Transient Downregulation Research Articles

Down-Regulation of Homeobox Gene Ventx Promotes Expansion of Human Bone Marrow Hematopoietic Stem Cells (HSC)

Abstract 1272Hematopoietic stem cells (HSC) give rise to mature cells of all lineages of blood and immune systems. HSC transplantation has shown great promise in the treatment of malignancies, reconstitution of hematopoietic systems and HSC-based gene therapy. Cell intrinsic factors/pathways have been the targets of intensive investigation for its potential application in HSC expansion. Over the past decades, several critical cell fate determination pathways, such as the Wnt signaling pathways and senescence pathways have been implicated in the proliferation and differentiation of HSC. Moreover, overexpression of HoxB4 and BMI1 was found to be able to expand human HSC 2∼3 folds. Nevertheless, the regulatory mechanisms of HSC proliferation and differentiation remain incompletely understood and safe and efficacious expansion of human HSC remains as a fundamental challenge that limits the clinical application of HSC-based therapy.VentX is a human homologue of the Xenopus homeobox protein Xom of the BMP4 signaling pathway. Using Xenopus model and methods of reverse genetics, our recent work showed that VentX is a LEF/TCF associated Wnt repressor and an activator of senescence pathways. VentX expression is highly regulated and restricted in hematopoietic cells and serves a major regulator of hematopoietic cell differentiation. To explore the potential role of VentX in proliferation and differentiation of HSC during hematopoiesis, we quantified VentX expression during hematopoiesis, using qRT-PCR methods and examined the effects of altered VentX expression on HSC properties in vitro and in vivo.Our data showed that VentX expression is significantly up-regulated during oncogenesis of hematopioetic cells. We demonstrated that lentiviral knockdown of VentX allowed for more than 5 fold ex vivo expansion of human HSC with balanced lineage development. Importantly, transient knockdown of VentX by siRNA also led to expansion of HSC. The effect of VentX down-regulation on the expansion of human HSC was also demonstrated by enhanced engraftment in the SCID/NODγ2null mouse model. Consistent with its role as a novel regulator of HSC, overexpression of VentX significantly inhibited clonal genesis of HSC. Mechanistically, we demonstrated that VentX controls the expression of cell cycle regulators downstream of the Wnt and senescence pathways, such as the C-myc, CyclinD1 and p21.In summary, using methods of reverse genetic and developmental modeling, we identified VentX as a novel regulator for expansion of human BM HSC. The results of our investigations provide novel insight in regulating HSC proliferation and differentiation. In addition, the findings that transient down-regulation of VentX by SiRNA lead to efficient expansion of bone marrow HSC suggests that VentX may serve as a novel target for safe expansion of HSC for its potential clinical applications. Disclosures:No relevant conflicts of interest to declare.

A New Protocol for Efficient and Non-Toxic Transfection of siRNA Into Leukemic Cell Lines and Primary Cells – a Direct Comparison Between Nucleofection and Accell Delivery Using Stem Cell Related Antigens As Validation Tools

