREB) was assessed. C1q treatment increased kappaB binding activity in freshly isolated human monocytes in a time-dependent fashion as assessed by electrophoretic mobility shift assays. In antibody supershift experiments, anti-p50 antibody supershifted the C1q-induced NFkappaB complex, whereas anti-p65 antibody had little effect, suggesting that C1q induced the translocation of NFkappaB p50p50 homodimers. This is in contrast to the dominant induction of p65 containing complexes in parallel monocyte cultures stimulated with LPS. C1q treatment also induced cAMP response element (CRE)-binding activity as demonstrated by electrophoretic mobility shift assay, increased phosphorylation of CREB, and induction of CRE driven gene expression. In contrast, CREB activation was not detected in LPS-treated monocytes. These results suggest that C1q may modulate the cytokine profile expressed in response to inflammatory stimuli (e.g. LPS), by triggering inhibitory and/or competing signals. Because C1q and other defense collagens have been shown to enhance clearance of apoptotic cells, this regulatory pathway may be beneficial in avoiding autoimmunity and/or resolving inflammation.

HCV NS5A inhibits interferon-α signaling through suppression of STAT1 phosphorylation in hepatocyte-derived cell lines

HCV NS5A appears to play an important role in HCV resistance to IFN-alpha but the molecular mechanism is not fully elucidated. Most studies regarding the involvement of signal transducer and activator of transcription 1 (STAT1) in inhibition of IFN-alpha signaling by NS5A were performed in non-hepatic cell lines and their relevance may be debatable. We analyzed the effects of NS5A on IFN-alpha signaling through STAT1 phosphorylation in three hepatocyte-derived cell lines, Hep3B, J5 and Huh7. Interaction of NS5A and STAT1 was also investigated with co-immunoprecipitation and confocal microscopy. IFN-alpha induces STAT1 activation in Hep3B cells in a dose- and time-dependent manner. Transient or stable NS5A expression inhibits STAT1 phosphorylation in a dose-dependent manner in hepatocyte-derived cell lines, whereas the levels of STAT1 phosphorylation remain unchanged in non-hepatocyte HeLa and COS7 cells despite increasing amounts of NS5A. The NS5A may interact with STAT1, specifically, the N-terminal 488 amino acids of STAT1. Furthermore, NS5A inhibits activation of interferon-stimulated gene factor 3 (ISGF3) and interferon-stimulated response element (ISRE)-driven gene expression, as demonstrated by electrophoretic mobility shift assay and luciferase assay, respectively. NS5A may interact with STAT1 and inhibit IFN-alpha signaling through suppression of STAT1 phosphorylation specifically in hepatocyte-derived cells.

Temporal and spatial control of transgene expression using laser induction of the hsp70promoter

BackgroundPrecise temporal and spatial regulation of transgene expression is a critical tool to investigate gene function in developing organisms. The most commonly used technique to achieve tight control of transgene expression, however, requires the use of specific DNA enhancers that are difficult to characterize in non-model organisms. Here, we sought to eliminate the need for this type of sequence-based gene regulation and to open the field of functional genetics to a broader range of organisms.ResultsWe have developed a new laser mediated method to heat shock groups of cells that provides precise spatio-temporal control of gene expression without requiring knowledge of specific enhancer sequences. We tested our laser-system in a transgenic line of Bicyclus anynana butterflies containing the EGFP reporter gene attached to the heat sensitive hsp70 promoter of Drosophila melanogaster. Whole organismal heat shocks demonstrated that this Drosophila promoter can drive gene expression in butterflies, and the subsequent laser heat shocks showed that it was possible to activate cell-specific gene expression in very precise patterns on developing pupal wings.ConclusionThis laser-mediated gene expression system will enable functional genetic investigations, i.e., the ectopic expression of genes and their knock-down in targeted groups of cells in model and non-model organisms with little or no available regulatory data, as long as a compatible heat-shock promoter is used and the target tissue is accessible to a laser beam. This technique will also be useful in evolutionary developmental biology as it will enable the study of the evolution of gene function across a variety of organisms.

