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Sample records for gene vector neurodegeneration

  1. The evolution of heart gene delivery vectors

    Science.gov (United States)

    Wasala, Nalinda B.; Shin, Jin-Hong; Duan, Dongsheng

    2012-01-01

    Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic materials for heart gene delivery. Several nonviral vectors (e.g. naked plasmids, plasmid lipid/polymer complexes and oligonucleotides) have been tested. Commonly used viral vectors include lentivirus, adenovirus and adeno-associated virus. Among these, adeno-associated virus has shown many attractive features for pre-clinical experimentation in animal models of heart diseases. We review the history and evolution of these vectors for heart gene transfer. PMID:21837689

  2. Modified montmorillonite as vector for gene delivery.

    Science.gov (United States)

    Lin, Feng-Huei; Chen, Chia-Hao; Cheng, Winston T K; Kuo, Tzang-Fu

    2006-06-01

    Currently, gene delivery systems can be divided into two parts: viral or non-viral vectors. In general, viral vectors have a higher efficiency on gene delivery. However, they may sometimes provoke mutagenesis and carcinogenesis once re-activating in human body. Lots of non-viral vectors have been developed that tried to solve the problems happened on viral vectors. Unfortunately, most of non-viral vectors showed relatively lower transfection rate. The aim of this study is to develop a non-viral vector for gene delivery system. Montmorillonite (MMT) is one of clay minerals that consist of hydrated aluminum with Si-O tetrahedrons on the bottom of the layer and Al-O(OH)2 octahedrons on the top. The inter-layer space is about 12 A. The room is not enough to accommodate DNA for gene delivery. In the study, the cationic hexadecyltrimethylammonium (HDTMA) will be intercalated into the interlayer of MMT as a layer expander to expand the layer space for DNA accommodation. The optimal condition for the preparation of DNA-HDTMA-MMT is as follows: 1 mg of 1.5CEC HDTMA-MMT was prepared under pH value of 10.7 and with soaking time for 2 h. The DNA molecules can be protected from nuclease degradation, which can be proven by the electrophoresis analysis. DNA was successfully transfected into the nucleus of human dermal fibroblast and expressed enhanced green fluorescent protein (EGFP) gene with green fluorescence emission. The HDTMA-MMT has a great potential as a vector for gene delivery in the future.

  3. Lentiviral Vector Gene Transfer to Porcine Airways

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    Patrick L Sinn

    2012-01-01

    Full Text Available In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE. Interestingly, feline immunodeficiency virus (FIV-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1–based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF.

  4. Missense variants in AIMP1 gene are implicated in autosomal recessive intellectual disability without neurodegeneration.

    Science.gov (United States)

    Iqbal, Zafar; Püttmann, Lucia; Musante, Luciana; Razzaq, Attia; Zahoor, Muhammad Yasir; Hu, Hao; Wienker, Thomas F; Garshasbi, Masoud; Fattahi, Zohreh; Gilissen, Christian; Vissers, Lisenka E L M; de Brouwer, Arjan P M; Veltman, Joris A; Pfundt, Rolph; Najmabadi, Hossein; Ropers, Hans-Hilger; Riazuddin, Sheikh; Kahrizi, Kimia; van Bokhoven, Hans

    2016-03-01

    AIMP1/p43 is a multifunctional non-catalytic component of the multisynthetase complex. The complex consists of nine catalytic and three non-catalytic proteins, which catalyze the ligation of amino acids to their cognate tRNA isoacceptors for use in protein translation. To date, two allelic variants in the AIMP1 gene have been reported as the underlying cause of autosomal recessive primary neurodegenerative disorder. Here, we present two consanguineous families from Pakistan and Iran, presenting with moderate to severe intellectual disability, global developmental delay, and speech impairment without neurodegeneration. By the combination of homozygosity mapping and next generation sequencing, we identified two homozygous missense variants, p.(Gly299Arg) and p.(Val176Gly), in the gene AIMP1 that co-segregated with the phenotype in the respective families. Molecular modeling of the variants revealed deleterious effects on the protein structure that are predicted to result in reduced AIMP1 function. Our findings indicate that the clinical spectrum for AIMP1 defects is broader than witnessed so far.

  5. Missense variants in AIMP1 gene are implicated in autosomal recessive intellectual disability without neurodegeneration

    Science.gov (United States)

    Iqbal, Zafar; Püttmann, Lucia; Musante, Luciana; Razzaq, Attia; Zahoor, Muhammad Yasir; Hu, Hao; Wienker, Thomas F; Garshasbi, Masoud; Fattahi, Zohreh; Gilissen, Christian; Vissers, Lisenka ELM; de Brouwer, Arjan PM; Veltman, Joris A; Pfundt, Rolph; Najmabadi, Hossein; Ropers, Hans-Hilger; Riazuddin, Sheikh; Kahrizi, Kimia; van Bokhoven, Hans

    2016-01-01

    AIMP1/p43 is a multifunctional non-catalytic component of the multisynthetase complex. The complex consists of nine catalytic and three non-catalytic proteins, which catalyze the ligation of amino acids to their cognate tRNA isoacceptors for use in protein translation. To date, two allelic variants in the AIMP1 gene have been reported as the underlying cause of autosomal recessive primary neurodegenerative disorder. Here, we present two consanguineous families from Pakistan and Iran, presenting with moderate to severe intellectual disability, global developmental delay, and speech impairment without neurodegeneration. By the combination of homozygosity mapping and next generation sequencing, we identified two homozygous missense variants, p.(Gly299Arg) and p.(Val176Gly), in the gene AIMP1 that co-segregated with the phenotype in the respective families. Molecular modeling of the variants revealed deleterious effects on the protein structure that are predicted to result in reduced AIMP1 function. Our findings indicate that the clinical spectrum for AIMP1 defects is broader than witnessed so far. PMID:26173967

  6. Gene Transfer of Brain-derived Neurotrophic Factor (BDNF) Prevents Neurodegeneration Triggered by FXN Deficiency.

    Science.gov (United States)

    Katsu-Jiménez, Yurika; Loría, Frida; Corona, Juan Carlos; Díaz-Nido, Javier

    2016-05-01

    Friedreich's ataxia is a predominantly neurodegenerative disease caused by recessive mutations that produce a deficiency of frataxin (FXN). Here, we have used a herpesviral amplicon vector carrying a gene encoding for brain-derived neurotrophic factor (BDNF) to drive its overexpression in neuronal cells and test for its effect on FXN-deficient neurons both in culture and in the mouse cerebellum in vivo. Gene transfer of BDNF to primary cultures of mouse neurons prevents the apoptosis which is triggered by the knockdown of FXN gene expression. This neuroprotective effect of BDNF is also observed in vivo in a viral vector-based knockdown mouse cerebellar model. The injection of a lentiviral vector carrying a minigene encoding for a FXN-specific short hairpin ribonucleic acid (shRNA) into the mouse cerebellar cortex triggers a FXN deficit which is accompanied by significant apoptosis of granule neurons as well as loss of calbindin in Purkinje cells. These pathological changes are accompanied by a loss of motor coordination of mice as assayed by the rota-rod test. Coinjection of a herpesviral vector encoding for BDNF efficiently prevents both the development of cerebellar neuropathology and the ataxic phenotype. These data demonstrate the potential therapeutic usefulness of neurotrophins like BDNF to protect FXN-deficient neurons from degeneration.

  7. Novel neuroprotective function of apical-basal polarity gene crumbs in amyloid beta 42 (aβ42 mediated neurodegeneration.

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    Andrew M Steffensmeier

    Full Text Available Alzheimer's disease (AD, OMIM: 104300, a progressive neurodegenerative disorder with no cure to date, is caused by the generation of amyloid-beta-42 (Aβ42 aggregates that trigger neuronal cell death by unknown mechanism(s. We have developed a transgenic Drosophila eye model where misexpression of human Aβ42 results in AD-like neuropathology in the neural retina. We have identified an apical-basal polarity gene crumbs (crb as a genetic modifier of Aβ42-mediated-neuropathology. Misexpression of Aβ42 caused upregulation of Crb expression, whereas downregulation of Crb either by RNAi or null allele approach rescued the Aβ42-mediated-neurodegeneration. Co-expression of full length Crb with Aβ42 increased severity of Aβ42-mediated-neurodegeneration, due to three fold induction of cell death in comparison to the wild type. Higher Crb levels affect axonal targeting from the retina to the brain. The structure function analysis identified intracellular domain of Crb to be required for Aβ42-mediated-neurodegeneration. We demonstrate a novel neuroprotective role of Crb in Aβ42-mediated-neurodegeneration.

  8. Genetically engineering adenoviral vectors for gene therapy.

    Science.gov (United States)

    Coughlan, Lynda

    2014-01-01

    Adenoviral (Ad) vectors are commonly used for various gene therapy applications. Significant advances in the genetic engineering of Ad vectors in recent years has highlighted their potential for the treatment of metastatic disease. There are several methods to genetically modify the Ad genome to incorporate retargeting peptides which will redirect the natural tropism of the viruses, including homologous recombination in bacteria or yeast. However, homologous recombination in yeast is highly efficient and can be achieved without the need for extensive cloning strategies. In addition, the method does not rely on the presence of unique restriction sites within the Ad genome and the reagents required for this method are widely available and inexpensive. Large plasmids containing the entire adenoviral genome (~36 kbp) can be modified within Saccharomyces cerevisiae yeast and genomes easily rescued in Escherichia coli hosts for analysis or amplification. A method for two-step homologous recombination in yeast is described in this chapter.

  9. Progresses towards safe and efficient gene therapy vectors.

    Science.gov (United States)

    Chira, Sergiu; Jackson, Carlo S; Oprea, Iulian; Ozturk, Ferhat; Pepper, Michael S; Diaconu, Iulia; Braicu, Cornelia; Raduly, Lajos-Zsolt; Calin, George A; Berindan-Neagoe, Ioana

    2015-10-13

    The emergence of genetic engineering at the beginning of the 1970's opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors.

  10. Mithramycin is a gene-selective Sp1 inhibitor that identifies a biological intersection between cancer and neurodegeneration.

    Science.gov (United States)

    Sleiman, Sama F; Langley, Brett C; Basso, Manuela; Berlin, Jill; Xia, Li; Payappilly, Jimmy B; Kharel, Madan K; Guo, Hengchang; Marsh, J Lawrence; Thompson, Leslie Michels; Mahishi, Lata; Ahuja, Preeti; MacLellan, W Robb; Geschwind, Daniel H; Coppola, Giovanni; Rohr, Jürgen; Ratan, Rajiv R

    2011-05-04

    Oncogenic transformation of postmitotic neurons triggers cell death, but the identity of genes critical for degeneration remain unclear. The antitumor antibiotic mithramycin prolongs survival of mouse models of Huntington's disease in vivo and inhibits oxidative stress-induced death in cortical neurons in vitro. We had correlated protection by mithramycin with its ability to bind to GC-rich DNA and globally displace Sp1 family transcription factors. To understand how antitumor drugs prevent neurodegeneration, here we use structure-activity relationships of mithramycin analogs to discover that selective DNA-binding inhibition of the drug is necessary for its neuroprotective effect. We identify several genes (Myc, c-Src, Hif1α, and p21(waf1/cip1)) involved in neoplastic transformation, whose altered expression correlates with protective doses of mithramycin or its analogs. Most interestingly, inhibition of one these genes, Myc, is neuroprotective, whereas forced expression of Myc induces Rattus norvegicus neuronal cell death. These results support a model in which cancer cell transformation shares key genetic components with neurodegeneration.

  11. Prospects for Foamy Viral Vector Anti-HIV Gene Therapy

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    Arun K. Nalla

    2016-03-01

    Full Text Available Stem cell gene therapy approaches for Human Immunodeficiency Virus (HIV infection have been explored in clinical trials and several anti-HIV genes delivered by retroviral vectors were shown to block HIV replication. However, gammaretroviral and lentiviral based retroviral vectors have limitations for delivery of anti-HIV genes into hematopoietic stem cells (HSC. Foamy virus vectors have several advantages including efficient delivery of transgenes into HSC in large animal models, and a potentially safer integration profile. This review focuses on novel anti-HIV transgenes and the potential of foamy virus vectors for HSC gene therapy of HIV.

  12. Targeted cancer gene therapy : the flexibility of adenoviral gene therapy vectors

    NARCIS (Netherlands)

    Rots, MG; Curiel, DT; Gerritsen, WR; Haisma, HJ

    2003-01-01

    Recombinant adenoviral vectors are promising reagents for therapeutic interventions in humans, including gene therapy for biologically complex diseases like cancer and cardiovascular diseases. In this regard, the major advantage of adenoviral vectors is their superior in vivo gene transfer

  13. AAV Vectorization of DSB-mediated Gene Editing Technologies.

    Science.gov (United States)

    Moser, Rachel J; Hirsch, Matthew L

    2016-01-01

    Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome. Additionally, AAV vectors are currently the most efficient format for in vivo gene delivery with no vector related complications in >100 clinical trials for diverse diseases. At the same time, advancements in the design of custom-engineered site-specific endonucleases and the utilization of elucidated endonuclease formats have resulted in efficient and facile genetic engineering for basic science and for clinical therapies. AAV vectors and gene editing technologies are an obvious marriage, using AAV for the delivery of repair substrate and/or a gene encoding a designer endonuclease; however, while efficient delivery and enhanced gene targeting by vector genomes are advantageous, other attributes of AAV vectors are less desirable for gene editing technologies. This review summarizes the various roles that AAV vectors play in gene editing technologies and provides insight into its trending applications for the treatment of genetic diseases.

  14. Identifying Cancer Driver Genes Using Replication-Incompetent Retroviral Vectors

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    Victor M. Bii

    2016-10-01

    Full Text Available Identifying novel genes that drive tumor metastasis and drug resistance has significant potential to improve patient outcomes. High-throughput sequencing approaches have identified cancer genes, but distinguishing driver genes from passengers remains challenging. Insertional mutagenesis screens using replication-incompetent retroviral vectors have emerged as a powerful tool to identify cancer genes. Unlike replicating retroviruses and transposons, replication-incompetent retroviral vectors lack additional mutagenesis events that can complicate the identification of driver mutations from passenger mutations. They can also be used for almost any human cancer due to the broad tropism of the vectors. Replication-incompetent retroviral vectors have the ability to dysregulate nearby cancer genes via several mechanisms including enhancer-mediated activation of gene promoters. The integrated provirus acts as a unique molecular tag for nearby candidate driver genes which can be rapidly identified using well established methods that utilize next generation sequencing and bioinformatics programs. Recently, retroviral vector screens have been used to efficiently identify candidate driver genes in prostate, breast, liver and pancreatic cancers. Validated driver genes can be potential therapeutic targets and biomarkers. In this review, we describe the emergence of retroviral insertional mutagenesis screens using replication-incompetent retroviral vectors as a novel tool to identify cancer driver genes in different cancer types.

  15. Malaria Prevention by New Technology: Vectored Delivery of Antibody Genes

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-15-1-0401 TITLE: Malaria Prevention by New Technology : Vectored Delivery of Antibody Genes PRINCIPAL INVESTIGATOR: Gary...CONTRACT NUMBER Malaria Prevention by New Technology : Vectored Delivery of Antibody Genes 5b. GRANT NUMBER W81XWH-15-1-0401 5c. PROGRAM ELEMENT...whole animals. Using a specific technology originally applied to expression of HIV antibodies, we demonstrated that mice can be protected from

  16. Construction of RNAi lentiviral vector targeting mouse Islet-1 gene

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    Shen-shen ZHI

    2011-02-01

    Full Text Available Objective To construct and select RNAi lentiviral vectors that can silence mouse Islet-1 gene effectively.Methods Three groups of RNAi-target of mouse Islet-1 gene were designed,and corresponding shRNA oligo(sh1,sh2 and sh3 were synthesized,and then they were respectively inserted to the PLVTHM vector that had been digested by endonuclease.Agarose gel electrophoresis and sequencing were used to select and indentify the positive clones.The positive clones were extracted and then mixed with E.coli to amplify positive clones.The amplified clones were then infected into 293T along with the other 3 helper plasmids to produce lentiviral vector.After the construction of the lentiviral vector,plaque formation test was performed to determine the titer of lentiviral vector.The lentiviral vectors were then infected into C3H10T1/2 cells.The transfect efficiency of the lentiviral vectors was determined with flow cytometry with detection of green fluorescent protein(GFP.Q-PCR was employed to detect the RNAi efficiency of the lentiviral vectors.Results Agarose gel electrophoresis analysis showed that the clones with right gene at the target size were successfully established;gene sequencing showed that the right DNA fragments had been inserted;plaque formation test showed that the titer of the virus solution was 3.87×108TU/ml;the transfect efficiency of the lentiviral vector infected into C3H10T1/2 cells was 90.36%.All the 3 groups of shRNA targets(sh1,sh2 and sh3 showed an inhibitory effect on Islet-1 gene,and the sh1 showed the highest inhibitory effect(76.8%,as compared with that of normal cells(P < 0.05.Conclusion The RNAi lentiviral vector that can effectively silence the mouse Islet-1 gene has been constructed successfully,which may lay a foundation for further investigation of Islet-1 gene.