Abstract 4802 BACKGROUND:Transient downregulation of genes in vitro by use of siRNA is a time-honored approach to study gene function. This can readily be applied in haematological malignancies, where many lesions derive from altered RNA activity arising e.g. from balanced translocations. PROBLEM FORMULATION:A crucial requirement to obtain this downregulation is an efficient and non-toxic delivery of the siRNA into the target cells, which has proven difficult to accomplish with some protocols; in particular, cells in suspension, such as leukemic blasts. HYPOTHESIS:Directly comparing the range of new transfection methodologies to unravel protocols suitable for cells in suspension. RESULTS:From pilot data, where a range of other methods were excluded due to either low transfection efficiencies or low viability of transfected cells, we compared the gold standard Nucleofection methodology to the novel non-viral based delivery method Accell in both suspension cell lines as well as primary patient AML and CML cells. As control the housekeeping gene Cyclophilin B was used, while hMICL and CD96, recently suggested to be associated with the putative leukemic stem cells, were chosen as target genes. Comparison of the two transfection methods yielded superior results using Accell methodology, as this technique not only yielded higher transfection rates but also retained superior cell viabilities for both cell lines and primary cells. Three AML and two CML samples were included in this study for the validation of transfection efficiency in primary cell employing the programs T-20 and U-15 for Nucleofection. Here, there was a large span in viabilities pre-experimentation among the five patient samples included in this study, probably reflecting the conditions of the patients. However, following transfection there was a significantly lower viability after Nucleofection (mean 37%, range 23%-65%) compared to Accell (mean 82%, range 57%-95%), in the two groups (p<0.001). Corroborating the cell line data transfection efficiencies were clearly superior after Accell transfection of primary patient cells (mean 85%, range 71%-97%) compared to Nucleofection transfection (mean 38%, range 23%-65%), p<0.01. Transfection efficiencies in primary cells after Accell delivery was 90–97% compared to 25–30% using Nucleofection for siRNA delivery. As a functional correlate to these data we performed CFU-GM assays on selected hMICL+ AML patients exposed to anti-hMICL siRNAs. While the AML blast growth of some patients were unaffected by the siRNA treatment others showed a clear inhibition of blast colony formation, suggesting a differential role of hMICL between AML patients, which needs to be further elucidated. CONCLUSION:Accell delivery is thus the method of choice for non-viral transfection of cells in suspension including primary AML and CML cells. These data should provide a platform for further studies of genes involved in early leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

Bortezomib Impairs Myeloma Cells (MM) Homologous Recombination Through Inhibition of the E2-Ubiquitin-Conjugating Enzyme UBC13

Abstract 1834 Background:Chromosomal instability is a defining feature of clonal MM plasma cells resulting in the perpetual accumulation of genomic aberrations. In addition to its role in protein homeostasis the ubiquitin-proteasome system is also involved in the regulation of DNA damage repair proteins. We have recently reported that proteasome inhibition induces a BRCAness state in MM cells (MM) resulting in a contextual synthetic lethality when combined with PARP inhibitors. We now report on the mechanisms by which bortezomib impairs homologous recombination (HR) mediated repair of DNA double stranded breaks (DNA-DSBs). Methods and results:Using the DR-GFP/SceI reporter assay in which cells are transfected with a plasmid tandemly expressing mutated GFP genes (5’-Sce/GFP – truncated IGFP-3’) and followed by infection with an adenovirus (AdNGUS24i) expressing the yeast Sce endonuclease, we have confirmed that treatment with bortezomib does severely impair HR in MM cells. At the transcriptional level, proteasome inhibition transiently (6–12 hours post treatment) reduced the mRNA expression of several HR and DNA damage repair genes (BRCA1, BRCA2, RAD51 and FANCD2) followed by recovery to baseline levels by 24 hours. This transient downregulation of these DNA damage repair genes was also confirmed in a promoter luciferase reporter screen. At the post-trancriptional level, examining the dynamics and kinetics of DNA-DSBs repair induced by radiation therapy or PARP inhibition, we have demonstrated that bortezomib does not alter the initial phase of DNA damage sensing (MRN complex recruitment, ATM and H2AX phosphorylation and MDC1 activation) but does impair the Lys63-poly-ubiquitylation of histones γH2AX and H2A. This histone ubiquitylation is a required modification for the second wave of DNA repair proteins recruitment and retention of BRCA1 and RAD51 at the sites of DNA-DSBs. Furthermore, we have shown that bortezomib treatment depleted the nuclear pools of ubiquitin and abrogated the poly-ubiquitylation (lack of co-localization poly-Ub FK2 foci with γH2AX), but not the phosphorylation and foci formation of histone H2AX. Therefore, bortezomib appears to impair histones (H2AX and H2A) poly-ubiquitylation, a process that is dependent on the activation and recruitment of the E3 ubiquitin-protein ligases RNF8 and RNF-168 as well as the ubiquitin-E2 conjugating enzyme UBC13 to the sites of DNA-DSBs. Western blot analysis of the nuclear and cytoplasmic fractions of cellular extracts, revealed that bortezomib dramatically reduced the nuclear accumulation of UBC13 in response to DNA-DSBs as well as the formation of UBC13-Ubiquitin thiolester bonds formation and hence impaired the transfer of ubiquitin to nucleosomal histones. Conclusion:Our studies demonstrate that proteasome inhibition with bortezomib severely impairs homology-mediated repair of DNA breaks in MM cells at the post-transcriptional level by altering the nuclear accumulation and function of the E2-ubiquitin conjugating enzyme UBC13. These results explain the observed clinical synergy between bortezomib and several DNA damaging agents and support further clinical investigation of the combination of these class of drugs in MM patients. Disclosures:Neri:Celgene: Honoraria, Research Funding. Bahlis:Celgene: Honoraria, Speakers Bureau.