Novel Function of Prothymosin Alpha as a Potent Inhibitor of Human Immunodeficiency Virus Type 1 Gene Expression in Primary Macrophages

CD8(+) T lymphocytes control human immunodeficiency virus type 1 (HIV-1) infection by a cytotoxic major histocompatibility complex-restricted pathway as well as by secretion of noncytotoxic soluble inhibitory factors. Several components of CD8(+) cell supernatants have been identified that contribute to the latter activity. In this study we report that prothymosin alpha (ProTalpha), a protein found in the cell culture medium of the herpesvirus saimiri-transformed CD8(+) T-cell line, K#1 50K, has potent HIV-1-inhibitory activity. Depletion of native ProTalpha from an HIV-1-inhibitory fraction of CD8(+) cell supernatants removes the inhibitory activity, supporting its role in inhibition via soluble mediators. ProTalpha is an abundant, acidic peptide that has been reported to be localized in the nucleus and associated with cell proliferation and activation of transcription. In this report we demonstrate that ProTalpha suppresses HIV-1 replication, its activity is target cell specific, and inhibition occurs following viral integration. Native and recombinant ProTalpha protein potently inhibit HIV-1 long terminal repeat (LTR)-driven gene expression in macrophages. Furthermore studies using different promoters in lentiviral vectors (cytomegalovirus and phosphoglycerate kinase) revealed that suppression of viral replication by ProTalpha is not HIV LTR specific.

Nuclear factor kappa B in the dorsal raphe of macaques: Anatomical link for steroids, cytokines and serotonin

Nuclear factor kappa B (NFkappaB) is a transcription factor that activates gene expression in response to proinflammatory cytokines, and elevated cytokines are associated with depression, which has a serotonergic component. We questioned (1) whether serotonin neurons contain NFkappaB, (2) whether NFkappaB detection with immunocytochemistry is changed in the dorsal raphe nucleus (DRN) by ovarian hormone treatment and (3) whether ovarian hormones regulate midbrain NFkappaB gene or protein expression. Monkeys were spayed and treated with placebo, estrogen (E), progesterone (P) or E+P for 1 month (n = 4 animals/treatment group), and the midbrain was harvested for immunocytochemistry and stereology. An antibody that detects nuclear location-specific (NLS)-NFkappaB p65 was applied, and the numbers of NLS-NFkappaB-immunopositive cells were counted in 9 sections of the DRN. Additional monkeys were used for Western blot analysis and quantitative reverse transcription-polymerase chain reaction (RT-PCR) for NFkappaB p65. In placebo-treated macaques, neurons were double-immunostained for serotonin and nuclear NFkappaB p65 throughout the DRN. The mean total number of NFkappaB-positive cells equalled 2178 (and standard error of the mean [SEM] 129) in the placebo group, 1631 (SEM 221) in the E-treated group, 2314 (SEM 186) in the P-treated group and 1162 (SEM 100) in the E+P-treated group (analysis of variance p = 0.003). The E-treated and E+P-treated groups had a significantly lower density of cells stained positive for NFkappaB than the placebo or P-treated groups (post hoc). Unmasking of NLS-NFkappaB immunostaining in the DRN revealed dense immunostaining in the cytoplasm of large dorsal raphe neurons. There was no difference between treatment groups in the amount of NFkappaB p65 detected by Western blot or in the relative expression of NFkappaB p65 mRNA with quantitative RT-PCR. These observations are consistent with the notion that gene and protein expression of NFkappaB are constitutive but that ovarian hormones can decrease the nuclear location of NFkappaB in dorsal raphe neurons and, thereby, decrease the ability of NFkappaB to drive gene expression in response to cytokines.

Activation of Human CYP2C9 Promoter and Regulation by CAR and PXR in Mouse Liver

The activity of various genomic segments at the 5'-flanking region of the human CYP2C9 gene in driving gene expression and their involvement in pregnane X receptor (PXR) and constitutive androstane receptor (CAR) mediated activation were evaluated in mouse hepatocytes. Using the genomic sequence of human CYP2C9 as a template, segments covering different regions of CYP2C9 5'-flanking sequences starting from the translation start site were amplified by PCR and inserted into a pGL-3 luciferase vector. Plasmid DNA containing the 0.2K, 1K, 2K, 3K, 5K, or 10K upstream sequences of the CYP2C9 gene were transfected into mouse liver by hydrodynamic delivery, and the activity of each fragment in driving reporter gene expression was assessed. With the exception of the 10K fragment, the level of luciferase activity in transfected mouse liver was similar among the constructs examined. Cotransfection of these reporter constructs with the pCMX-PXR or pCMX-CAR plasmids resulted in a slight increase in luciferase gene expression that could be significantly enhanced by chemical inducers. In mice cotransfected with pCMX-PXR, pregnenolone-16 alpha-carbonitrile (PCN) induced a 20-fold increase in the luciferase level compared to a 70-fold increase induced by rifampicin. Similarly, when animals were cotransfected with the pCMX-CAR plasmid, phenobarbital and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene enhanced luciferase gene expression by 10- and 57-fold, respectively. The element responsible for PXR- and CAR-mediated activation of luciferase gene expression by chemical inducers was found to reside in the -2000 to -1000 bp region of the 5'-flanking sequence of the CYP2C9 gene. These results prove that PXR and CAR are transcription factors regulating CYP2C9 gene expression.