  17. Simian virus 40 vectors for pulmonary gene therapy

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    Oppenheim Ariella

    2007-10-01

    Full Text Available Abstract Background Sepsis remains the leading cause of death in critically ill patients. One of the primary organs affected by sepsis is the lung, presenting as the Acute Respiratory Distress Syndrome (ARDS. Organ damage in sepsis involves an alteration in gene expression, making gene transfer a potential therapeutic modality. This work examines the feasibility of applying simian virus 40 (SV40 vectors for pulmonary gene therapy. Methods Sepsis-induced ARDS was established by cecal ligation double puncture (2CLP. SV40 vectors carrying the luciferase reporter gene (SV/luc were administered intratracheally immediately after sepsis induction. Sham operated (SO as well as 2CLP rats given intratracheal PBS or adenovirus expressing luciferase served as controls. Luc transduction was evaluated by in vivo light detection, immunoassay and luciferase mRNA detection by RT-PCR in tissue harvested from septic rats. Vector abundance and distribution into alveolar cells was evaluated using immunostaining for the SV40 VP1 capsid protein as well as by double staining for VP1 and for the surfactant protein C (proSP-C. Immunostaining for T-lymphocytes was used to evaluate the cellular immune response induced by the vector. Results Luc expression measured by in vivo light detection correlated with immunoassay from lung tissue harvested from the same rats. Moreover, our results showed vector presence in type II alveolar cells. The vector did not induce significant cellular immune response. Conclusion In the present study we have demonstrated efficient uptake and expression of an SV40 vector in the lungs of animals with sepsis-induced ARDS. These vectors appear to be capable of in vivo transduction of alveolar type II cells and may thus become a future therapeutic tool.

  18. Missense variants in AIMP1 gene are implicated in autosomal recessive intellectual disability without neurodegeneration

    OpenAIRE

    Iqbal, Zafar; P?ttmann, Lucia; Musante, Luciana; Razzaq, Attia; Zahoor, Muhammad Yasir; Hu, Hao; Wienker, Thomas F; Garshasbi, Masoud; Fattahi, Zohreh; Gilissen, Christian; Vissers, Lisenka ELM; de Brouwer, Arjan PM; Veltman, Joris A; Pfundt, Rolph; Najmabadi, Hossein

    2015-01-01

    AIMP1/p43 is a multifunctional non-catalytic component of the multisynthetase complex. The complex consists of nine catalytic and three non-catalytic proteins, which catalyze the ligation of amino acids to their cognate tRNA isoacceptors for use in protein translation. To date, two allelic variants in the AIMP1 gene have been reported as the underlying cause of autosomal recessive primary neurodegenerative disorder. Here, we present two consanguineous families from Pakistan and Iran, presenti...

  19. Molecular Aspects of Dopaminergic Neurodegeneration: Gene-Environment Interaction in Parkin Dysfunction

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    Syed Z. Imam

    2011-12-01

    Full Text Available Parkinson’s disease (PD is a common neurodegenerative movement disorder that is characterized pathologically by a progressive loss of midbrain dopaminergic neurons and by protein inclusions, designated Lewy bodies and Lewy neurites. PD is one of the most common neurodegenerative diseases, affecting almost 1% of the population over 60 years old. Although the symptoms and neuropathology of PD have been well characterized, the underlying mechanisms and causes of the disease are still not clear. Genetic mutations can provide important clues to disease mechanism, but most PD cases are sporadic rather than familial; environmental factors have long been suspected to contribute to the disease. Although more than 90% of PD cases occur sporadically and are thought to be due, in part, to oxidative stress and mitochondrial dysfunction, the study of genetic mutations has provided great insight into the molecular mechanisms of PD. Furthermore, rotenone, a widely used pesticide, and paraquat and maneb cause a syndrome in rats and mice that mimics, both behaviorally and neurologically, the symptoms of PD. In the current review, we will discuss various aspects of gene-environment interaction that lead to progressive dopaminergic neurodegenration, mainly focusing on our current finding based on stress-mediated parkin dysfunction.

  20. Environmental factors as modulators of neurodegeneration: insights from gene-environment interactions in Huntington's disease.

    Science.gov (United States)

    Mo, Christina; Hannan, Anthony J; Renoir, Thibault

    2015-05-01

    Unlike many other neurodegenerative diseases with established gene-environment interactions, Huntington's disease (HD) is viewed as a disorder governed by genetics. The cause of the disease is a highly penetrant tandem repeat expansion encoding an extended polyglutamine tract in the huntingtin protein. In the year 2000, a pioneering study showed that the disease could be delayed in transgenic mice by enriched housing conditions. This review describes subsequent human and preclinical studies identifying environmental modulation of motor, cognitive, affective and other symptoms found in HD. Alongside the behavioral observations we also discuss potential mechanisms and the relevance to other neurodegenerative disorders, including Alzheimer's and Parkinson's disease. In mouse models of HD, increased sensorimotor and cognitive stimulation can delay or ameliorate various endophenotypes. Potential mechanisms include increased trophic support, synaptic plasticity, adult neurogenesis, and other forms of experience-dependent cellular plasticity. Subsequent clinical investigations support a role for lifetime activity levels in modulating the onset and progression of HD. Stress can accelerate memory and olfactory deficits and exacerbate cellular dysfunctions in HD mice. In the absence of effective treatments to slow the course of HD, environmental interventions offer feasible approaches to delay the disease, however further preclinical and human studies are needed in order to generate clinical recommendations. Environmental interventions could be combined with future pharmacological therapies and stimulate the identification of enviromimetics, drugs which mimic or enhance the beneficial effects of cognitive stimulation and physical activity. Copyright © 2015. Published by Elsevier Ltd.

  1. Chromosome preference of disease genes and vectorization for the prediction of non-coding disease genes.

    Science.gov (United States)

    Peng, Hui; Lan, Chaowang; Liu, Yuansheng; Liu, Tao; Blumenstein, Michael; Li, Jinyan

    2017-10-03

    Disease-related protein-coding genes have been widely studied, but disease-related non-coding genes remain largely unknown. This work introduces a new vector to represent diseases, and applies the newly vectorized data for a positive-unlabeled learning algorithm to predict and rank disease-related long non-coding RNA (lncRNA) genes. This novel vector representation for diseases consists of two sub-vectors, one is composed of 45 elements, characterizing the information entropies of the disease genes distribution over 45 chromosome substructures. This idea is supported by our observation that some substructures (e.g., the chromosome 6 p-arm) are highly preferred by disease-related protein coding genes, while some (e.g., the 21 p-arm) are not favored at all. The second sub-vector is 30-dimensional, characterizing the distribution of disease gene enriched KEGG pathways in comparison with our manually created pathway groups. The second sub-vector complements with the first one to differentiate between various diseases. Our prediction method outperforms the state-of-the-art methods on benchmark datasets for prioritizing disease related lncRNA genes. The method also works well when only the sequence information of an lncRNA gene is known, or even when a given disease has no currently recognized long non-coding genes.

  2. Gene therapy of Fanconi anemia: preclinical efficacy using lentiviral vectors.

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    Galimi, Francesco; Noll, Meenakshi; Kanazawa, Yoshiyuki; Lax, Timothy; Chen, Cindy; Grompe, Markus; Verma, Inder M

    2002-10-15

    Fanconi anemia (FA) is an inherited cancer susceptibility syndrome caused by mutations in a DNA repair pathway including at least 6 genes (FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG). The clinical course of the disease is dominated by progressive, life-threatening bone marrow failure and high incidence of acute myelogenous leukemia and solid tumors. Allogeneic bone marrow transplantation (BMT) is a therapeutic option but requires HLA-matched donors. Gene therapy holds great promise for FA, but previous attempts to use retroviral vectors in humans have proven ineffective given the impaired proliferation potential of human FA hematopoietic progenitors (HPCs). In this work, we show that using lentiviral vectors efficient genetic correction can be achieved in quiescent hematopoietic progenitors from Fanca(-/-) and Fancc(-/-) mice. Long-term repopulating HPCs were transduced by a single exposure of unfractionated bone marrow mononuclear cells to lentivectors carrying the normal gene. Notably, no cell purification or cytokine prestimulation was necessary. Resistance to DNA- damaging agents was fully restored by lentiviral transduction, allowing for in vivo selection of the corrected cells with nonablative doses of cyclophosphamide. This study strongly supports the use of lentiviral vectors for FA gene therapy in humans.

  3. Bacteria as vectors for gene therapy of cancer.

    LENUS (Irish Health Repository)

    Baban, Chwanrow K

    2012-01-31

    Anti-cancer therapy faces major challenges, particularly in terms of specificity of treatment. The ideal therapy would eradicate tumor cells selectively with minimum side effects on normal tissue. Gene or cell therapies have emerged as realistic prospects for the treatment of cancer, and involve the delivery of genetic information to a tumor to facilitate the production of therapeutic proteins. However, there is still much to be done before an efficient and safe gene medicine is achieved, primarily developing the means of targeting genes to tumors safely and efficiently. An emerging family of vectors involves bacteria of various genera. It has been shown that bacteria are naturally capable of homing to tumors when systemically administered resulting in high levels of replication locally. Furthermore, invasive species can deliver heterologous genes intra-cellularly for tumor cell expression. Here, we review the use of bacteria as vehicles for gene therapy of cancer, detailing the mechanisms of action and successes at preclinical and clinical levels.

  4. Development of Viral Vectors for Gene Therapy for Chronic Pain

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    Yu Huang

    2011-01-01

    Full Text Available Chronic pain is a major health concern that affects millions of people. There are no adequate long-term therapies for chronic pain sufferers, leading to significant cost for both society and the individual. The most commonly used therapy for chronic pain is the application of opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to addiction and may cause side effects. Further studies of the mechanisms of chronic pain have opened the way for development of new treatment strategies, one of which is gene therapy. The key to gene therapy is selecting safe and highly efficient gene delivery systems that can deliver therapeutic genes to overexpress or suppress relevant targets in specific cell types. Here we review several promising viral vectors that could be applied in gene transfer for the treatment of chronic pain and further discuss the possible mechanisms of genes of interest that could be delivered with viral vectors for the treatment of chronic pain.

  5. Novel redox nanomedicine improves gene expression of polyion complex vector

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    Kazuko Toh, Toru Yoshitomi, Yutaka Ikeda and Yukio Nagasaki

    2011-01-01

    Full Text Available Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP as an ROS scavenger. When polyethyleneimine (PEI/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems.

  6. A multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors for functional gene analysis.

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    Weber, Kristoffer; Bartsch, Udo; Stocking, Carol; Fehse, Boris

    2008-04-01

    Functional gene analysis requires the possibility of overexpression, as well as downregulation of one, or ideally several, potentially interacting genes. Lentiviral vectors are well suited for this purpose as they ensure stable expression of complementary DNAs (cDNAs), as well as short-hairpin RNAs (shRNAs), and can efficiently transduce a wide spectrum of cell targets when packaged within the coat proteins of other viruses. Here we introduce a multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors designed according to the "building blocks" principle. Using a wide spectrum of different fluorescent markers, including drug-selectable enhanced green fluorescent protein (eGFP)- and dTomato-blasticidin-S resistance fusion proteins, LeGO vectors allow simultaneous analysis of multiple genes and shRNAs of interest within single, easily identifiable cells. Furthermore, each functional module is flanked by unique cloning sites, ensuring flexibility and individual optimization. The efficacy of these vectors for analyzing multiple genes in a single cell was demonstrated in several different cell types, including hematopoietic, endothelial, and neural stem and progenitor cells, as well as hepatocytes. LeGO vectors thus represent a valuable tool for investigating gene networks using conditional ectopic expression and knock-down approaches simultaneously.

  7. The feasibility of incorporating Vpx into lentiviral gene therapy vectors

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    Samantha A McAllery

    2016-01-01

    Full Text Available While current antiretroviral therapy has significantly improved, challenges still remain in life-long targeting of HIV-1 reservoirs. Lentiviral gene therapy has the potential to deliver protective genes into the HIV-1 reservoir. However, inefficient reverse transcription (RT occurs in HIV-1 reservoirs during lentiviral gene delivery. The viral protein Vpx is capable of increasing lentiviral RT by antagonizing the restriction factor SAMHD1. Incorporating Vpx into lentiviral vectors could substantially increase gene delivery into the HIV-1 reservoir. The feasibility of this Vpx approach was tested in resting cell models utilizing macrophages and dendritic cells. Our results showed Vpx exposure led to increased permissiveness of cells over a period that exceeded 2 weeks. Consequently, significant lower potency of HIV-1 antiretrovirals inhibiting RT and integration was observed. When Vpx was incorporated with anti-HIV-1 genes inhibiting either pre-RT or post-RT stages of the viral life-cycle, transduction levels significantly increased. However, a stronger antiviral effect was only observed with constructs that inhibit pre-RT stages of the viral life cycle. In conclusion this study demonstrates a way to overcome the major delivery obstacle of gene delivery into HIV-1 reservoir cell types. Importantly, incorporating Vpx with pre-RT anti-HIV-1 genes, demonstrated the greatest protection against HIV-1 infection.

  8. Recombinant vectors construction for cellobiohydrolase encoding gene constitutive expression

    Directory of Open Access Journals (Sweden)

    Leontina GURGU

    2012-12-01

    Full Text Available Cellobiohydrolases (EC 3.2.1.91 are important exo enzymes involved in cellulose hydrolysis alongside endoglucanases (EC 3.2.1.4 and β-glucosidases (EC 3.2.1.21. Heterologous cellobiohydrolase gene expression under constitutive promoter control using Saccharomyces cerevisiae as host system is of great importance for a successful SSF process. From this point of view, the main objective of the work was to use Yeplac181 expression vector as a recipient for cellobiohdrolase - cbhB encoding gene expression under the control of the actin promoter, in Saccharomyces cerevisiae. Two hybridvectors, YEplac-Actp and YEplac-Actp-CbhB, were generated usingEscherichia coli XLI Blue for the cloning experiments. Constitutive cbhB gene expression was checked by proteine gel electrophoresis (SDS-PAGE after insertion of these constructs into Saccharomyces cerevisiae.

  9. An efficient marker-free vector for clean gene transfer into plants ...

    African Journals Online (AJOL)

    A marker-free vector, pBINMF, for clean gene transfer was constructed based on the binary vector pBINPLUS. Vector pBINMF, carrying only a multiple cloning site (MCS) between the left and the right T-DNA border, was suitable to directly generate marker-free transgenic plants (MFTPs) without any vector sequences ...

  10. HIV-derived vectors for gene therapy targeting dendritic cells.

    Science.gov (United States)

    Rossetti, Maura; Cavarelli, Mariangela; Gregori, Silvia; Scarlatti, Gabriella

    2013-01-01

    Human immunodeficiency virus type 1 (HIV-1)-derived lentiviral vectors (LV) have the potential to mediate stable therapeutic gene transfer. However, similarly to other viral vectors, their benefit is compromised by the induction of an immune response toward transgene-expressing cells that closely mimics antiviral immunity. LV share with the parental HIV the ability to activate dendritic cells (DC), while lack the peculiar ability of subverting DC functions, which is responsible for HIV immune escape. Understanding the interaction between LV and DC, with plasmacytoid and myeloid DC playing fundamental and distinct roles, has paved the way to novel approaches aimed at regulating transgene-specific immune responses. Thanks to the ability to target either DC subsets LV might be a powerful tool to induce immunity (i.e., gene therapy of cancer), cell death (i.e., in HIV/AIDS infection), or tolerance (i.e., gene therapy strategies for monogenic diseases). In this chapter, similarities and differences between the LV-mediated and HIV-mediated induction of immune responses, with specific focus on their interactions with DC, are discussed.