A novel role for Slit2/Robo1 axis in modulating HIV-1 replication in T cells

Slit2 is a secreted glycoprotein that has been shown to possess anti-inflammatory properties. In addition, Slit2 has been shown to modulate CXCR4-mediated functional effects in T cells. However, its role in HIV-1 pathogenesis is not known. The objective of the current study is to analyze the role of Slit2 in modulating HIV-1 replication in T cells. The effect of endogenous Slit2 expression of HIV-1 replication in T cells was studied by transient overexpression or downregulation of Slit2. The role of exogenous Slit2 was studied by analyzing the effect of soluble Slit2 protein on HIV-1 replication in T-cell lines and peripheral blood mononuclear cells (PBMCs). Studies on T-cell lines revealed a higher expression of Slit2 in Jurkat T cells compared with MT4 cells. We observed that downregulation of Slit2 in Jurkat T cells using Slit2-specific small inhibitor RNA enhanced HIV-1 replication. However, overexpression of Slit2 in MT4 cells and PBMCs reduced HIV-1 replication. As Slit2 is a secretory protein, we further analyzed the role of soluble Slit2 on HIV-1 virus replication using various cell lines and PBMCs. Our data indicated that exogenous Slit2 inhibited replication of both X4-tropic and R5-tropic HIV-1 viruses. Further studies revealed that Slit2 mediated its functional effects by binding to Robo1 receptor. Taken together, our results describe Slit2/Robo1 axis as a novel modulator of HIV-1 replication in T cells. These studies may contribute to the understanding of the immunopathogenesis of HIV-1 infection.

Alterations of A-Type Potassium Channels in Hippocampal Neurons after Traumatic Brain Injury

Traumatic brain injury (TBI) is associated with cognitive deficits, memory impairment, and epilepsy. Previous studies have reported neuronal loss and neuronal hyperexcitability in the post-traumatic hippocampus. A-type K+ currents (I(A)) play a critical role in modulating the intrinsic membrane excitability of hippocampal neurons. The disruption of I(A) is reportedly linked to hippocampal dysfunction. The present study investigates the changes of I(A) in the hippocampus after TBI. TBI in rats was induced by controlled cortical impact. The impact induced a reproducible lesion in the cortex and an obvious neuronal death in the ipsilateral hippocampus CA3 region. At one week after TBI, immunohistochemical staining and Western blotting showed that the expression of I(A) channel subunit Kv4.2 was markedly decreased in the ipsilateral hippocampus, but remained unchanged in the contralateral hippocampus. Meanwhile, electrophysiological recording showed that I(A) currents in ipsilateral CA1 pyramidal neurons were significantly reduced, which was associated with an increased neuronal excitability. Furthermore, there was an increased sensitivity to bicuculline-induced seizures in TBI rats. At 8 weeks after TBI, immunohistochemical staining and electrophysiological recording indicated that I(A) returned to control levels. These findings suggest that TBI causes a transient downregulation of I(A) in hippocampal CA1 neurons, which might be associated with the hyperexcitability in the post-traumatic hippocampus, and in turn leads to seizures and epilepsy.