Primary cilia as biosensors for blood flow: lessons from cardiovascular development

Background Blood flow induced fluid shear stress is a determinative factor in cardiovascular development and pathology, as it drives gene expression in endothelial cells. Extensive remodeling during development alters patterns of this hemodynamic force considerably. The mechanism by which endothelial cells sense these alterations was unclear. We postulate a role for primary cilia as fluid shear stress sensors on endothelial cells. Such a function has already been attributed to cilia on epithelial cells of the adult kidney and Hensen’s node in the embryo. Methods and Results We show shear stress related distribution of cellular protrusions within the cardiovasculature of the chicken embryo with field emission scanning electron microscopy. We identify one of these protrusions as a cilium by immunofluorescence. The ciliary distribution is compared to the expression pattern of the high shear stress marker; Krüppel-like factor-2. We demonstrate the presence of cilia on endocardial cells in low or disturbed shear stress areas. Conclusion We postulate that the cilium is part of the mechanosensing mechanism of endothelial cells. In low shear stress areas cilia transduce the forces to the cytoskeleton, which in turn generates a response. This mechanism is already operational during the early phases of cardiovascular development and is involved in flow directed remodeling events. We are extending our study to adult mice and we are performing functional experiments, which confirm a role for cilia in mechanosensing.

Comparison of promoter region constructs forin vivointramuscular expression

High transgene expression is generally expected after gene transfer. However, different level, kinetics and localization of expression might be needed for relevant therapeutic applications. Former studies have compared various promoter regions driving gene expression leading to conflicting results. In the present work, two promoter families have been compared using the efficient in vivo intramuscular electrotransfer technique. Three promoter regions were constructed by associating the strong ubiquitous cytomegalovirus (CMV) enhancer-promoter to its homologous intron A or to a heterologous intron, or to a hybrid intron. Promoter regions derived from the muscle creatine kinase (MCK) promoter were also studied. The expression of the same transgene (SeAP or neurotrophin-3) under control of these different promoters was compared after plasmid electrotransfer in mouse tibialis-cranialis skeletal muscle. Heterologous intron association to the CMV promoter did not modify gene expression kinetics nor increase gene expression level. Usefulness of intron A or hybrid intron association to the CMV promoter depended on the gene. The various MCK promoters drove efficient gene expression but lower than that obtained with the CMV promoter. Furthermore, peak value was reached earlier with MCK promoter regions (14 days). For applications of gene transfer restricted to skeletal muscle, the MCK promoter or a MCK promoter variant would be a promising alternative to the CMV promoter. Indeed, it has been demonstrated that the use of MCK promoter limits humoral and cell-mediated immune responses. Furthermore, the MCK promoter decreases the initial expression peak that may be detrimental, drives a sustained gene expression, and improves gene transfer safety.

Inhibition of human immunodeficiency virus type 1 replication in latently infected cells by a novel IkappaB kinase inhibitor.

In human immunodeficiency virus type 1 (HIV-1) latently infected cells, NF-kappaB plays a major role in the transcriptional induction of HIV-1 replication. Hence, downregulation of NF-kappaB activation has long been sought for effective anti-HIV therapy. Tumor necrosis factor alpha (TNF-alpha) stimulates IkappaB kinase (IKK) complex, a critical regulator in the NF-kappaB signaling pathway. A novel IKK inhibitor, ACHP {2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl-nicotinonitrile}, was developed and evaluated as a potent and specific inhibitor for IKK-alpha and IKK-beta. In this study, we examined the ability of this compound to inhibit HIV-1 replication in OM10.1 cells latently infected with HIV. When these cells were pretreated with ACHP, TNF-alpha-induced HIV-1 replication was dramatically inhibited, as measured by the HIV p24 antigen levels in the culture supernatants. Its 50% effective concentration was approximately 0.56 microM, whereas its 50% cytotoxic concentration was about 15 microM. Western blot analysis revealed inhibition of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 nuclear translocation, and p65 phosphorylation. ACHP was also found to suppress HIV-1 long terminal repeat (LTR)-driven gene expression through the inhibition of NF-kappaB activation. Furthermore, ACHP inhibited TNF-alpha-induced NF-kappaB (p65) recruitment to the HIV-1 LTR, as assessed by chromatin immunoprecipitation assay. These findings suggest that ACHP acts as a potent suppressor of TNF-alpha-induced HIV replication in latently infected cells and that this inhibition is mediated through suppression of IKK activity.