  11. Gene Expression Profiling as a Tool to Investigate the Molecular Machinery Activated during Hippocampal Neurodegeneration Induced by Trimethyltin (TMT Administration

    Directory of Open Access Journals (Sweden)

    Maria Concetta Geloso

    2013-08-01

    Full Text Available Trimethyltin (TMT is an organotin compound exhibiting neurotoxicant effects selectively localized in the limbic system and especially marked in the hippocampus, in both experimental animal models and accidentally exposed humans. TMT administration causes selective neuronal death involving either the granular neurons of the dentate gyrus or the pyramidal cells of the Cornu Ammonis, with a different pattern of localization depending on the different species studied or the dosage schedule. TMT is broadly used to realize experimental models of hippocampal neurodegeneration associated with cognitive impairment and temporal lobe epilepsy, though the molecular mechanisms underlying the associated selective neuronal death are still not conclusively clarified. Experimental evidence indicates that TMT-induced neurodegeneration is a complex event involving different pathogenetic mechanisms, probably acting differently in animal and cell models, which include neuroinflammation, intracellular calcium overload, and oxidative stress. Microarray-based, genome-wide expression analysis has been used to investigate the molecular scenario occurring in the TMT-injured brain in different in vivo and in vitro models, producing an overwhelming amount of data. The aim of this review is to discuss and rationalize the state-of-the-art on TMT-associated genome wide expression profiles in order to identify comparable and reproducible data that may allow focusing on significantly involved pathways.

  12. Metals and Neurodegeneration

    Science.gov (United States)

    Chen, Pan; Miah, Mahfuzur Rahman; Aschner, Michael

    2016-01-01

    Metals play important roles in the human body, maintaining cell structure and regulating gene expression, neurotransmission, and antioxidant response, to name a few. However, excessive metal accumulation in the nervous system may be toxic, inducing oxidative stress, disrupting mitochondrial function, and impairing the activity of numerous enzymes. Damage caused by metal accumulation may result in permanent injuries, including severe neurological disorders. Epidemiological and clinical studies have shown a strong correlation between aberrant metal exposure and a number of neurological diseases, including Alzheimer’s disease, amyotrophic lateral sclerosis, autism spectrum disorders, Guillain–Barré disease, Gulf War syndrome, Huntington’s disease, multiple sclerosis, Parkinson’s disease, and Wilson’s disease. Here, we briefly survey the literature relating to the role of metals in neurodegeneration. PMID:27006759

  13. Herpes simplex virus type 1 (HSV-1)-derived recombinant vectors for gene transfer and gene therapy.

    Science.gov (United States)

    Marconi, Peggy; Fraefel, Cornel; Epstein, Alberto L

    2015-01-01

    Herpes simplex virus type 1 (HSV-1 ) is a human pathogen whose lifestyle is based on a long-term dual interaction with the infected host, being able to establish both lytic and latent infections. The virus genome is a 153-kilobase pair (kbp) double-stranded DNA molecule encoding more than 80 genes. The interest of HSV-1 as gene transfer vector stems from its ability to infect many different cell types, both quiescent and proliferating cells, the very high packaging capacity of the virus capsid, the outstanding neurotropic adaptations that this virus has evolved, and the fact that it never integrates into the cellular chromosomes, thus avoiding the risk of insertional mutagenesis. Two types of vectors can be derived from HSV-1, recombinant vectors and amplicon vectors, and different methodologies have been developed to prepare large stocks of each type of vector. This chapter summarizes the approach most commonly used to prepare recombinant HSV-1 vectors through homologous recombination, either in eukaryotic cells or in bacteria.

  14. Perinatal systemic gene delivery using adeno-associated viral vectors

    Directory of Open Access Journals (Sweden)

    Rajvinder eKarda

    2014-11-01

    Full Text Available Neurodegenerative monogenic diseases can also affect a broad range of tissues and organs throughout the body. An effective treatment would require a systemic approach. The intravenous administration of novel therapies is ideal but is hampered by the inability of such drugs to cross the blood-brain barrier and precludes efficacy in the central nervous system. A number of these early lethal intractable diseases also present devastating irreversible pathology at birth or soon after. Therefore, any therapy would ideally be administered during the perinatal period to prevent, stop or ameliorate disease progression. The concept of perinatal gene therapy has moved a step further towards being a feasible approach to treating such disorders. This has primarily been driven by the recent discoveries that particular serotypes of adeno-associated virus (AAV gene delivery vectors have the ability to cross the blood-brain barrier following intravenous administration. Furthermore, this has been safely demonstrated in perinatal mice and non-human primates. This review focuses on the progress made in using AAV to achieve systemic transduction and what this means for developing perinatal gene therapy for early lethal neurodegenerative diseases.

  15. Gene expression profiling for human iPS-derived motor neurons from sporadic ALS patients reveals a strong association between mitochondrial functions and neurodegeneration

    Science.gov (United States)

    Alves, Chrystian J.; Dariolli, Rafael; Jorge, Frederico M.; Monteiro, Matheus R.; Maximino, Jessica R.; Martins, Roberto S.; Strauss, Bryan E.; Krieger, José E.; Callegaro, Dagoberto; Chadi, Gerson

    2015-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that leads to widespread motor neuron death, general palsy and respiratory failure. The most prevalent sporadic ALS form is not genetically inherited. Attempts to translate therapeutic strategies have failed because the described mechanisms of disease are based on animal models carrying specific gene mutations and thus do not address sporadic ALS. In order to achieve a better approach to study the human disease, human induced pluripotent stem cell (hiPSC)-differentiated motor neurons were obtained from motor nerve fibroblasts of sporadic ALS and non-ALS subjects using the STEMCCA Cre-Excisable Constitutive Polycistronic Lentivirus system and submitted to microarray analyses using a whole human genome platform. DAVID analyses of differentially expressed genes identified molecular function and biological process-related genes through Gene Ontology. REVIGO highlighted the related functions mRNA and DNA binding, GTP binding, transcription (co)-repressor activity, lipoprotein receptor binding, synapse organization, intracellular transport, mitotic cell cycle and cell death. KEGG showed pathways associated with Parkinson's disease and oxidative phosphorylation, highlighting iron homeostasis, neurotrophic functions, endosomal trafficking and ERK signaling. The analysis of most dysregulated genes and those representative of the majority of categorized genes indicates a strong association between mitochondrial function and cellular processes possibly related to motor neuron degeneration. In conclusion, iPSC-derived motor neurons from motor nerve fibroblasts of sporadic ALS patients may recapitulate key mechanisms of neurodegeneration and may offer an opportunity for translational investigation of sporadic ALS. Large gene profiling of differentiated motor neurons from sporadic ALS patients highlights mitochondrial participation in the establishment of autonomous mechanisms associated with sporadic ALS

  16. PCR-based detection of gene transfer vectors: application to gene doping surveillance.

    Science.gov (United States)

    Perez, Irene C; Le Guiner, Caroline; Ni, Weiyi; Lyles, Jennifer; Moullier, Philippe; Snyder, Richard O

    2013-12-01

    Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR.

  17. Mesoporous silica nanoparticles as vectors for gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Crapina, Laura Cipriano; Bizeto, Marcos, E-mail: lauracrapina@hotmail.com [Universidade Federal de Sao Paulo (UNIFESP), SP (Brazil)

    2016-07-01

    Full text: Mesoporous silica nanoparticles present unique physical-chemical properties, such as high surface area, tunable pore size, easy surface chemical modification, good biocompatibility and low toxicology. Those properties make this class of inorganic materials promising for several potential applications in the biomedical field. This work seeks to develop mesoporous silica nanoparticles with characteristics suitable to the transport of nucleic acids, such as plasmid DNA and microRNA, with the aim of substituting viral vectors in gene therapy. A successful nanocarrier must have positive charge at physiological conditions and pore diameter larger than 30 Å. The mesoporous silica was synthesized according to the method described by Bein and collaborators [1]. Based on a cocondensation synthetic route, positively charged nanoparticles were obtained through the insertion of N-3-(trimethoxysilyl)propyldiethylenetriamine in the silica walls. Pore expansion was achieved through the incorporation of 1,2,4- trimethylbenzene into the hexadecyltrimethylammonium micellar aggregates, which are a structure-directing agent for the mesopores. The resulting nanoparticles were characterized by DLS, ζ potential, XRD, FTIR, SEM, TEM, TGA and elemental analysis. In addition, the capability of nucleic acid adsorption was tested and confirmed by gel electrophoresis. Discovery of a non-viral therapeutic agent would aid the viability of gene therapy, which is a treatment for chronic ischemia, metabolic and genetic disorders. Reference: [1] K. Moeller, J. Kobler, T. Bein, Journal of Materials Chemistry, 17, 624-631, (2007). (author)

  18. A sight on the current nanoparticle-based gene delivery vectors

    Science.gov (United States)

    Dizaj, Solmaz Maleki; Jafari, Samira; Khosroushahi, Ahmad Yari

    2014-05-01

    Nowadays, gene delivery for therapeutic objects is considered one of the most promising strategies to cure both the genetic and acquired diseases of human. The design of efficient gene delivery vectors possessing the high transfection efficiencies and low cytotoxicity is considered the major challenge for delivering a target gene to specific tissues or cells. On this base, the investigations on non-viral gene vectors with the ability to overcome physiological barriers are increasing. Among the non-viral vectors, nanoparticles showed remarkable properties regarding gene delivery such as the ability to target the specific tissue or cells, protect target gene against nuclease degradation, improve DNA stability, and increase the transformation efficiency or safety. This review attempts to represent a current nanoparticle based on its lipid, polymer, hybrid, and inorganic properties. Among them, hybrids, as efficient vectors, are utilized in gene delivery in terms of materials (synthetic or natural), design, and in vitro/ in vivo transformation efficiency.

  19. Vectors

    DEFF Research Database (Denmark)

    Boeriis, Morten; van Leeuwen, Theo

    2017-01-01

    should be taken into account in discussing ‘reactions’, which Kress and van Leeuwen link only to eyeline vectors. Finally, the question can be raised as to whether actions are always realized by vectors. Drawing on a re-reading of Rudolf Arnheim’s account of vectors, these issues are outlined......This article revisits the concept of vectors, which, in Kress and van Leeuwen’s Reading Images (2006), plays a crucial role in distinguishing between ‘narrative’, action-oriented processes and ‘conceptual’, state-oriented processes. The use of this concept in image analysis has usually focused...

  20. Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ishii, Jun; Kondo, Takashi; Makino, Harumi; Ogura, Akira; Matsuda, Fumio; Kondo, Akihiko

    2014-05-01

    Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  1. Extensive innate immune gene activation accompanies brain aging, increasing vulnerability to cognitive decline and neurodegeneration: a microarray study

    Science.gov (United States)

    2012-01-01

    Background This study undertakes a systematic and comprehensive analysis of brain gene expression profiles of immune/inflammation-related genes in aging and Alzheimer’s disease (AD). Methods In a well-powered microarray study of young (20 to 59 years), aged (60 to 99 years), and AD (74 to 95 years) cases, gene responses were assessed in the hippocampus, entorhinal cortex, superior frontal gyrus, and post-central gyrus. Results Several novel concepts emerge. First, immune/inflammation-related genes showed major changes in gene expression over the course of cognitively normal aging, with the extent of gene response far greater in aging than in AD. Of the 759 immune-related probesets interrogated on the microarray, approximately 40% were significantly altered in the SFG, PCG and HC with increasing age, with the majority upregulated (64 to 86%). In contrast, far fewer immune/inflammation genes were significantly changed in the transition to AD (approximately 6% of immune-related probesets), with gene responses primarily restricted to the SFG and HC. Second, relatively few significant changes in immune/inflammation genes were detected in the EC either in aging or AD, although many genes in the EC showed similar trends in responses as in the other brain regions. Third, immune/inflammation genes undergo gender-specific patterns of response in aging and AD, with the most pronounced differences emerging in aging. Finally, there was widespread upregulation of genes reflecting activation of microglia and perivascular macrophages in the aging brain, coupled with a downregulation of select factors (TOLLIP, fractalkine) that when present curtail microglial/macrophage activation. Notably, essentially all pathways of the innate immune system were upregulated in aging, including numerous complement components, genes involved in toll-like receptor signaling and inflammasome signaling, as well as genes coding for immunoglobulin (Fc) receptors and human leukocyte antigens I

  2. Extensive innate immune gene activation accompanies brain aging, increasing vulnerability to cognitive decline and neurodegeneration: a microarray study

    Directory of Open Access Journals (Sweden)

    Cribbs David H

    2012-07-01

    Full Text Available Abstract Background This study undertakes a systematic and comprehensive analysis of brain gene expression profiles of immune/inflammation-related genes in aging and Alzheimer’s disease (AD. Methods In a well-powered microarray study of young (20 to 59 years, aged (60 to 99 years, and AD (74 to 95 years cases, gene responses were assessed in the hippocampus, entorhinal cortex, superior frontal gyrus, and post-central gyrus. Results Several novel concepts emerge. First, immune/inflammation-related genes showed major changes in gene expression over the course of cognitively normal aging, with the extent of gene response far greater in aging than in AD. Of the 759 immune-related probesets interrogated on the microarray, approximately 40% were significantly altered in the SFG, PCG and HC with increasing age, with the majority upregulated (64 to 86%. In contrast, far fewer immune/inflammation genes were significantly changed in the transition to AD (approximately 6% of immune-related probesets, with gene responses primarily restricted to the SFG and HC. Second, relatively few significant changes in immune/inflammation genes were detected in the EC either in aging or AD, although many genes in the EC showed similar trends in responses as in the other brain regions. Third, immune/inflammation genes undergo gender-specific patterns of response in aging and AD, with the most pronounced differences emerging in aging. Finally, there was widespread upregulation of genes reflecting activation of microglia and perivascular macrophages in the aging brain, coupled with a downregulation of select factors (TOLLIP, fractalkine that when present curtail microglial/macrophage activation. Notably, essentially all pathways of the innate immune system were upregulated in aging, including numerous complement components, genes involved in toll-like receptor signaling and inflammasome signaling, as well as genes coding for immunoglobulin (Fc receptors and human

  3. Pseudotyped Lentiviral Vectors for Retrograde Gene Delivery into Target Brain Regions

    Directory of Open Access Journals (Sweden)

    Kenta Kobayashi

    2017-08-01

    Full Text Available Gene transfer through retrograde axonal transport of viral vectors offers a substantial advantage for analyzing roles of specific neuronal pathways or cell types forming complex neural networks. This genetic approach may also be useful in gene therapy trials by enabling delivery of transgenes into a target brain region distant from the injection site of the vectors. Pseudotyping of a lentiviral vector based on human immunodeficiency virus type 1 (HIV-1 with various fusion envelope glycoproteins composed of different combinations of rabies virus glycoprotein (RV-G and vesicular stomatitis virus glycoprotein (VSV-G enhances the efficiency of retrograde gene transfer in both rodent and nonhuman primate brains. The most recently developed lentiviral vector is a pseudotype with fusion glycoprotein type E (FuG-E, which demonstrates highly efficient retrograde gene transfer in the brain. The FuG-E–pseudotyped vector permits powerful experimental strategies for more precisely investigating the mechanisms underlying various brain functions. It also contributes to the development of new gene therapy approaches for neurodegenerative disorders, such as Parkinson’s disease, by delivering genes required for survival and protection into specific neuronal populations. In this review article, we report the properties of the FuG-E–pseudotyped vector, and we describe the application of the vector to neural circuit analysis and the potential use of the FuG-E vector in gene therapy for Parkinson’s disease.