Direct and indirect impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on adult mouse Leydig cells: An in vitro study

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related substances are ubiquitous environmental pollutants that exert adverse effects on reproductive processes. In testis, Leydig cells which produce testosterone are under hormonal and local control exerted by cytokines including TNFα. Using mouse Leydig primary cell cultures as a model, we studied the effects of TCDD on the steroidogenic outcome of Leydig cells and the gene expression levels of Ccl5 and Cxcl4, previously shown to be target genes of TCDD in testis. We found that TCDD did not alter the steroidogenic outcome of Leydig cells but that it up-regulated Cxcl4 gene expression levels. TCDD also impacted Ccl5 gene expression when cells had been co-treated with TNFα. TCDD action probably initiated with binding to the aryl hydrocarbon receptor (AhR) present on Leydig cells. TCDD regulated the gene expression levels of AhR (transient down-regulation) and its repressor AhRR and Cyp1b1 (up-regulation). The trophic human chorionic gonadotropin (hCG) hormone did not impact AhR, its repressor AhRR or Cyp1b1 but it opposed the TCDD-enhanced AhRR mRNA levels. Conversely, TNFα stimulated AhR gene expression levels. Collectively, it is suggested that the impact of TCDD on expression of target genes in Leydig cells may operate under the complex network of hormones and cytokines.

Transient silencing of the grapevine gene VvPGIP1 by agroinfiltration with a construct for RNA interference

Grapevine is an economically important crop, and the recent completion of its genome makes it possible to study the function of specific genes through reverse genetics. However, the analysis of gene function by RNA interference (RNAi) in grapevine is difficult, because the generation of stable transgenic plants has low efficiency and is time consuming. Recently, transient expression of genes in grapevine leaves has been obtained by Agrobacterium tumefaciens infiltration (agroinfiltration). We therefore tested the possibility to silence grapevine genes by agroinfiltration of RNAi constructs. A construct to express a double strand RNA (dsRNA) corresponding to the defense-related gene VvPGIP1, encoding a polygalacturonase-inhibiting protein (PGIP), was obtained and transiently expressed by agroinfiltration in leaves of grapevine plants grown in vitro. Expression of VvPGIP1 and accumulation of PGIP activity were strongly induced by infiltration with control bacteria, but not with bacteria carrying the dsRNA construct, indicating that the gene was efficiently silenced. In contrast, expression of another defense-related gene, VST1, encoding a stilbene synthase, was unaffected by the dsRNA construct. We have therefore demonstrated the possibility of transient down-regulation of grapevine genes by agroinfiltration of constructs for the expression of dsRNA. This system can be employed to evaluate the effectiveness of constructs that can be subsequently used to generate stable RNAi transgenic plants.

Linoleic acid induces an EMT-like process in mammary epithelial cells MCF10A

Epidemiological studies and animal models suggest an association between high levels of dietary fat intake and an increased risk of developing breast cancer. Epithelial–mesenchymal-transition (EMT) is a process, by which epithelial cells are transdifferentiated to a mesenchymal state, and it has been implicated in cancer progression, including invasion and metastasis. Linoleic acid (LA) induces proliferation and invasion in breast cancer cells. However, the role of LA on the EMT process in human mammary epithelial cells remains to be studied. In the present study, we demonstrate that LA induces a transient down-regulation of E-cadherin expression, accompanied with an increase of Snail1, Snail2, Twist1, Twist2 and Sip1 expressions. Furthermore, LA induces FAK and NFκB activation, MMP-2 and -9 secretions, migration and invasion. In summary, our findings demonstrate, for the first time, that LA promotes an EMT-like process in MCF10A human mammary epithelial cells.

Expression of BDNF and NT‐3 during the ontogeny and regeneration of the lacertidian (Gallotia galloti) visual system

Retinal ganglion cell (RGC) axons regrow spontaneously after optic nerve (ON) transection in G. galloti. Because brain-derived neurotrophic factor (BDNF) is considered the major neurotrophin participating in vertebrate visual system development and promotes RGC survival, we investigated its distribution using dual-labeling immunohistochemistry for neuronal and glial markers. We examined the developing and regenerating lizard visual system at 1, 3, 6, 9, and 12 months postlesion to comparatively evaluate BDNF expression patterns. BDNF was detected from midembryonic stages (E35) in both retinal plexiform layers, and in radial glial processes in the tectum. Moreover, RGC axon staining was detected at late prenatal stages (E39), showing a transient punctate staining which progressed in a temporo-spatial pattern that was similar to myelination. Strong expression in RGC axons was maintained in adults. However, transient downregulation of BDNF staining occurred on the experimental side one month after ON transection followed by a gradual recovery with extensive punctate/swelling distribution and persistent upregulation at 12 months. Conversely, quantitative PCR analysis for 1 and 12 months regenerate lizards showed downregulation of the ratio of BDNF mRNA expression at 12 months and nonsignificant changes of NT-3 transcripts. In summary, we demonstrate that BDNF and NT-3 are abundantly expressed during lizard visual system ontogeny and regeneration suggesting their participation in the development, maintenance and plasticity of the system.

Ca2+/Calmodulin-Dependent Kinase (CaMK) Signaling via CaMKI and AMP-Activated Protein Kinase Contributes to the Regulation of WIPI-1 at the Onset of Autophagy

Autophagy is initiated by multimembrane vesicle (autophagosome) formation upon mammalian target of rapamycin inhibition and phosphatidylinositol 3-phosphate [PtdIns(3)P] generation. Upstream of microtubule-associated protein 1 light chain 3 (LC3), WD-repeat proteins interacting with phosphoinositides (WIPI proteins) specifically bind PtdIns(3)P at forming autophagosomal membranes and become membrane-bound proteins of generated autophagosomes. Here, we applied automated high-throughput WIPI-1 puncta analysis, paralleled with LC3 lipidation assays, to investigate Ca(2+)-mediated autophagy modulation. We imposed cellular stress by starvation or administration of etoposide (0.5-50 μM), sorafenib (1-40 μM), staurosporine (20-500 nM), or thapsigargin (20-500 nM) (1, 2, or 3 h) and measured the formation of WIPI-1 positive autophagosomal membranes. Automated analysis of up to 5000 individual cells/treatment demonstrated that Ca(2+) chelation by BAPTA-AM (10 and 30 μM) counteracted starvation or pharmacological compound-induced WIPI-1 puncta formation and LC3 lipidation. Application of selective Ca(2+)/calmodulin-dependent kinase kinase (CaMKK) α/β and calmodulin-dependent kinase (CaMK) I/II/IV inhibitors 7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid acetate (STO-609; 10-30 μg/ml) and 2-(N-[2-hydroxyethyl])-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylamine (KN-93; 1-10 μM), respectively, significantly reduced starvation-induced autophagosomal membrane formation, suggesting that Ca(2+) mobilization upon autophagy induction involves CaMKI/IV. By small interefering RNA (siRNA)-mediated down-regulation of CaMKI or CaMKIV, we demonstrate that CaMKI contributes to stimulation of WIPI-1. In line, WIPI-1 positive autophagosomal membranes were formed in AMP-activated protein kinase (AMPK) α(1)/α(2)-deficient mouse embryonic fibroblasts upon nutrient starvation, whereas basal autophagy was prominently reduced. However, transient down-regulation of AMPK by siRNA resulted in an increased basal level of both WIPI-1 puncta and LC3 lipidation, and nutrient-starvation induced autophagy was sensitive to STO-609/KN-93. Our data provide evidence that pharmacological compound-modulated and starvation-induced autophagy involves Ca(2+)-dependent signaling, including CaMKI independent of AMPKα(1)/α(2). Our data also suggest that AMPKα(1)/α(2) might differentially contribute to the regulation of WIPI-1 at the onset of autophagy.

Targeted mutagenesis of zebrafish: Use of zinc finger nucleases

The modeling of human disease in the zebrafish (Danio rerio) is moving away from chemical mutagensis and transient downregulation using morpholino oligomers to more targeted and stable transgenic methods. In this respect, zinc finger nucleases offer a means of introducing mutations at targeted sites at high efficiency. We describe here the development of zinc finger nucleases and their general use in model systems with a focus on the zebrafish.