  4. Preparation and characterization of magnetic gene vectors for targeting gene delivery

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, S.W.; Liu, G. [College of Chemistry, Chemical Engineering and Materials Science and Key Laboratory of Organic Synthesis of Jiangsu Province, Soochow University, SIP, Suzhou 215123 (China); Hong, R.Y., E-mail: rhong@suda.edu.cn [College of Chemistry, Chemical Engineering and Materials Science and Key Laboratory of Organic Synthesis of Jiangsu Province, Soochow University, SIP, Suzhou 215123 (China); State Key Laboratory of Multi-phase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080 (China); Li, H.Z. [State Key Laboratory of Multi-phase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080 (China); Li, Y.G., E-mail: ilguoliang@sohu.com [Department of radiology, the First Affiliated Hospital of Soochow University, Suzhou 215007 (China); Wei, D.G., E-mail: dougwei@deas.harvard.edu [Center for Nanoscale Systems, School of Engineering and Applied Science, Harvard University, 11 Oxford Street, Cambridge, MA 02139 (United States)

    2012-10-15

    Highlights: Black-Right-Pointing-Pointer PEI is ideal candidate polymer for the design of gene delivery systems. Black-Right-Pointing-Pointer PEI-CMD-MNPs exhibited a typical superparamagnetic behavior. Black-Right-Pointing-Pointer PEI-CMD-MNPs were well stable over the entire range of pH and NaCl concentration. Black-Right-Pointing-Pointer DNA-PEI-CMD-MNPs transfected cells by a magnet have higher transfection efficiency and gene expression efficiency. - Abstract: The PEI-CMD-MNPs were successfully prepared by the surface modification of magnetic Fe{sub 3}O{sub 4} nanoparticles with carboxymethyl dextran (CMD) and polyethyleneimine (PEI). The PEI-CMD-MNPs polyplexes exhibited a typical superparamagnetic behavior and were well stable over the entire range of pH and NaCl concentration. These PEI-CMD-MNPs were used as magnetic gene vectors for targeting gene delivery. The prepared MNPs at different surface modification stages were characterized using Fourier transform infrared (FT-IR), thermogravimetric analysis (TGA), field emissions canning electron microscopy (FE-SEM), powder X-ray diffraction (XRD) and dynamic laser light scattering (DLS) analysis. The magnetic properties were studied by vibrating sample magnetometer (VSM). To evaluate the performance of the magnetic nanoparticles as gene transfer vector, the PEI-CMD-MNPs were used to delivery green fluorescent protein (GFP) gene into BHK21 cells. The expression of GFP gene was detected by fluorescence microscope. DNA-PEI-CMD-MNPs polyplexes absorbed by the cells were also monitored by Magnetic resonance imaging (MRI). The transfection efficiency and gene expression efficiency of that transfected with a magnet were much higher than that of standard transfection.

  5. A novel binary T-vector with the GFP reporter gene for promoter characterization.

    Directory of Open Access Journals (Sweden)

    Shu-Ye Jiang

    Full Text Available Several strategies have been developed to clone PCR fragments into desired vectors. However, most of commercially available T-vectors are not binary vectors and cannot be directly used for Agrobacterium-mediated plant genetic transformation. In this study, a novel binary T-vector was constructed by integrating two AhdI restriction sites into the backbone vector pCAMBIA 1300. The T-vector also contains a GFP reporter gene and thus, can be used to analyze promoter activity by monitoring the reporter gene. On the other hand, identification and characterization of various promoters not only benefit the functional annotation of their genes but also provide alternative candidates to be used to drive interesting genes for plant genetic improvement by transgenesis. More than 1,000 putative pollen-specific rice genes have been identified in a genome-wide level. Among them, 67 highly expressed genes were further characterized. One of the pollen-specific genes LOC_Os10g35930 was further surveyed in its expression patterns with more details by quantitative real-time reverse-transcription PCR (qRT-PCR analysis. Finally, its promoter activity was further investigated by analyzing transgenic rice plants carrying the promoter::GFP cassette, which was constructed from the newly developed T-vector. The reporter GFP gene expression in these transgenic plants showed that the promoter was active only in mature but not in germinated pollens.

  6. In situ preparation of magnetic nonviral gene vectors and magnetofection in vitro

    International Nuclear Information System (INIS)

    Shi Yunfeng; Pang Yan; Su Yue; Zhu Xinyuan; Yan Deyue; Zhou Linzhu; Xiao Wangchuan; Wang Xiaoliang; Gu Hongchen; Wang Ruibin; Li Huiqin; Zhu Bangshang

    2010-01-01

    Magnetic nonviral gene vectors were in situ prepared in the presence of ferrous salts and hyperbranched poly(ethylenimine)s (HPEI) with different molecular weights. HPEI, one of the most promising nonviral vectors, was not only utilized as the nanoreactor and stabilizer to prepare magnetic nanoparticles, but also skillfully used as a base supplier to avoid introducing alkali hydroxide or ammonia. Magnetic nonviral gene vectors with various magnetite contents and saturation magnetizations were obtained by changing the weight ratio of HPEI to FeSO 4 ·7H 2 O and the molecular weight of HPEI. MTT assays suggested that the resulting magnetite/HPEI gene vectors had lower cytotoxicity compared with pure HPEI. The magnetite/HPEI nonviral gene vectors were used for magnetofection. It was found that the luciferase expression level mediated by magnetite/HPEI in COS-7 cells under a magnetic gradient field was approximately 13-fold greater than that of standard HPEI transfection.

  7. Graphene materials as 2D non-viral gene transfer vector platforms.

    Science.gov (United States)

    Vincent, M; de Lázaro, I; Kostarelos, K

    2017-03-01

    Advances in genomics and gene therapy could offer solutions to many diseases that remain incurable today, however, one of the critical reasons halting clinical progress is due to the difficulty in designing efficient and safe delivery vectors for the appropriate genetic cargo. Safety and large-scale production concerns counter-balance the high gene transfer efficiency achieved with viral vectors, while non-viral strategies have yet to become sufficiently efficient. The extraordinary physicochemical, optical and photothermal properties of graphene-based materials (GBMs) could offer two-dimensional components for the design of nucleic acid carrier systems. We discuss here such properties and their implications for the optimization of gene delivery. While the design of such vectors is still in its infancy, we provide here an exhaustive and up-to-date analysis of the studies that have explored GBMs as gene transfer vectors, focusing on the functionalization strategies followed to improve vector performance and on the biological effects attained.

  8. Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

    OpenAIRE

    Podsakoff, G; Wong, K K; Chatterjee, S

    1994-01-01

    Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthe...

  9. An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer.

    Directory of Open Access Journals (Sweden)

    Julius W Kim

    Full Text Available Vectors based on human adenovirus serotype 5 (HAdV-5 continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting.As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4. This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells.These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.

  10. Highly efficient retrograde gene transfer into motor neurons by a lentiviral vector pseudotyped with fusion glycoprotein.

    Directory of Open Access Journals (Sweden)

    Miyabi Hirano

    Full Text Available The development of gene therapy techniques to introduce transgenes that promote neuronal survival and protection provides effective therapeutic approaches for neurological and neurodegenerative diseases. Intramuscular injection of adenoviral and adeno-associated viral vectors, as well as lentiviral vectors pseudotyped with rabies virus glycoprotein (RV-G, permits gene delivery into motor neurons in animal models for motor neuron diseases. Recently, we developed a vector with highly efficient retrograde gene transfer (HiRet by pseudotyping a human immunodeficiency virus type 1 (HIV-1-based vector with fusion glycoprotein B type (FuG-B or a variant of FuG-B (FuG-B2, in which the cytoplasmic domain of RV-G was replaced by the corresponding part of vesicular stomatitis virus glycoprotein (VSV-G. We have also developed another vector showing neuron-specific retrograde gene transfer (NeuRet with fusion glycoprotein C type, in which the short C-terminal segment of the extracellular domain and transmembrane/cytoplasmic domains of RV-G was substituted with the corresponding regions of VSV-G. These two vectors afford the high efficiency of retrograde gene transfer into different neuronal populations in the brain. Here we investigated the efficiency of the HiRet (with FuG-B2 and NeuRet vectors for retrograde gene transfer into motor neurons in the spinal cord and hindbrain in mice after intramuscular injection and compared it with the efficiency of the RV-G pseudotype of the HIV-1-based vector. The main highlight of our results is that the HiRet vector shows the most efficient retrograde gene transfer into both spinal cord and hindbrain motor neurons, offering its promising use as a gene therapeutic approach for the treatment of motor neuron diseases.

  11. Progress in developing cationic vectors for non-viral systemic gene therapy against cancer.

    Science.gov (United States)

    Morille, Marie; Passirani, Catherine; Vonarbourg, Arnaud; Clavreul, Anne; Benoit, Jean-Pierre

    2008-01-01

    Initially, gene therapy was viewed as an approach for treating hereditary diseases, but its potential role in the treatment of acquired diseases such as cancer is now widely recognized. The understanding of the molecular mechanisms involved in cancer and the development of nucleic acid delivery systems are two concepts that have led to this development. Systemic gene delivery systems are needed for therapeutic application to cells inaccessible by percutaneous injection and for multi-located tumor sites, i.e. metastases. Non-viral vectors based on the use of cationic lipids or polymers appear to have promising potential, given the problems of safety encountered with viral vectors. Using these non-viral vectors, the current challenge is to obtain a similarly effective transfection to viral ones. Based on the advantages and disadvantages of existing vectors and on the hurdles encountered with these carriers, the aim of this review is to describe the "perfect vector" for systemic gene therapy against cancer.

  12. Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

    OpenAIRE

    Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

    1990-01-01

    Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacc...

  13. Recent Advances in Non-viral Vectors for Gene Delivery

    Science.gov (United States)

    Guo, Xia; Huang, Leaf

    2011-01-01

    CONSPECTUS Non-viral vectors, typically based on cationic lipids or polymers, are preferred due to safety concerns with viral vectors. So far, non-viral vectors can proficiently transfect cells in culture, but obtaining efficient nanomedicines is far from evident. To overcome the hurdles associated with non-viral vectors is significant for improving delivery efficiency and therapeutic effect of nucleic acid. The drawbacks include the strong interaction of cationic delivery vehicles with blood components, uptake by the reticuloendothelial system (RES), toxicity, targeting ability of the carriers to the cells of interest, and so on. PEGylation is the predominant method used to reduce the binding of plasma proteins with non-viral vectors and minimize the clearance by RES after intravenous administration. The nanoparticles that are not rapidly cleared from the circulation accumulate in the tumors due to the enhanced permeability and retention effect, and the targeting ligands attached to the distal end of the PEGylated components allow binding to the receptors on the target cell surface. Neutral or anionic liposomes have been also developed for systemic delivery of nucleic acids in experimental animal model. Designing and synthesizing novel cationic lipids and polymers, and binding nucleic acid with peptides, targeting ligands, polymers, or environmentally sensitive moieties also attract many attentions for resolving the problems encountered by non-viral vectors. The application of inorganic nanoparticles in nucleic acid delivery is an emerging field, too. Recently, different classes of non-viral vectors appear to be converging and the features of different classes of non-viral vectors could be combined in one strategy. More hurdles associated with efficient nucleic acid delivery therefore might be expected to be overcome. In this account, we will focus on these novel non-viral vectors, which are classified into multifunctional hybrid nucleic acid vectors, novel

  14. Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption

    Science.gov (United States)

    2013-01-01

    Background The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker. Results We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered. Conclusions Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain. PMID:23497512

  15. Sleeping Beauty-baculovirus hybrid vectors for long-term gene expression in the eye.

    Science.gov (United States)

    Turunen, Tytteli Anni Kaarina; Laakkonen, Johanna Päivikki; Alasaarela, Laura; Airenne, Kari Juhani; Ylä-Herttuala, Seppo

    2014-01-01

    A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid-based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors. We constructed and optimized SB-baculovirus hybrid vectors that bear either SB100x transposase or SB transposon in the forward or reverse orientations with respect to the viral backbone The functionality of the novel hybrid vectors was investigated in cell cultures and in a proof-of-concept study in the mouse eye. The hybrid vectors showed high and sustained transgene expression that remained stable and demonstrated no signs of decline during the 2 months follow-up in vitro. These results were verified in the mouse eye where persistent transgene expression was detected two months after intravitreal injection. Our results confirm that (i) SB-baculovirus hybrid vectors mediate long-term gene expression in vitro and in vivo, and (ii) the hybrid vectors are potential new tools for the treatment of ocular diseases. Copyright © 2014 John Wiley & Sons, Ltd.

  16. Transcriptional Enhancers Induce Insertional Gene Deregulation Independently From the Vector Type and Design

    Science.gov (United States)

    Maruggi, Giulietta; Porcellini, Simona; Facchini, Giulia; Perna, Serena K; Cattoglio, Claudia; Sartori, Daniela; Ambrosi, Alessandro; Schambach, Axel; Baum, Christopher; Bonini, Chiara; Bovolenta, Chiara; Mavilio, Fulvio; Recchia, Alessandra

    2009-01-01

    The integration characteristics of retroviral (RV) vectors increase the probability of interfering with the regulation of cellular genes, and account for a tangible risk of insertional mutagenesis in treated patients. To assess the potential genotoxic risk of conventional or self-inactivating (SIN) γ-RV and lentiviral (LV) vectors independently from the biological consequences of the insertion event, we developed a quantitative assay based on real-time reverse transcriptase—PCR on low-density arrays to evaluate alterations of gene expression in individual primary T-cell clones. We show that the Moloney leukemia virus long terminal repeat (LTR) enhancer has the strongest activity in both a γ-RV and a LV vector context, while an internal cellular promoter induces deregulation of gene expression less frequently, at a shorter range and to a lower extent in both vector types. Downregulation of gene expression was observed only in the context of LV vectors. This study indicates that insertional gene activation is determined by the characteristics of the transcriptional regulatory elements carried by the vector, and is largely independent from the vector type or design. PMID:19293778

  17. Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy.

    Science.gov (United States)

    Vink, Conrad A; Counsell, John R; Perocheau, Dany P; Karda, Rajvinder; Buckley, Suzanne M K; Brugman, Martijn H; Galla, Melanie; Schambach, Axel; McKay, Tristan R; Waddington, Simon N; Howe, Steven J

    2017-08-02

    Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wild-type genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  18. Introduction of optical reporter gene into cancer and immune cells using lentiviral vector

    International Nuclear Information System (INIS)

    Min, Jung Joon; Le, Uyenchi N.; Moon, Sung Min; Heo, Young Jun; Song, Ho Chun; Bom, Hee Seung; Kim, Yeon Soo

    2004-01-01

    For some applications such as gene therapy or reporter gene imaging, a gene has to be introduced into the organism of interest. Adenoviral vectors are capable of transducing both replicating and non-dividing cells. The adenoviral vectors do not integrate their DNA into host DNA, but do lead to an immune response. Lentiviruses belong to the retrovirus family and are capable of infecting both dividing and non-dividing cells. The human immunodeficiency virus (HIV) is an example of a lentavirus. A disabled HIV virus has been developed and could be used for in vivo gene delivery. A portion of the viral genome which encodes for accessory proteins canbe deleted without affecting production of the vector and efficiency of infection. Lentiviral delivery into various rodent tissues shows sustained expression of the transgene of up to six months. Furthermore, there seems to be little or no immune response with these vectors. These lentiviral vectors hold significant promise for in vivo gene delivery. We constructed lentiviral vector encoding firefly luciferase (Fluc) and eGFP. Fluc-eGFP fusion gene was inserted into multiple cloning sites of pLentiM1.3 vector. Reporter gene (Fluc-eGFP) was designed to be driven by murine CMV promoter with enhanced efficacy of transgene expression as compared to human CMV promoter. We transfected pLenti1.3-Fluc into human cervix cancer cell line (HeLa) and murine T lymphocytes. We also constructed adenovirus encoding Fluc and transfected to HeLa and T cells. This LentiM1.3-Fluc was transfected into HeLa cells and murine T lymphocytes in vitro, showing consistent expression of eGFP under the fluorescence microscopy from the 2nd day of transfection. Firefly luciferase reporter gene was not expressed in immune cells when it is mediated by adenovirus. Lentivirus was validated as a useful vector for both immune and cancer cells

  19. Viral vectors for cystic fibrosis gene therapy: What does the future hold?

    Directory of Open Access Journals (Sweden)

    Uta Griesenbach

    2010-12-01

    Full Text Available Uta Griesenbach1, Makoto Inoue2, Mamoru Hasegawa2, Eric WFW Alton11Department of Gene Therapy, Imperial College London, UK; The UK Cystic Fibrosis Gene Therapy Consortium; 2DNAVEC Corporation, Tsukuba, JapanAbstract: Gene transfer to the airway epithelium has been more difficult than originally anticipated, largely because of significant extra- and intracellular barriers in the lung. In general, viral vectors are more adapted to overcoming these barriers than nonviral gene transfer agents and are, therefore, more efficient in transferring genes into recipient cells. Viral vectors derived from adenovirus, adeno-associated virus, and Sendai virus, which all have a natural tropism for the airway epithelium, have been evaluated for cystic fibrosis (CF gene therapy. Although these vectors transduce airway epithelial cells efficiently, gene expression is transient and repeated administration is inefficient. They are, therefore, unlikely to be suitable for CF gene therapy. More recently, lentiviruses (LV have been assessed for lung gene transfer. In contrast to retroviruses, they transduce nondividing cells and randomly integrate into the genome. However, LVs do not have a natural tropism for the lung, and a significant amount of effort has been put into pseudotyping these vectors with proteins suitable for airway gene transfer. Several studies have shown that LV-mediated transduction leads to persistent gene expression (for the lifetime of the animal in the airways and, importantly, repeated administration is feasible. Thus, appropriately pseudotyped LV vectors are promising candidates for CF gene therapy. Here, we will review preclinical and clinical research related to viral CF gene therapy.Keywords: cystic fibrosis, gene therapy, adenovirus, AAV, lentivirus, Sendai virus

  20. Hobo-like transposable elements as non-drosophilid gene vectors

    International Nuclear Information System (INIS)

    O'Brochta, D.A.; Warren, W.D.; Saville, K.J.; Whyard, S.; Mende, H.A.; Pinkerton, A.C.; Coates, C.J.; Atkinson, P.W.