Nonviral transfection of leukemic primary cells and cells lines by siRNA—a direct comparison between Nucleofection and Accell delivery

Transient downregulation of genes invitro employing short interfering RNA (siRNA) is a time-honored approach to study gene function. A crucial prerequisite to obtain a downregulation is an efficient and nontoxic delivery of the siRNA into the target cells. However, this has proven difficult to accomplish, particular in cells in suspension. Thus, there is a need for a systematic evaluation of different methodologies to identify the most suitable protocol. We compared Nucleofection with Accell, a novel nonviral-based delivery system in the setting of leukemic blasts from patients with myeloid leukemias. Two cell surface proteins, human inhibitory C-type lectin-like receptor and CD96, both believed to be associated with leukemic stem cells, were chosen as target genes. Accell not only yielded higher transfection rates, but also retained superior cell viabilities for both cell lines and primary leukemic cells. Thus, transfection efficiencies in primary cells after Accell delivery was 85% (range, 71-97%) compared to 38% (23-65%) using Nucleofection for siRNA delivery. Preliminary studies of clonal growth of primary acute myeloid leukemia cells indicated growth inhibition after siRNA transfection. Our results reveal that Accell delivery is suitable for nonviral transfection of cells in suspension, including primary leukemic cells. These data should provide a platform for further studies of genes involved in early leukemogenesis.

Downregulation of the Posterior Medial Frontal Cortex Prevents Social Conformity

We often change our behavior to conform to real or imagined group pressure. Social influence on our behavior has been extensively studied in social psychology, but its neural mechanisms have remained largely unknown. Here we demonstrate that the transient downregulation of the posterior medial frontal cortex by theta-burst transcranial magnetic stimulation reduces conformity, as indicated by reduced conformal adjustments in line with group opinion. Both the extent and probability of conformal behavioral adjustments decreased significantly relative to a sham and a control stimulation over another brain area. The posterior part of the medial frontal cortex has previously been implicated in behavioral and attitudinal adjustments. Here, we provide the first interventional evidence of its critical role in social influence on human behavior.

The transcription factor Mxd4 controls the proliferation of the first blood precursors at the onset of hematopoietic development in vitro

The balance between proliferation and differentiation during hematopoietic development in the embryo is a complex process, the detailed molecular mechanisms of which remain to be fully characterized. The transcription factor Mxd4, a member of the Myc-Max-Mad network, was identified in a global gene expression profiling screen as being tightly regulated at the onset of hematopoietic lineage specification upon invitro differentiation of mouse embryonic stem cells. Our study investigated the Mxd4 expression pattern at the onset of hematopoiesis and the biological relevance of its sharp and transient downregulation. To study the expression pattern and role of Mxd4 at the onset of hematopoiesis, the invitro differentiation of embryonic stem cells was used as a model system. Gain of function assays were performed using a doxycycline-inducible embryonic stem cell system. We show here that Mxd4 expression is transiently downregulated at an early stage of commitment to the hematopoietic lineage. Enforced expression of Mxd4 at this period of differentiation results in a defect in hematopoietic progenitor development, with impaired development of both primitive and definitive blood lineages. This effect is due to a severe decrease in cell proliferation, with an increased frequency of cells in the G(0)/G(1) phase of the cell cycle, alongside a reduced frequency of cells in the S phase. Together our results indicate that during embryonic hematopoietic differentiation Mxd4 is an important player in the regulation of blood progenitor proliferation, and suggest that downregulation of its expression might be required for a proliferative burst preceding lineage specification.

Reversal of Stathmin-Mediated Microtubule Destabilization Sensitizes Retinoblastoma Cells to A Low Dose of Antimicrotubule Agents: A Novel Synergistic Therapeutic Intervention