    1998-01-01

    Using genetic and physical methods we discovered short-inverted repeat type transposable elements in non-drosophilid insects including, Bactorcera tryoni, Musca domestica, Musca vetustissima and Lucilia cuprina. These elements are related to hobo, Ac and Tam3. The Hermes element from M domestica is 2749 bp in length and has terminal inverted repeats and a transposase coding region very similar to those in hobo. Hermes is functional in M Domestic and can act as a gene vector in this species. When Hermes is introduced into D. melanogaster it is hyperactive, relative to existing vector systems used in this species. Hermes will be useful as a gene vector. (author)

  1. Comparative genomic analysis of Drosophila melanogaster and vector mosquito developmental genes.

    Directory of Open Access Journals (Sweden)

    Susanta K Behura

    Full Text Available Genome sequencing projects have presented the opportunity for analysis of developmental genes in three vector mosquito species: Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae. A comparative genomic analysis of developmental genes in Drosophila melanogaster and these three important vectors of human disease was performed in this investigation. While the study was comprehensive, special emphasis centered on genes that 1 are components of developmental signaling pathways, 2 regulate fundamental developmental processes, 3 are critical for the development of tissues of vector importance, 4 function in developmental processes known to have diverged within insects, and 5 encode microRNAs (miRNAs that regulate developmental transcripts in Drosophila. While most fruit fly developmental genes are conserved in the three vector mosquito species, several genes known to be critical for Drosophila development were not identified in one or more mosquito genomes. In other cases, mosquito lineage-specific gene gains with respect to D. melanogaster were noted. Sequence analyses also revealed that numerous repetitive sequences are a common structural feature of Drosophila and mosquito developmental genes. Finally, analysis of predicted miRNA binding sites in fruit fly and mosquito developmental genes suggests that the repertoire of developmental genes targeted by miRNAs is species-specific. The results of this study provide insight into the evolution of developmental genes and processes in dipterans and other arthropods, serve as a resource for those pursuing analysis of mosquito development, and will promote the design and refinement of functional analysis experiments.

  2. Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

    Science.gov (United States)

    Podsakoff, G; Wong, K K; Chatterjee, S

    1994-09-01

    Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.

  3. Analyzing the Genotoxicity of Retroviral Vectors in Hematopoietic Cell Gene Therapy

    Directory of Open Access Journals (Sweden)

    Luca Biasco

    2018-03-01

    Full Text Available Retroviral vectors, including those derived from gammaretroviruses and lentiviruses, have found their way into the clinical arena and demonstrated remarkable efficacy for the treatment of immunodeficiencies, leukodystrophies, and globinopathies. Despite these successes, gene therapy unfortunately also has had to face severe adverse events in the form of leukemias and myelodysplastic syndromes, related to the semi-random vector integration into the host cell genome that caused deregulation of neighboring proto-oncogenes. Although improvements in vector design clearly lowered the risk of this insertional mutagenesis, analysis of potential genotoxicity and the consequences of vector integration remain important parameters for basic and translational research and most importantly for the clinic. Here, we review current assays to analyze biodistribution and genotoxicity in the pre-clinical setting and describe tools to monitor vector integration sites in vector-treated patients as a biosafety readout.

  4. Glycoprotein is enough for sindbis virus-derived DNA vector to express heterogenous genes

    Directory of Open Access Journals (Sweden)

    Fu Juanjuan

    2011-07-01

    Full Text Available Abstract To investigate the necessity and potential application of structural genes for expressing heterogenous genes from Sindbis virus-derived vector, the DNA-based expression vector pVaXJ was constructed by placing the recombinant genome of sindbis-like virus XJ-160 under the control of the human cytomegalovirus (CMV promoter of the plasmid pVAX1, in which viral structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes. The defect helper plasmids pVaE or pVaC were developed by cloning the gene of glycoprotein E3E26KE1 or capsid protein of XJ-160 virus into pVAX1, respectively. The report gene cassette pVaXJ-EGFP or pV-Gluc expressing enhanced green fluorescence protein (EGFP or Gaussia luciferase (G.luc were constructed by cloning EGFP or G.luc gene into pVaXJ. EGFP or G.luc was expressed in the BHK-21 cells co-transfected with report gene cassettes and pVaE at levels that were comparable to those produced by report gene cassettes, pVaC and pVaE and were much higher than the levels produced by report gene cassette and pVaC, suggesting that glycoprotein is enough for Sindbis virus-derived DNA vector to express heterogenous genes in host cells. The method of gene expression from Sindbis virus-based DNA vector only co-transfected with envelop E gene increase the conveniency and the utility of alphavirus-based vector systems in general.

  5. Adeno-associated viral vectors as agents for gene delivery : application in disorders and trauma of the central nervous system

    NARCIS (Netherlands)

    Ruitenberg, Marc J; Eggers, Ruben; Boer, Gerard J; Verhaagen, J.

    2002-01-01

    The use of viral vectors as agents for gene delivery provides a direct approach to manipulate gene expression in the mammalian central nervous system (CNS). The present article describes in detail the methodology for the injection of viral vectors, in particular adeno-associated virus (AAV) vectors,

  6. Genetic stability of gene targeted immunoglobulin loci. I. Heavy chain isotype exchange induced by a universal gene replacement vector.

    Science.gov (United States)

    Kardinal, C; Selmayr, M; Mocikat, R

    1996-11-01

    Gene targeting at the immunoglobulin loci of B cells is an efficient tool for studying immunoglobulin expression or generating chimeric antibodies. We have shown that vector integration induced by human immunoglobulin G1 (IgG1) insertion vectors results in subsequent vector excision mediated by the duplicated target sequence, whereas replacement events which could be induced by the same constructs remain stable. We could demonstrate that the distribution of the vector homology strongly influences the genetic stability obtained. To this end we developed a novel type of a heavy chain replacement vector making use of the heavy chain class switch recombination sequence. Despite the presence of a two-sided homology this construct is universally applicable irrespective of the constant gene region utilized by the B cell. In comparison to an integration vector the frequency of stable incorporation was strongly increased, but we still observed vector excision, although at a markedly reduced rate. The latter events even occurred with circular constructs. Linearization of the construct at various sites and the comparison with an integration vector that carries the identical homology sequence, but differs in the distribution of homology, revealed the following features of homologous recombination of immunoglobulin genes: (i) the integration frequency is only determined by the length of the homology flank where the cross-over takes place; (ii) a 5' flank that does not meet the minimum requirement of homology length cannot be complemented by a sufficient 3' flank; (iii) free vector ends play a role for integration as well as for replacement targeting; (iv) truncating recombination events are suppressed in the presence of two flanks. Furthermore, we show that the switch region that was used as 3' flank is non-functional in an inverted orientation.

  7. Development of a gene silencing DNA vector derived from a broad host range geminivirus

    Directory of Open Access Journals (Sweden)

    Hancock Leandria C

    2009-07-01

    Full Text Available Abstract Background Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems. Results The construction of a gene silencing vector derived from the geminivirus Beet curly top virus (BCTV, named pWSRi, is reported. Two versions of the vector have been developed to allow application by biolistic techniques or by agro-infiltration. We demonstrate its ability to silence nuclear genes including ribulose bisphosphate carboxylase small subunit (rbcS, transketolase, the sulfur allele of magnesium chelatase (ChlI, and two homeotic transcription factors in spinach or tomato by generating gene-specific knock-down phenotypes. Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application. The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation. However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant. Conclusion The unique properties of the pWSRi vector, the ability to silence genes in meristem tissue, the separation of virus and silencing phenotypes, and the broad natural host range of BCTV, suggest that it will have wide utility.

  8. Gene Therapy with Helper-Dependent Adenoviral Vectors: Current Advances and Future Perspectives

    Directory of Open Access Journals (Sweden)

    Philip Ng

    2010-09-01

    Full Text Available Recombinant Adenoviral vectors represent one of the best gene transfer platforms due to their ability to efficiently transduce a wide range of quiescent and proliferating cell types from various tissues and species. The activation of an adaptive immune response against the transduced cells is one of the major drawbacks of first generation Adenovirus vectors and has been overcome by the latest generation of recombinant Adenovirus, the Helper-Dependent Adenoviral (HDAd vectors. HDAds have innovative features including the complete absence of viral coding sequences and the ability to mediate high level transgene expression with negligible chronic toxicity. This review summarizes the many aspects of HDAd biology and structure with a major focus on in vivo gene therapy application and with an emphasis on the unsolved issues that these vectors still presents toward clinical application.

  9. Oxidative Stress in Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Varsha Shukla

    2011-01-01

    Full Text Available It has been demonstrated that oxidative stress has a ubiquitous role in neurodegenerative diseases. Major source of oxidative stress due to reactive oxygen species (ROS is related to mitochondria as an endogenous source. Although there is ample evidence from tissues of patients with neurodegenerative disorders of morphological, biochemical, and molecular abnormalities in mitochondria, it is still not very clear whether the oxidative stress itself contributes to the onset of neurodegeneration or it is part of the neurodegenerative process as secondary manifestation. This paper begins with an overview of how oxidative stress occurs, discussing various oxidants and antioxidants, and role of oxidative stress in diseases in general. It highlights the role of oxidative stress in neurodegenerative diseases like Alzheimer's, Parkinson's, and Huntington's diseases and amyotrophic lateral sclerosis. The last part of the paper describes the role of oxidative stress causing deregulation of cyclin-dependent kinase 5 (Cdk5 hyperactivity associated with neurodegeneration.

  10. Development of new USER-based cloning vectors for multiple genes expression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kildegaard, Kanchana Rueksomtawin; Jensen, Niels Bjerg; Maury, Jerome

    2013-01-01

    auxotrophic and dominant markers for convenience of use. Our vector set also contains both integrating and multicopy vectors for stability of protein expression and high expression level. We will make the new vector system available to the yeast community and provide a comprehensive protocol for cloning...... the production strain with the proper phenotype and product yield. However, the sequential number of metabolic engineering is time-consuming. Furthermore, the number of available selectable markers is also limiting the number of genetic modifications. To overcome these limitations, we have developed a new set...... of shuttle vectors for convenience of use for high-throughput cloning and selectable marker recycling. The new USER-based cloning vectors consist of a unique USER site and a CRE-loxP-mediated marker recycling system. The USER site allows insertion of genes of interest along with a bidirectional promoter...

  11. Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

    Science.gov (United States)

    Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

    1990-01-01

    Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.

  12. A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize.

    Science.gov (United States)

    Mei, Yu; Zhang, Chunquan; Kernodle, Bliss M; Hill, John H; Whitham, Steven A

    2016-06-01

    Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. © 2016 American Society of Plant Biologists. All Rights Reserved.

  13. A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize1[OPEN

    Science.gov (United States)

    Mei, Yu; Kernodle, Bliss M.; Hill, John H.

    2016-01-01

    Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. PMID:27208311

  14. Reporter gene expression in fish following cutaneous infection with pantropic retroviral vectors.

    Science.gov (United States)

    Paul, T A; Burns, J C; Shike, H; Getchell, R; Bowser, P R; Whitlock, K E; Casey, J W

    2001-06-01

    A central issue in gene delivery systems is choosing promoters that will direct defined and sustainable levels of gene expression. Pantropic retroviral vectors provide a means to insert genes into either somatic or germline cells. In this study, we focused on somatic cell infection by evaluating the activity of 3 promoters inserted by vectors into fish cell lines and fish skin using pantropic retroviruses. In bluegill and zebrafish cell lines, the highest levels of luciferase expression were observed from the 5' murine leukemia virus long terminal repeat of the retroviral vector. The Rous sarcoma virus long terminal repeat and cytomegalovirus early promoter, as internal promoters, generated lower levels of luciferase. Luciferase reporter vectors infected zebrafish skin, as measured by the presence of viral DNA, and expressed luciferase. We infected developing walleye dermal sarcomas with retroviral vectors to provide an environment with enhanced cell proliferation, a condition necessary for integration of the provirus into the host genome. We demonstrated a 4-fold to 7-fold increase in luciferase gene expression in tumor tissue over infections in normal walleye skin.

  15. Adenovirus-derived vectors for prostate cancer gene therapy

    Czech Academy of Sciences Publication Activity Database

    de Vrij, J.; Willemsen, R. A.; Lindholm, L.; Hoeben, R. C.; Bangma, Ch. H.; Barber, Ch.; Behr, J.-P.; Briggs, S.; Carlisle, R.; Cheng, W.-S.; Dautzenberg, I. J. C.; de Ridder, C.; Dzojic, H.; Erbacher, P.; Essand, M.; Fisher, K.; Frazier, A.; Georgopoulos, L. J.; Jennings, I.; Kochanek, S.; Koppers-Lalic, D.; Kraaij, R.; Kreppel, F.; Magnusson, M.; Maitland, N.; Neuberg, P.; Nugent, R.; Ogris, M.; Remy, J.-S.; Scaife, M.; Schenk, E.; Schooten, E.; Seymour, L.; Slade, M.; Szyjanowicz, P.; Totterman, T.; Uil, T. G.; Ulbrich, Karel; van der Weel, L.; van Weerden, W.; Wagner, E.; Zuber, G.

    2010-01-01

    Roč. 21, č. 7 (2010), s. 795-805 ISSN 1043-0342 EU Projects: European Commission(XE) 512087 - GIANT Keywords : adenovirus * gene delivery * prostate cancer Subject RIV: CD - Macromolecular Chemistry Impact factor: 4.829, year: 2010

  16. Improved osteogenic vector for non-viral gene therapy

    Directory of Open Access Journals (Sweden)

    ARA Hacobian

    2016-03-01

    Full Text Available Therapeutic compensation of deficient bone regeneration is a challenging task and a topic of on-going search for novel treatment strategies. One promising approach for improvement involves non-viral gene delivery using the bone morphogenetic protein-2 (BMP-2 gene to provide transient, local and sustained expression of the growth factor. However, since efficiency of non-viral gene delivery is low, this study focused on the improvement of a BMP-2 gene expression system, aiming for compensation of poor transfection efficiency. First, the native BMP-2 gene sequence was modified by codon optimisation and altered by inserting a highly truncated artificial intron (96 bp. Transfection of multiple cell lines and rat adipose-derived mesenchymal stem cells with plasmids harbouring the improved BMP-2 sequence led to a several fold increased expression rate and subsequent osteogenic differentiation. Additionally, comparing expression kinetics of elongation factor 1 alpha (EF1α promoter with a state of the art CMV promoter revealed significantly higher BMP-2 expression when under the influence of the EF1α promoter. Results obtained by quantification of bone markers as well as osteogenic assays showed reduced sensitivity to promoter silencing effects of the EF1α promoter in rat adipose-derived mesenchymal stem cells. Finally, screening of several protein secretion signals using either luciferase or BMP-2 as reporter protein revealed no superior candidates for potential replacement of the native BMP-2 secretion signal. Taken together, by enhancing the exogenous BMP-2 expression system, low transfection efficiencies in therapeutic applications can be compensated, making safe non-viral systems even more suitable for tissue regeneration approaches.

  17. Gene Therapy Vectors with Enhanced Transfection Based on Hydrogels Modified with Affinity Peptides

    Science.gov (United States)

    Shepard, Jaclyn A.; Wesson, Paul J.; Wang, Christine E.; Stevans, Alyson C.; Holland, Samantha J.; Shikanov, Ariella; Grzybowski, Bartosz A.; Shea, Lonnie D.

    2011-01-01

    Regenerative strategies for damaged tissue aim to present biochemical cues that recruit and direct progenitor cell migration and differentiation. Hydrogels capable of localized gene delivery are being developed to provide a support for tissue growth, and as a versatile method to induce the expression of inductive proteins; however, the duration, level, and localization of expression isoften insufficient for regeneration. We thus investigated the modification of hydrogels with affinity peptides to enhance vector retention and increase transfection within the matrix. PEG hydrogels were modified with lysine-based repeats (K4, K8), which retained approximately 25% more vector than control peptides. Transfection increased 5- to 15-fold with K8 and K4 respectively, over the RDG control peptide. K8- and K4-modified hydrogels bound similar quantities of vector, yet the vector dissociation rate was reduced for K8, suggesting excessive binding that limited transfection. These hydrogels were subsequently applied to an in vitro co-culture model to induce NGF expression and promote neurite outgrowth. K4-modified hydrogels promoted maximal neurite outgrowth, likely due to retention of both the vector and the NGF. Thus, hydrogels modified with affinity peptides enhanced vector retention and increased gene delivery, and these hydrogels may provide a versatile scaffold for numerous regenerative medicine applications. PMID:21514659

  18. Neuroinflammation Induces Neurodegeneration.

    Science.gov (United States)

    Kempuraj, D; Thangavel, R; Natteru, P A; Selvakumar, G P; Saeed, D; Zahoor, H; Zaheer, S; Iyer, S S; Zaheer, A

    2016-01-01

    Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Multiple Sclerosis (MS) are characterized by neuronal degeneration and neuronal death in specific regions of the central nervous system (CNS). In AD, neurons of the hippocampus and entorhinal cortex are the first to degenerate, whereas in PD, dopaminergic neurons in the substantia nigra degenerate. MS patients show destruction of the myelin sheath. Once the CNS neurons are damaged, they are unable to regenerate unlike any other tissue in the body. Neurodegeneration is mediated by inflammatory and neurotoxic mediators such as interleukin-1beta (IL-1β), IL-6, IL-8, IL-33, tumor necrosis factor-alpha (TNF-α), chemokine (C-C motif) ligand 2 (CCL2), CCL5, matrix metalloproteinase (MMPs), granulocyte macrophage colony-stimulating factor (GM-CSF), glia maturation factor (GMF), substance P, reactive oxygen species (ROS), reactive nitrogen species (RNS), mast cells-mediated histamine and proteases, protease activated receptor-2 (PAR-2), CD40, CD40L, CD88, intracellular Ca + elevation, and activation of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa-B (NF-kB). Activated microglia, astrocytes, neurons, T-cells and mast cells release these inflammatory mediators and mediate neuroinflammation and neurodegeneration in a vicious manner. Further, immune and inflammatory cells and inflammatory mediators from the periphery cross the defective blood-brain-barrier (BBB) and augment neuroinflammation. Though inflammation is crucial in the onset and the progression of neurodegenerative diseases, anti-inflammatory drugs do not provide significant therapeutic effects in these patients till date, as the disease pathogenesis is not yet clearly understood. In this review, we discuss the possible factors involved in neuroinflammation-mediated neurodegeneration.

  19. Targeted Adenoviral Vector Demonstrates Enhanced Efficacy for In Vivo Gene Therapy of Uterine Leiomyoma.

    Science.gov (United States)

    Abdelaziz, Mohamed; Sherif, Lotfy; ElKhiary, Mostafa; Nair, Sanjeeta; Shalaby, Shahinaz; Mohamed, Sara; Eziba, Noura; El-Lakany, Mohamed; Curiel, David; Ismail, Nahed; Diamond, Michael P; Al-Hendy, Ayman

    2016-04-01

    Gene therapy is a potentially effective non-surgical approach for the treatment of uterine leiomyoma. We demonstrated that targeted adenovirus vector, Ad-SSTR-RGD-TK/GCV, was highly effective in selectively inducing apoptosis and inhibiting proliferation of human leiomyoma cells in vitro while sparing normal myometrial cells. An in-vivo study, to compare efficacy and safety of modified adenovirus vector Ad-SSTR-RGD-TK/GCV versus untargeted vector for treatment of leiomyoma. Female nude mice were implanted with rat leiomyoma cells subcutaneously. Then mice were randomized into three groups. Group 1 received Ad-LacZ (marker gene), Group 2 received untargeted Ad-TK, and Group 3 received the targeted Ad-SSTR-RGD-TK. Tumors were measured weekly for 4 weeks. Then mice were sacrificed and tissue samples were collected. Evaluation of markers of apoptosis, proliferation, extracellular matrix, and angiogenesis was performed using Western Blot & Immunohistochemistry. Statistical analysis was done using ANOVA. Dissemination of adenovirus was assessed by PCR. In comparison with the untargeted vector, the targeted adenoviral vector significantly shrank leiomyoma size (P leiomyoma lesions with both targeted and untargeted adenovirus. Targeted adenovirus, effectively reduces tumor size in leiomyoma without dissemination to other organs. Further evaluation of this localized targeted strategy for gene therapy is needed in appropriate preclinical humanoid animal models in preparation for a future pilot human trial. © The Author(s) 2016.

  20. Gene therapy in the inner ear using adenovirus vectors.

    Science.gov (United States)

    Husseman, Jacob; Raphael, Yehoash

    2009-01-01

    Therapies for the protection and regeneration of auditory hair cells are of great interest given the significant monetary and lifestyle impact of hearing loss. The past decade has seen tremendous advances in the use of adenoviral vectors to achieve these aims. Preliminary data demonstrated the functional capacity of this technique as adenoviral-induced expression of neurotrophic and growth factors protected hair cells and spiral ganglion neurons from ototoxic insults. Subsequent efforts confirmed the feasibility of adenoviral transfection of cells in the auditory neuroepithelium via cochleostomy into the scala media. Most recently, efforts have focused on regeneration of depleted hair cells. Mammalian hearing loss is generally considered a permanent insult as the auditory epithelium lacks a basal layer capable of producing new hair cells. Recently, the transcription factor Atoh1 has been found to play a critical role in hair cell differentiation. Adenoviral-mediated overexpression of Atoh1 in culture and in vivo have shown the ability to regenerate auditory and vestibular hair cells by causing transdifferentiation of neighboring epithelial-supporting cells. Functional recovery of both the auditory and vestibular systems has been documented following adenoviral induced Atoh1 overexpression. Copyright (c) 2009 S. Karger AG, Basel.

  1. Generation of a lentiviral vector producer cell clone for human Wiskott-Aldrich syndrome gene therapy

    Directory of Open Access Journals (Sweden)

    Matthew M Wielgosz

    Full Text Available We have developed a producer cell line that generates lentiviral vector particles of high titer. The vector encodes the Wiskott-Aldrich syndrome (WAS protein. An insulator element has been added to the long terminal repeats of the integrated vector to limit proto-oncogene activation. The vector provides high-level, stable expression of WAS protein in transduced murine and human hematopoietic cells. We have also developed a monoclonal antibody specific for intracellular WAS protein. This antibody has been used to monitor expression in blood and bone marrow cells after transfer into lineage negative bone marrow cells from WAS mice and in a WAS negative human B-cell line. Persistent expression of the transgene has been observed in transduced murine cells 12–20 weeks following transplantation. The producer cell line and the specific monoclonal antibody will facilitate the development of a clinical protocol for gene transfer into WAS protein deficient stem cells.

  2. Recombinant human parvovirus B19 vectors: erythroid cell-specific delivery and expression of transduced genes.

    Science.gov (United States)

    Ponnazhagan, S; Weigel, K A; Raikwar, S P; Mukherjee, P; Yoder, M C; Srivastava, A

    1998-06-01

    A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562-566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111-1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and

  3. Recombinant Human Parvovirus B19 Vectors: Erythroid Cell-Specific Delivery and Expression of Transduced Genes

    Science.gov (United States)

    Ponnazhagan, Selvarangan; Weigel, Kirsten A.; Raikwar, Sudhanshu P.; Mukherjee, Pinku; Yoder, Mervin C.; Srivastava, Arun

    1998-01-01

    A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562–566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111–1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited

  4. Non-Primate Lentiviral Vectors and Their Applications in Gene Therapy for Ocular Disorders

    Directory of Open Access Journals (Sweden)

    Vincenzo Cavalieri

    2018-06-01

    Full Text Available Lentiviruses have a number of molecular features in common, starting with the ability to integrate their genetic material into the genome of non-dividing infected cells. A peculiar property of non-primate lentiviruses consists in their incapability to infect and induce diseases in humans, thus providing the main rationale for deriving biologically safe lentiviral vectors for gene therapy applications. In this review, we first give an overview of non-primate lentiviruses, highlighting their common and distinctive molecular characteristics together with key concepts in the molecular biology of lentiviruses. We next examine the bioengineering strategies leading to the conversion of lentiviruses into recombinant lentiviral vectors, discussing their potential clinical applications in ophthalmological research. Finally, we highlight the invaluable role of animal organisms, including the emerging zebrafish model, in ocular gene therapy based on non-primate lentiviral vectors and in ophthalmology research and vision science in general.

  5. Neonatal Gene Therapy for Hemophilia B by a Novel Adenovirus Vector Showing Reduced Leaky Expression of Viral Genes.

    Science.gov (United States)

    Iizuka, Shunsuke; Sakurai, Fuminori; Tachibana, Masashi; Ohashi, Kazuo; Mizuguchi, Hiroyuki

    2017-09-15

    Gene therapy during neonatal and infant stages is a promising approach for hemophilia B, a congenital disorder caused by deficiency of blood coagulation factor IX (FIX). An adenovirus (Ad) vector has high potential for use in neonatal or infant gene therapy for hemophilia B due to its superior transduction properties; however, leaky expression of Ad genes often reduces the transduction efficiencies by Ad protein-mediated tissue damage. Here, we used a novel Ad vector, Ad-E4-122aT, which exhibits a reduction in the leaky expression of Ad genes in liver, in gene therapy studies for neonatal hemophilia B mice. Ad-E4-122aT exhibited significantly higher transduction efficiencies than a conventional Ad vector in neonatal mice. In neonatal hemophilia B mice, a single neonatal injection of Ad-E4-122aT expressing human FIX (hFIX) (Ad-E4-122aT-AHAFIX) maintained more than 6% of the normal plasma hFIX activity levels for approximately 100 days. Sequential administration of Ad-E4-122aT-AHAFIX resulted in more than 100% of the plasma hFIX activity levels for more than 100 days and rescued the bleeding phenotypes of hemophilia B mice. In addition, immunotolerance to hFIX was induced by Ad-E4-122aT-AHAFIX administration in neonatal hemophilia B mice. These results indicated that Ad-E4-122aT is a promising gene delivery vector for neonatal or infant gene therapy for hemophilia B.

  6. Transient gene transfer to neurons and glia : analysis of adenoviral vector performance in the CNS and PNS

    NARCIS (Netherlands)

    Hermens, W.T.J.M.C.; Giger, Roman J; Holtmaat, Anthony J D G; Dijkhuizen, Paul A; Houweling, D A; Verhaagen, J

    In this paper a detailed protocol is presented for neuroscientists planning to start work on first generation recombinant adenoviral vectors as gene transfer agents for the nervous system. The performance of a prototype adenoviral vector encoding the bacterial lacZ gene as a reporter was studied,

  7. Efficient adenoviral vector directed expression of a foreign gene to neurons and sustentacular cells in the mouse olfactory neuroepithelium

    NARCIS (Netherlands)

    Gispen, W.H.; Holtmaat, A.J.G.D.; Hermens, W.T.J.M.C.; Oestreicher, A.B.; Kaplitt, M.G.; Verhaagen, J.

    1996-01-01

    Replication deficient recombinant adenoviral vectors are efficient gene transfer agents for postmitotic cells, including neurons and glial cells. In this paper we have examined the effectiveness of adenoviral vector-mediated gene transfer to the olfactory epithelium of adult mice. We show that

  8. Efficient adenoviral vector-directed expression of a foreign gene to neurons and sustentacular cells in the mouse olfactory neuroepithelium

    NARCIS (Netherlands)

    Holtmaat, Anthony J D G; Hermens, W.T.J.M.C.; Oestreicher, A B; Gispen, Willem Hendrik; Kaplitt, M G; Verhaagen, J

    1996-01-01

    Replication deficient recombinant adenoviral vectors are efficient gene transfer agents for postmitotic cells, including neurons and glial cells. In this paper we have examined the effectiveness of adenoviral vector-mediated gene transfer to the olfactory epithelium of adult mice. We show that

  9. Adenoviral vector-mediated gene transfer and neurotransplantation : possibilities and limitations in grafting of the fetal rat suprachiasmatic nucleus

    NARCIS (Netherlands)

    van Esseveldt, K E; Liu, R.; Hermens, W.T.J.M.C.; Verhaagen, J; Boer, G J

    Several studies have reported on the use of primary neural cells transduced by adenoviral vectors as donor cells in neurotransplantation. In the present investigation, we examined whether adenoviral vector-mediated gene transfer could be used to introduce and express a foreign gene in solid neural

  10. Cloning and Expression Vector Construction of Glutamate Decarboxylase Gene from Lactobacillus Plantarum

    Directory of Open Access Journals (Sweden)

    B Arabpour

    2016-06-01

    Full Text Available BACKGROUND AND OBJECTIVE: Gamma-aminobutyric acid (GABA is a four-carbon non-protein amino acid used in the treatment of hypertension, diabetes, inflammation, and depression. GABA is synthesized by glutamic acid decarboxylase (GAD enzyme in many organisms, including bacteria. Therefore, cloning of this enzyme is essential to the optimization of GABA production. This study aimed to clone and construct the expression vector of GAD gene from Lactobacillus plantarum PTCC 1058 bacterium. METHODS: In this experimental study, we investigated the morphological, biochemical, genetic and 16s rDNA sequencing of L. plantarum PTCC 1058 strain. Genomic DNA of the bacterium was isolated and amplified using the GAD gene via polymerase chain reaction (PCR. Afterwards, the gene was inserted into the pJET1.2/blunt cloning vector and subcloned in vector pET32a. Plasmid pET32a-gad expression vector was transformed in Escherichia coli BL21 strain, and protein expression was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE. FINDINGS: Morphological, biochemical and genetic analyses of 16s rDNA sequencing indicated that the studied substrain was of the L. plantarum strain. In addition, results of nucleotide sequencing of the fragmented segment via PCR showed the presence of GAD gene. Results of colony PCR and SDS-PAGE analysis confirmed the accuracy of the cloning and gene expression of the recombinant Escherichia coli BL21 strain. CONCLUSION: According to the results of this study, cloning of GAD gene from L. plantarum PTCC 1058 was successful. These cloned genes could grow rapidly in prokaryotic and eukaryotic systems and be used in cost-effective culture media and even non-recyclable waste.

  11. Episomal Nonviral Gene Therapy Vectors Slow Progression of Atherosclerosis in a Model of Familial Hypercholesterolemia

    Directory of Open Access Journals (Sweden)

    Alastair G Kerr

    2016-01-01

    Full Text Available Familial hypercholesterolemia (FH is a life-threatening genetic disorder characterized by elevated levels of plasma low-density lipoprotein cholesterol (LDL-cholesterol. Current attempts at gene therapy for FH have been limited by the use of strong heterologous promoters which lack genomic DNA elements essential for regulated expression. Here, we have combined a minigene vector expressing the human LDLR cDNA from a 10 kb native human LDLR locus genomic DNA promoter element, with an efficient miRNA targeting 3-hydroxy-3-methylgutaryl-coenzyme A reductase (Hmgcr, to further enhance LDLR expression. We show that the combined vector suppresses endogenous Hmgcr transcripts in vivo, leading to an increase in LDLR transgene expression. In a diet-induced Ldlr-/- mouse model of FH, we show that administration of the combined vector reduces atherogenic plasma lipids by ≃32%. Finally, we demonstrate that our episomal nonviral vectors are able to reduce atherosclerosis by ≃40% after 12 weeks in vivo. Taken together, the vector system we describe exploits the normal cellular regulation of the LDLR to provide prolonged expression of LDLR through targeted knockdown of Hmgcr. This novel gene therapy system could act alone, or in synergy with current therapies that modulate intracellular cholesterol, such as statins, greatly enhancing its therapeutic application for FH.

  12. Gene therapy for human glioblastoma using neurotropic JC virus-like particles as a gene delivery vector.

    Science.gov (United States)

    Chao, Chun-Nun; Yang, Yu-Hsuan; Wu, Mu-Sheng; Chou, Ming-Chieh; Fang, Chiung-Yao; Lin, Mien-Chun; Tai, Chien-Kuo; Shen, Cheng-Huang; Chen, Pei-Lain; Chang, Deching; Wang, Meilin

    2018-02-02

    Glioblastoma multiforme (GBM), the most common malignant brain tumor, has a short period of survival even with recent multimodality treatment. The neurotropic JC polyomavirus (JCPyV) infects glial cells and oligodendrocytes and causes fatal progressive multifocal leukoencephalopathy in patients with AIDS. In this study, a possible gene therapy strategy for GBM using JCPyV virus-like particles (VLPs) as a gene delivery vector was investigated. We found that JCPyV VLPs were able to deliver the GFP reporter gene into tumor cells (U87-MG) for expression. In an orthotopic xenograft model, nude mice implanted with U87 cells expressing the near-infrared fluorescent protein and then treated by intratumoral injection of JCPyV VLPs carrying the thymidine kinase suicide gene, combined with ganciclovir administration, exhibited significantly prolonged survival and less tumor fluorescence during the experiment compared with controls. Furthermore, JCPyV VLPs were able to protect and deliver a suicide gene to distal subcutaneously implanted U87 cells in nude mice via blood circulation and inhibit tumor growth. These findings show that metastatic brain tumors can be targeted by JCPyV VLPs carrying a therapeutic gene, thus demonstrating the potential of JCPyV VLPs to serve as a gene therapy vector for the far highly treatment-refractory GBM.