To explore the possibility of stathmin as an effective therapeutic target and to evaluate the synergistic combination of stathmin RNAi and the antimicrotubule agents paclitaxel and vincristine to retinoblastoma Y79 cells. RNAi-mediated specific inhibition of stathmin expression in Y79 cells was shown by real-time quantitative RT-PCR (RT-Q-PCR), its effect on cell proliferation by MTT assay, cell invasion using matrigel, microtubule polymerization by immunohistochemistry, apoptosis, cell cycle analysis by flow cytometry analysis, and the changes in FOXM1 protein expression were studied by Western blot. The effect of combination treatment of stathmin siRNA and paclitaxel/vincristine was studied by assessing cell viability and apoptosis. Short interfering RNA-mediated transient stathmin downregulation resulted in a marked inhibition of retinoblastoma cell proliferation and cell invasion in vitro. Stathmin inhibition promoted Y79 cells to G2/M phase, and ultimately there were increased apoptotic events as evidenced by higher caspase-3 activation and cleaved poly(ADP-ribose) polymerase expression. Cells transfected with stathmin siRNA showed long and bundled microtubule polymers and sensitized the Y79 cells significantly to paclitaxel and vincristine. Stathmin may be a pivotal determinant for retinoblastoma tumorigenesis and chemosensitivity. Strategies to inhibit stathmin will help to enhance the cytotoxic effect of paclitaxel while reducing toxicity (or side effects) to normal cells caused by high doses.

Transient suppression of MLH1 allows effective single-nucleotide substitution by single-stranded DNA oligonucleotides

Short synthetic single-stranded oligodeoxyribonucleotides (ssODNs) can be used to introduce subtle modifications into the genome of mouse embryonic stem cells (ESCs). We have previously shown that effective application of ssODN-mediated gene targeting in ESC requires (transient) suppression of DNA mismatch repair (MMR). However, whereas transient down-regulation of the mismatch recognition protein MSH2 allowed substitution of 3 or 4 nucleotides, 1 or 2 nucleotide substitutions were still suppressed. We now demonstrate that single- or dinucleotide substitution can effectively be achieved by transient down-regulation of the downstream MMR protein MLH1. By exploiting highly specific real-time PCR, we demonstrate the feasibility of substituting a single basepair in a non-selectable gene. However, disabling the MMR machinery may lead to inadvertent mutations. To obtain insight into the mutation rate associated with transient MMR suppression, we have compared the impact of transient and constitutive MMR deficiency on the repair of frameshift intermediates at mono- and dinucleotide repeats. Repair at these repeats relied on the substrate specificity and functional redundancy of the MSH2/MSH6 and MSH2/MSH3 MMR complexes. MLH1 knockdown increased the level of spontaneous mutagenesis, but modified ESCs remained germ line competent. Thus, transient MLH1 suppression provides a valuable extension of the MSH2 knockdown strategy, allowing rapid generation of mice carrying single basepair alterations in their genome.

Retinoic Acid-inducible Gene I-inducible miR-23b Inhibits Infections by Minor Group Rhinoviruses through Down-regulation of the Very Low Density Lipoprotein Receptor

In mammals, viral infections are detected by innate immune receptors, including Toll-like receptor and retinoic acid inducible gene I (RIG-I)-like receptor (RLR), which activate the type I interferon (IFN) system. IFN essentially activates genes encoding antiviral proteins that inhibit various steps of viral replication as well as facilitate the subsequent activation of acquired immune responses. In this study, we investigated the expression of non-coding RNA upon viral infection or RLR activation. Using a microarray, we identified several microRNAs (miRNA) specifically induced to express by RLR signaling. As suggested by Bioinformatics (miRBase Target Data base), one of the RLR-inducible miRNAs, miR-23b, actually knocked down the expression of very low density lipoprotein receptor (VLDLR) and LDLR-related protein 5 (LRP5). Transfection of miR-23b specifically inhibited infection of rhinovirus 1B (RV1B), which utilizes the low density lipoprotein receptor (LDLR) family for viral entry. Conversely, introduction of anti-miRNA-23b enhanced the viral yield. Knockdown experiments using small interfering RNA (siRNA) revealed that VLDLR, but not LRP5, is critical for an efficient infection by RV1B. Furthermore, experiments with the transfection of infectious viral RNA revealed that miR-23b did not affect post-entry viral replication. Our results strongly suggest that RIG-I signaling results in the inhibitions of infections of RV1B through the miR-23b-mediated down-regulation of its receptor VLDLR.