  13. Dopaminergic expression of the Parkinsonian gene LRRK2-G2019S leads to non-autonomous visual neurodegeneration, accelerated by increased neural demands for energy

    Science.gov (United States)

    Hindle, Samantha; Afsari, Farinaz; Stark, Meg; Middleton, C. Adam; Evans, Gareth J.O.; Sweeney, Sean T.; Elliott, Christopher J.H.

    2013-01-01

    Parkinson's disease (PD) is associated with loss of dopaminergic signalling, and affects not just movement, but also vision. As both mammalian and fly visual systems contain dopaminergic neurons, we investigated the effect of LRRK2 mutations (the most common cause of inherited PD) on Drosophila electroretinograms (ERGs). We reveal progressive loss of photoreceptor function in flies expressing LRRK2-G2019S in dopaminergic neurons. The photoreceptors showed elevated autophagy, apoptosis and mitochondrial disorganization. Head sections confirmed extensive neurodegeneration throughout the visual system, including regions not directly innervated by dopaminergic neurons. Other PD-related mutations did not affect photoreceptor function, and no loss of vision was seen with kinase-dead transgenics. Manipulations of the level of Drosophila dLRRK suggest G2019S is acting as a gain-of-function, rather than dominant negative mutation. Increasing activity of the visual system, or of just the dopaminergic neurons, accelerated the G2019S-induced deterioration of vision. The fly visual system provides an excellent, tractable model of a non-autonomous deficit reminiscent of that seen in PD, and suggests that increased energy demand may contribute to the mechanism by which LRRK2-G2019S causes neurodegeneration. PMID:23396536

  14. Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi

    DEFF Research Database (Denmark)

    Frandsen, Rasmus John Normand; Andersson, Jens A.; Kristensen, Matilde Bylov

    2008-01-01

    Background: The rapid increase in whole genome fungal sequence information allows large scale functional analyses of target genes. Efficient transformation methods to obtain site-directed gene replacement, targeted over-expression by promoter replacement, in-frame epitope tagging or fusion...... of coding sequences with fluorescent markers such as GFP are essential for this process. Construction of vectors for these experiments depends on the directional cloning of two homologous recombination sequences on each side of a selection marker gene. Results: Here, we present a USER Friendly cloning based...

  15. Elimination of contaminating cap genes in AAV vector virions reduces immune responses and improves transgene expression in a canine gene therapy model.

    Science.gov (United States)

    Wang, Z; Halbert, C L; Lee, D; Butts, T; Tapscott, S J; Storb, R; Miller, A D

    2014-04-01

    Animal and human gene therapy studies utilizing AAV vectors have shown that immune responses to AAV capsid proteins can severely limit transgene expression. The main source of capsid antigen is that associated with the AAV vectors, which can be reduced by stringent vector purification. A second source of AAV capsid proteins is that expressed from cap genes aberrantly packaged into AAV virions during vector production. This antigen source can be eliminated by the use of a cap gene that is too large to be incorporated into an AAV capsid, such as a cap gene containing a large intron (captron gene). Here, we investigated the effects of elimination of cap gene transfer and of vector purification by CsCl gradient centrifugation on AAV vector immunogenicity and expression following intramuscular injection in dogs. We found that both approaches reduced vector immunogenicity and that combining the two produced the lowest immune responses and highest transgene expression. This combined approach enabled the use of a relatively mild immunosuppressive regimen to promote robust micro-dystrophin gene expression in Duchenne muscular dystrophy-affected dogs. Our study shows the importance of minimizing AAV cap gene impurities and indicates that this improvement in AAV vector production may benefit human applications.

  16. Multicomponent nanoparticles as nonviral vectors for the treatment of Fabry disease by gene therapy

    Directory of Open Access Journals (Sweden)

    Ruiz de Garibay AP

    2012-10-01

    Full Text Available Aritz Pérez Ruiz de Garibay, Diego Delgado, Ana del Pozo-Rodríguez, María Ángeles Solinís, Alicia Rodríguez GascónPharmacokinetics, Nanotechnology and Gene Therapy Group, Pharmacy Faculty, University of the Basque Country UPV/EHU, Vitoria-Gasteiz, SpainPurpose: Gene-mediated enzyme replacement is a reasonable and highly promising approach for the treatment of Fabry disease (FD. The objective of the present study was to demonstrate the potential applications of solid lipid nanoparticle (SLN-based nonviral vectors for the treatment of FD.Methods: SLNs containing the pR-M10-αGal A plasmid that encodes the α-Galactosidase A (α-Gal A enzyme were prepared and their in vitro transfection efficacy was studied in Hep G2 cells. We also studied the cellular uptake of the vectors and the intracellular disposition of the plasmid.Results: The enzymatic activity of the cells treated with the vectors increased significantly relative to the untreated cells, regardless of the formulation assayed. When the SLNs were prepared with protamine or dextran and protamine, the activity of the α-Gal A enzyme by the transfected Hep G2 cells increased up to 12-fold compared to that of untreated cells.Conclusion: With this work we have revealed in Hep G2 cells the ability of a multicomponent system based on SLNs to act as efficient nonviral vectors to potentially correct low α-Gal A activity levels in FD with gene therapy.Keywords: solid lipid nanoparticles, Fabry disease, nonviral vectors, gene therapy

  17. Peptides Against Autoimmune Neurodegeneration.

    Science.gov (United States)

    Stepanov, Alexey; Lomakin, Yakov; Gabibov, Alexander; Belogurov, Alexey

    2017-01-01

    The mammalian immune system is a nearly perfect defensive system polished by a hundred million years of evolution. Unique flexibility and adaptivity have created a virtually impenetrable barrier to numerous exogenous pathogens that are assaulting us every moment. Unfortunately, triggers that remain mostly enigmatic will sometimes persuade the immune system to retarget against self-antigens. This civil war remains underway, showing no mercy and taking no captives, eventually leading to irreversible pathological changes in the human body. Research that has emerged during the last two decades has given us hope that we may have a chance to overcome autoimmune diseases using a variety of techniques to "reset" the immune system. In this report, we summarize recent advances in utilizing short polypeptides - mostly fragments of autoantigens - in the treatment of autoimmune neurodegeneration. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  18. Gateway binary vectors with the bialaphos resistance gene, bar, as a selection marker for plant transformation.

    Science.gov (United States)

    Nakamura, Shinya; Mano, Shoji; Tanaka, Yuji; Ohnishi, Masato; Nakamori, Chihiro; Araki, Masami; Niwa, Tomoko; Nishimura, Mikio; Kaminaka, Hironori; Nakagawa, Tsuyoshi; Sato, Yutaka; Ishiguro, Sumie

    2010-01-01

    We constructed two series of Gateway binary vectors, pGWBs and R4pGWBs, possessing the bialaphos resistance gene (bar) as a selection marker for plant transformation. The reporters and tags employed in this system are sGFP, GUS, LUC, EYFP, ECFP, G3GFP, mRFP, TagRFP, 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7 and TAP. Selection of Arabidopsis transformants with BASTA was successfully carried out using both plate-grown and soil-grown seedlings. Transformed rice calli and suspension-cultured tobacco cells were selected on plates containing BASTA or glufosinate-ammonium. These vectors are compatible with existing pGWB and R4pGWB vectors carrying kanamycin and hygromycin B resistance.

  19. Safe and Effective Gene Therapy for Murine Wiskott-Aldrich Syndrome Using an Insulated Lentiviral Vector

    Directory of Open Access Journals (Sweden)

    Swati Singh

    2017-03-01

    Full Text Available Wiskott-Aldrich syndrome (WAS is a life-threatening immunodeficiency caused by mutations within the WAS gene. Viral gene therapy to restore WAS protein (WASp expression in hematopoietic cells of patients with WAS has the potential to improve outcomes relative to the current standard of care, allogeneic bone marrow transplantation. However, the development of viral vectors that are both safe and effective has been problematic. While use of viral transcriptional promoters may increase the risk of insertional mutagenesis, cellular promoters may not achieve WASp expression levels necessary for optimal therapeutic effect. Here we evaluate a self-inactivating (SIN lentiviral vector combining a chromatin insulator upstream of a viral MND (MPSV LTR, NCR deleted, dl587 PBS promoter driving WASp expression. Used as a gene therapeutic in Was−/− mice, this vector resulted in stable WASp+ cells in all hematopoietic lineages and rescue of T and B cell defects with a low number of viral integrations per cell, without evidence of insertional mutagenesis in serial bone marrow transplants. In a gene transfer experiment in non-human primates, the insulated MND promoter (driving GFP expression demonstrated long-term polyclonal engraftment of GFP+ cells. These observations demonstrate that the insulated MND promoter safely and efficiently reconstitutes clinically effective WASp expression and should be considered for future WAS therapy.

  20. Efficient Gene Delivery to Pig Airway Epithelia and Submucosal Glands Using Helper-Dependent Adenoviral Vectors

    Directory of Open Access Journals (Sweden)

    Huibi Cao

    2013-01-01

    Full Text Available Airway gene delivery is a promising strategy to treat patients with life-threatening lung diseases such as cystic fibrosis (CF. However, this strategy has to be evaluated in large animal preclinical studies in order to translate it to human applications. Because of anatomic and physiological similarities between the human and pig lungs, we utilized pig as a large animal model to examine the safety and efficiency of airway gene delivery with helper-dependent adenoviral vectors. Helper-dependent vectors carrying human CFTR or reporter gene LacZ were aerosolized intratracheally into pigs under bronchoscopic guidance. We found that the LacZ reporter and hCFTR transgene products were efficiently expressed in lung airway epithelial cells. The transgene vectors with this delivery can also reach to submucosal glands. Moreover, the hCFTR transgene protein localized to the apical membrane of both ciliated and nonciliated epithelial cells, mirroring the location of wild-type CF transmembrane conductance regulator (CFTR. Aerosol delivery procedure was well tolerated by pigs without showing systemic toxicity based on the limited number of pigs tested. These results provide important insights into developing clinical strategies for human CF lung gene therapy.

  1. Targeting of breast metastases using a viral gene vector with tumour-selective transcription.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2012-01-31

    BACKGROUND: Adeno-associated virus (AAV) vectors have significant potential as gene delivery vectors for cancer gene therapy. However, broad AAV2 tissue tropism results in nonspecific gene expression. MATERIALS AND METHODS: We investigated use of the C-X-C chemokine receptor type 4 (CXCR4) promoter to restrict AAV expression to tumour cells, in subcutaneous MCF-7 xenograft mouse models of breast cancer and in patient samples, using bioluminescent imaging and flow cytometric analysis. RESULTS: Higher transgene expression levels were observed in subcutaneous MCF-7 tumours relative to normal tissue (muscle) using the CXCR4 promoter, unlike a ubiquitously expressing Cytomegalovirus promoter construct, with preferential AAVCXCR4 expression in epithelial tumour and CXCR4-positive cells. Transgene expression following intravenously administered AAVCXCR4 in a model of liver metastasis was detected specifically in livers of tumour bearing mice. Ex vivo analysis using patient samples also demonstrated higher AAVCXCR4 expression in tumour compared with normal liver tissue. CONCLUSION: This study demonstrates for the first time, the potential for systemic administration of AAV2 vector for tumour-selective gene therapy.

  2. Effects of Vector Backbone and Pseudotype on Lentiviral Vector-mediated Gene Transfer: Studies in Infant ADA-Deficient Mice and Rhesus Monkeys

    Science.gov (United States)

    Carbonaro Sarracino, Denise; Tarantal, Alice F; Lee, C Chang I.; Martinez, Michele; Jin, Xiangyang; Wang, Xiaoyan; Hardee, Cinnamon L; Geiger, Sabine; Kahl, Christoph A; Kohn, Donald B

    2014-01-01

    Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options. PMID:24925206

  3. Effects of vector backbone and pseudotype on lentiviral vector-mediated gene transfer: studies in infant ADA-deficient mice and rhesus monkeys.

    Science.gov (United States)

    Carbonaro Sarracino, Denise; Tarantal, Alice F; Lee, C Chang I; Martinez, Michele; Jin, Xiangyang; Wang, Xiaoyan; Hardee, Cinnamon L; Geiger, Sabine; Kahl, Christoph A; Kohn, Donald B

    2014-10-01

    Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options.

  4. 'Molecular switch' vectors for hypoxia- and radiation-mediated gene therapy of cancer

    International Nuclear Information System (INIS)

    Greco, O.; Marples, B.; Joiner, M.C.; Scott, S.D.

    2003-01-01

    Intratumoral areas of low oxygen concentration are known to be refractive to radiotherapy treatment. However, this physiological condition can be exploited for selective cancer gene therapy. We have developed a series of synthetic promoters selectively responsive to both hypoxia and ionizing radiation (IR). These promoters contain hypoxia regulatory elements (HREs) from the erythropoietin (Epo), the phosphoglycerate kinase1(PGK1) and vascular endothelial growth factor (VEGF) genes, and/or IR-responsive CArG elements from the Early Growth Response 1 (Egr1) gene. The HRE and CArG promoters were able to regulate expression of reporter and suicide genes in human tumor cells, following corresponding stimulation with hypoxia (0.1% O2) or X-irradiation (5Gy) [Greco et al, 2002, Gene Therapy 9:1403]. Furthermore, the chimeric HRE + CArG promoters could be activated by these stimuli independently or even more significantly when given in combination, with the Epo HRE/CArG promoter proving to be the most responsive and robust. In order to amplify and maintain transgene expression even following withdrawal of the triggering stimuli, we have developed a 'molecular switch' system [Scott et al, 2000, Gene Therapy 7:1121]. This 'switch' system has now been engineered as a single vector molecule, containing HRE and CArG promoters. This new series of HRE/CArG switch vectors have been tested in a herpes simplex thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene assay. Results indicate that a) higher and more selective tumor cell kill is achieved with the switch when compared with the HRE and CArG promoters directly driving HSVtk expression and b) the Epo HRE/CArG switch vectors appear to function as efficiently as the strong constitutive cytomegalovirus (CMV) promoter construct

  5. Self-focusing therapeutic gene delivery with intelligent gene vector swarms: intra-swarm signalling through receptor transgene expression in targeted cells.

    Science.gov (United States)

    Tolmachov, Oleg E

    2015-01-01

    Gene delivery in vivo that is tightly focused on the intended target cells is essential to maximize the benefits of gene therapy and to reduce unwanted side-effects. Cell surface markers are immediately available for probing by therapeutic gene vectors and are often used to direct gene transfer with these vectors to specific target cell populations. However, it is not unusual for the choice of available extra-cellular markers to be too scarce to provide a reliable definition of the desired therapeutically relevant set of target cells. Therefore, interrogation of intra-cellular determinants of cell-specificity, such as tissue-specific transcription factors, can be vital in order to provide detailed cell-guiding information to gene vector particles. An important improvement in cell-specific gene delivery can be achieved through auto-buildup in vector homing efficiency using intelligent 'self-focusing' of swarms of vector particles on target cells. Vector self-focusing was previously suggested to rely on the release of diffusible chemo-attractants after a successful target-specific hit by 'scout' vector particles. I hypothesize that intelligent self-focusing behaviour of swarms of cell-targeted therapeutic gene vectors can be accomplished without the employment of difficult-to-use diffusible chemo-attractants, instead relying on the intra-swarm signalling through cells expressing a non-diffusible extra-cellular receptor for the gene vectors. In the proposed model, cell-guiding information is gathered by the 'scout' gene vector particles, which: (1) attach to a variety of cells via a weakly binding (low affinity) receptor; (2) successfully facilitate gene transfer into these cells; (3) query intra-cellular determinants of cell-specificity with their transgene expression control elements and (4) direct the cell-specific biosynthesis of a vector-encoded strongly binding (high affinity) cell-surface receptor. Free members of the vector swarm loaded with therapeutic cargo

  6. Design and application of cationic amphiphilic β-cyclodextrin derivatives as gene delivery vectors

    Science.gov (United States)

    Wan, Ning; Huan, Meng-Lei; Ma, Xi-Xi; Jing, Zi-Wei; Zhang, Ya-Xuan; Li, Chen; Zhou, Si-Yuan; Zhang, Bang-Le

    2017-11-01

    The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic β-cyclodextrin (β-CD) derivatives were designed and synthesized by using 6-mono-OTs-β-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of β-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed β-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.