Differential regulation of the expression of neurotrophin receptors in rat extraocular motoneurons after lesion

Neurotrophins acting through high-affinity tyrosine kinase receptors (trkA, trkB, and trkC) play a crucial role in regulating survival and maintenance of specific neuronal functions after injury. Adult motoneurons supplying extraocular muscles survive after disconnection from the target, but suffer dramatic changes in morphological and physiological properties, due in part to the loss of their trophic support from the muscle. To investigate the dependence of the adult rat extraocular motoneurons on neurotrophins, we examined trkA, trkB, and trkC mRNA expression after axotomy by in situ hybridization. trkA mRNA expression was detectable at low levels in unlesioned motoneurons, and its expression was downregulated 1 and 3 days after injury. Expression of trkB and trkC mRNAs was stronger, and after axotomy a simultaneous, but inverse regulation of both receptors was observed. Thus, whereas a considerable increase in trkB expression was seen about 2 weeks after axotomy, the expression of trkC mRNA had decreased at the same post-lesion period. Injured extraocular motoneurons also experienced an initial induction in expression of calcitonin gene-related peptide and a transient downregulation of cholinergic characteristics, indicating a switch in the phenotype from a transmitter-specific to a regenerative state. These results suggest that specific neurotrophins may contribute differentially to the survival and regenerative responses of extraocular motoneurons after lesion.

Hyperinsulinemia leads to uncoupled insulin regulation of the GLUT4 glucose transporter and the FoxO1 transcription factor

Insulin resistance is a component of the metabolic syndrome and Type 2 diabetes. It has been recently shown that in liver insulin resistance is not complete. This so-called selective insulin resistance is characterized by defective insulin inhibition of hepatic glucose output while insulin-induced lipogenesis is maintained. How this occurs and whether uncoupled insulin action develops in other tissues is unknown. Here we show in a model of chronic hyperinsulinemia that adipocytes develop selective insulin resistance in which translocation of the GLUT4 glucose transporter to the cell surface is blunted yet nuclear exclusion of the FoxO1 transcription factor is preserved, rendering uncoupled insulin-controlled carbohydrate and lipid metabolisms. We found that in adipocytes FoxO1 nuclear exclusion has a lower half-maximal insulin dose than GLUT4 translocation, and it is because of this inherent greater sensitivity that control of FoxO1 by physiological insulin concentrations is maintained in adipocytes with compromised insulin signaling. Pharmacological and genetic interventions revealed that insulin regulates GLUT4 and FoxO1 through the PI3-kinase isoform p110α, although FoxO1 showed higher sensitivity to p110α activity than GLUT4. Transient down-regulation and overexpression of Akt isoforms in adipocytes demonstrated that insulin-activated PI3-kinase signals to GLUT4 primarily through Akt2 kinase, whereas Akt1 and Akt2 signal to FoxO1. We propose that the lower threshold of insulin activity for FoxO1's nuclear exclusion is in part due to its regulation by both Akt isoforms. Identification of uncoupled insulin action in adipocytes suggests this condition might be a general phenomenon of insulin target tissues contributing to insulin resistance's pathophysiology.

Expression of Aux/IAA genes during development of sylleptic and proleptic buds in white poplar

In this study, the genes of theAux/IAA family were used as functional markers to characterise bud break stages in a white poplar clone. In the first experiment, under greenhouse conditions, the sprouting of repressed sylleptic buds was obtained by pruning the shoot tip during growing season. Buds were collected at 0, 6, 24 and 48 h after pruning for molecular analyses. A decrease in transcript level ofIAA4 andIAA8 genes was observed in the first and second bud below the cut after 6 and 24 h, respectively. In the second experiment, bud break of post-dormant proleptic buds was induced by forcing in climatic chamber. The first 6 and the following 6 buds below the apical one were sampled every 48 h during forcing. Anatomical studies were also carried out on buds and plants were equipped with stem and bud radial growth sensors to check their swelling. In both experiments, gene expression patterns showed a decrease and a successive increase in expression ofIAA4 andIAA8 genes during bud break. The transient down-regulation of these genes was observed only in buds that formed new branches. Thus, similar molecular mechanisms are involved in bud break of both sylleptic and proleptic buds.