  7. [Construction and identification of Nogo extra cellular peptide residues 1-40 gene lentiviral vector].

    Science.gov (United States)

    Yuan, Haifeng; Song, Yueming; Liu, Hao; Zhou, Chunguang; Kong, Qingquan; Liu, Liming; Gong, Quan

    2012-02-01

    To construct a lentiviral expression vector carrying Nogo extra cellular peptide residues 1-40 (NEP1-40) and to obtain NEP1-40 efficient and stable expression in mammalian cells. The DNA fragment of NEP1-40 coding sequence was amplified by PCR with designed primer from the cDNA library including NEP1-40 gene, and then subcloned into pGC-FU vector with in-fusion technique to generate the lentiviral expression vector, pGC-FU-NEP1-40. The positive clones were screened by PCR and the correct NEP1-40 was confirmed by sequencing. Recombinant lentiviruses were produced in 293T cells after the cotransfection of pGC-FU-NEP1-40, and packaging plasmids of pHelper 1.0 and pHelper 2.0. Green fluorescent protein (GFP) expression of infected 293T cells was observed to evaluate gene delivery efficiency. NEP1-40 protein expression in 293T cells was detected by Western blot. The lentiviral expression vector carrying NEP1-40 was successfully constructed by GFP observation, and NEP1-40 protein expression was detected in 293T cells by Western blot. The recombinant lentivirus pGC-FU-NEP1-40 is successfully constructed and it lays a foundation for further molecular function study of NEP 1-40.

  8. Analysis of the role of homology arms in gene-targeting vectors in human cells.

    Directory of Open Access Journals (Sweden)

    Ayako Ishii

    Full Text Available Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ, a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4, the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.

  9. Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.

    Directory of Open Access Journals (Sweden)

    Gideon Hen

    Full Text Available The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV, into the chorioallantoic membrane (CAM of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP or recombinant alpha-melanocyte-stimulating hormone (α-MSH genes, driven by the cytomegalovirus (CMV promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP nick end labeling (TUNEL assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA, and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.

  10. Construction of novel shuttle expression vectors for gene expression in Bacillus subtilis and Bacillus pumilus.

    Science.gov (United States)

    Shao, Huanhuan; Cao, Qinghua; Zhao, Hongyan; Tan, Xuemei; Feng, Hong

    2015-01-01

    A native plasmid (pSU01) was detected by genome sequencing of Bacillus subtilis strain S1-4. Two pSU01-based shuttle expression vectors pSU02-AP and pSU03-AP were constructed enabling stable replication in B. subtilis WB600. These vectors contained the reporter gene aprE, encoding an alkaline protease from Bacillus pumilus BA06. The expression vector pSU03-AP only possessed the minimal replication elements (rep, SSO, DSO) and exhibited more stability on structure, suggesting that the rest of the genes in pSU01 (ORF1, ORF2, mob, hsp) were unessential for the structural stability of plasmid in B. subtilis. In addition, recombinant production of the alkaline protease was achieved more efficiently with pSU03-AP whose copy number was estimated to be more than 100 per chromosome. Furthermore, pSU03-AP could also be used to transform and replicate in B. pumilus BA06 under selective pressure. In conclusion, pSU03-AP is expected to be a useful tool for gene expression in Bacillus subtilis and B. pumilus.

  11. PGMA-Based Cationic Nanoparticles with Polyhydric Iodine Units for Advanced Gene Vectors.

    Science.gov (United States)

    Sun, Yue; Hu, Hao; Yu, Bingran; Xu, Fu-Jian

    2016-11-16

    It is crucial for successful gene delivery to develop safe, effective, and multifunctional polycations. Iodine-based small molecules are widely used as contrast agents for CT imaging. Herein, a series of star-like poly(glycidyl methacrylate) (PGMA)-based cationic vectors (II-PGEA/II) with abundant flanking polyhydric iodine units are prepared for multifunctional gene delivery systems. The proposed II-PGEA/II star vector is composed of one iohexol intermediate (II) core and five ethanolamine (EA) and II-difunctionalized PGMA arms. The amphipathic II-PGEA/II vectors readily self-assemble into well-defined cationic nanoparticles, where massive hydroxyl groups can establish a hydration shell to stabilize the nanoparticles. The II introduction improves cell viabilities of polycations. Moreover, by controlling the suitable amount of introduced II units, the resultant II-PGEA/II nanoparticles can produce fairly good transfection performances in different cell lines. Particularly, the II-PGEA/II nanoparticles induce much better in vitro CT imaging abilities in tumor cells than iohexol (one commonly used commercial CT contrast agent). The present design of amphipathic PGMA-based nanoparticles with CT contrast agents would provide useful information for the development of new multifunctional gene delivery systems.

  12. Polydnaviruses of Parasitic Wasps: Domestication of Viruses To Act as Gene Delivery Vectors

    Directory of Open Access Journals (Sweden)

    Michael R. Strand

    2012-01-01

    Full Text Available Symbiosis is a common phenomenon in which associated organisms can cooperate in ways that increase their ability to survive, reproduce, or utilize hostile environments. Here, we discuss polydnavirus symbionts of parasitic wasps. These viruses are novel in two ways: (1 they have become non-autonomous domesticated entities that cannot replicate outside of wasps; and (2 they function as a delivery vector of genes that ensure successful parasitism of host insects that wasps parasitize. In this review we discuss how these novelties may have arisen, which genes are potentially involved, and what the consequences have been for genome evolution.

  13. Fetal muscle gene transfer is not enhanced by an RGD capsid modification to high-capacity adenoviral vectors.

    Science.gov (United States)

    Bilbao, R; Reay, D P; Hughes, T; Biermann, V; Volpers, C; Goldberg, L; Bergelson, J; Kochanek, S; Clemens, P R

    2003-10-01

    High levels of alpha(v) integrin expression by fetal muscle suggested that vector re-targeting to integrins could enhance adenoviral vector-mediated transduction, thereby increasing safety and efficacy of muscle gene transfer in utero. High-capacity adenoviral (HC-Ad) vectors modified by an Arg-Gly-Asp (RGD) peptide motif in the HI loop of the adenoviral fiber (RGD-HC-Ad) have demonstrated efficient gene transfer through binding to alpha(v) integrins. To test integrin targeting of HC-Ad vectors for fetal muscle gene transfer, we compared unmodified and RGD-modified HC-Ad vectors. In vivo, unmodified HC-Ad vector transduced fetal mouse muscle with four-fold higher efficiency compared to RGD-HC-Ad vector. Confirming that the difference was due to muscle cell autonomous factors and not mechanical barriers, transduction of primary myogenic cells isolated from murine fetal muscle in vitro demonstrated a three-fold better transduction by HC-Ad vector than by RGD-HC-Ad vector. We hypothesized that the high expression level of coxsackievirus and adenovirus receptor (CAR), demonstrated in fetal muscle cells both in vitro and in vivo, was the crucial variable influencing the relative transduction efficiencies of HC-Ad and RGD-HC-Ad vectors. To explore this further, we studied transduction by HC-Ad and RGD-HC-Ad vectors in paired cell lines that expressed alpha(v) integrins and differed only by the presence or absence of CAR expression. The results increase our understanding of factors that will be important for retargeting HC-Ad vectors to enhance gene transfer to fetal muscle.

  14. Interaction between subclinical doses of the Parkinson's disease associated gene, α-synuclein, and the pesticide, rotenone, precipitates motor dysfunction and nigrostriatal neurodegeneration in rats.

    Science.gov (United States)

    Naughton, Carol; O'Toole, Daniel; Kirik, Deniz; Dowd, Eilís

    2017-01-01

    In most patients, Parkinson's disease is thought to emerge after a lifetime of exposure to, and interaction between, various genetic and environmental risk factors. One of the key genetic factors linked to this condition is α-synuclein, and the α-synuclein protein is pathologically associated with idiopathic cases. However, α-synuclein pathology is also present in presymptomatic, clinically "normal" individuals suggesting that environmental factors, such as Parkinson's disease-linked agricultural pesticides, may be required to precipitate Parkinson's disease in these individuals. In this context, the aim of this study was to assess the behavioural and neuropathological impact of exposing rats with a subclinical load of α-synuclein to subclinical doses of the organic pesticide, rotenone. Rats were randomly assigned to two groups for intra-nigral infusion of AAV 2/5- GFP or AAV 2/5 -α-synuclein. Post viral motor function was assessed at 8, 10 and 12 weeks in the Corridor, Stepping and Whisker tests of lateralised motor function. At week 12, animals were performance-matched to receive a subsequent intra-striatal challenge of the organic pesticide rotenone (or its vehicle) to yield four final groups (Control, Rotenone, AAV 2/5 -α-synuclein and Combined). Behavioural testing resumed one week after rotenone surgery and continued for 5 weeks. We found that, when administered alone, neither intra-nigral AAV-α-synuclein nor intra-striatal rotenone caused sufficient nigrostriatal neurodegeneration to induce a significant motor impairment in their own right. However, when these were administered sequentially to the same rats, the interaction between the two Parkinsonian challenges significantly exacerbated nigrostriatal neurodegeneration which precipitated a pronounced impairment in motor function. These results indicate that exposing rats with a subclinical α-synuclein-induced pathology to the pesticide, rotenone, profoundly exacerbates their Parkinsonian

  15. Virus-induced gene silencing in diverse maize lines using the Brome Mosaic virus-based silencing vector

    Science.gov (United States)

    Virus-induced gene silencing (VIGS) is a widely used tool for gene function studies in many plant species, though its use in monocots has been limited. Using a Brome mosaic virus (BMV) vector designed to silence the maize phytoene desaturase gene, a genetically diverse set of maize inbred lines was ...

  16. A compact dual promoter adeno-associated viral vector for efficient delivery of two genes to dorsal root ganglion neurons

    NARCIS (Netherlands)

    Fagoe, N D; Eggers, R; Verhaagen, J; Mason, M R J

    Adeno-associated viral (AAV) vectors based on serotype 5 are an efficient means to target dorsal root ganglia (DRG) to study gene function in the primary sensory neurons of the peripheral nervous system. In this study, we have developed a compact AAV dual promoter vector composed of the

  17. Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection.

    Science.gov (United States)

    Saito, Shinta; Ura, Kiyoe; Kodama, Miho; Adachi, Noritaka

    2015-06-30

    Targeted gene modification by homologous recombination provides a powerful tool for studying gene function in cells and animals. In higher eukaryotes, non-homologous integration of targeting vectors occurs several orders of magnitude more frequently than does targeted integration, making the gene-targeting technology highly inefficient. For this reason, negative-selection strategies have been employed to reduce the number of drug-resistant clones associated with non-homologous vector integration, particularly when artificial nucleases to introduce a DNA break at the target site are unavailable or undesirable. As such, an exon-trap strategy using a promoterless drug-resistance marker gene provides an effective way to counterselect non-homologous integrants. However, constructing exon-trapping targeting vectors has been a time-consuming and complicated process. By virtue of highly efficient att-mediated recombination, we successfully developed a simple and rapid method to construct plasmid-based vectors that allow for exon-trapping gene targeting. These exon-trap vectors were useful in obtaining correctly targeted clones in mouse embryonic stem cells and human HT1080 cells. Most importantly, with the use of a conditionally cytotoxic gene, we further developed a novel strategy for negative selection, thereby enhancing the efficiency of counterselection for non-homologous integration of exon-trap vectors. Our methods will greatly facilitate exon-trapping gene-targeting technologies in mammalian cells, particularly when combined with the novel negative selection strategy.

  18. CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

    Science.gov (United States)

    Ehrke-Schulz, Eric; Schiwon, Maren; Leitner, Theo; Dávid, Stephan; Bergmann, Thorsten; Liu, Jing; Ehrhardt, Anja

    2017-12-07

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

  19. Viral vectors for gene modification of plants as chem/bio sensors.

    Energy Technology Data Exchange (ETDEWEB)

    Manginell, Monica; Harper, Jason C.; Arango, Dulce C.; Brozik, Susan Marie; Dolan, Patricia L.

    2006-11-01

    Chemical or biological sensors that are specific, sensitive, and robust allowing intelligence gathering for verification of nuclear non-proliferation treaty compliance and detouring production of weapons of mass destruction are sorely needed. Although much progress has been made in the area of biosensors, improvements in sensor lifetime, robustness, and device packaging are required before these devices become widely used. Current chemical and biological detection and identification techniques require less-than-covert sample collection followed by transport to a laboratory for analysis. In addition to being expensive and time consuming, results can often be inconclusive due to compromised sample integrity during collection and transport. We report here a demonstration of a plant based sensor technology which utilizes mature and seedling plants as chemical sensors. One can envision genetically modifying native plants at a site of interest that can report the presence of specific toxins or chemicals. In this one year project we used a developed inducible expression system to show the feasibility of plant sensors. The vector was designed as a safe, non-infectious vector which could be used to invade, replicate, and introduce foreign genes into mature host plants that then allow the plant to sense chem/bio agents. The genes introduced through the vector included a reporter gene that encodes for green fluorescent protein (GFP) and a gene that encodes for a mammalian receptor that recognizes a chemical agent. Specifically, GFP was induced by the presence of 17-{beta}-Estradiol (estrogen). Detection of fluorescence indicated the presence of the target chemical agent. Since the sensor is a plant, costly device packaging development or manufacturing of the sensor were not required. Additionally, the biological recognition and reporting elements are maintained in a living, natural environment and therefore do not suffer from lifetime disadvantages typical of most biosensing

  20. Have we found an optimal insertion site in a Newcastle disease virus vector to express a foreign gene for vaccine and gene therapy purposes?

    Science.gov (United States)

    Using reverse genetics technology, many strains of Newcastle disease virus (NDV) have been developed as vectors to express foreign genes for vaccine and gene therapy purposes. The foreign gene is usually inserted into a non-coding region of the NDV genome as an independent transcription unit. Eval...

  1. Study on constructing retroviral vector carrying HSV-tk gene and its antitumor effect in vitro

    International Nuclear Information System (INIS)

    Pan Yujun; Hui Guozhen; Hu Jin

    1997-01-01

    The author reports the construction of retroviral vector PLNTK carrying HsV-tk gene driven by pgk promoter and the successful transferring into cells NBA 2 and SHG 44 respectively as shown by PCR. In vitro study, HSV-tk-expressed-cells prove to be more sensitive to ACV than parent cells. The sensitivity of SHGLNTK and NBALNTK to ACV is 1000 and 500 times that of their parent cells respectively. 3 H-TdR test demonstrated that the DNA replication in gene modified cells is more suppressed than that of parent cells when treated with ACV. Moreover, the ACV sensitivity level of parent cells is enhanced when co-cultured with gene modified cells, which suggests the existence of the bystander effect

  2. Low-Dose Gene Therapy for Murine PKU Using Episomal Naked DNA Vectors Expressing PAH from Its Endogenous Liver Promoter

    Directory of Open Access Journals (Sweden)

    Hiu Man Grisch-Chan

    2017-06-01

    Full Text Available Limited duration of transgene expression, insertional mutagenesis, and size limitations for transgene cassettes pose challenges and risk factors for many gene therapy vectors. Here, we report on physiological expression of liver phenylalanine hydroxylase (PAH by delivery of naked DNA/minicircle (MC-based vectors for correction of homozygous enu2 mice, a model of human phenylketonuria (PKU. Because MC vectors lack a defined size limit, we constructed a MC vector expressing a codon-optimized murine Pah cDNA that includes a truncated intron and is under the transcriptional control of a 3.6-kb native Pah promoter/enhancer sequence. This vector, delivered via hydrodynamic injection, yielded therapeutic liver PAH activity and sustained correction of blood phenylalanine comparable to viral or synthetic liver promoters. Therapeutic efficacy was seen with vector copy numbers of 95% loss of vector genomes and PAH activity in liver, demonstrating that MC vectors had not integrated into the liver genome. In conclusion, MC vectors, which do not have a defined size-limitation, offer a favorable safety profile for hepatic gene therapy due to their non-integration in combination with native promoters.

  3. aeGEPUCI: a database of gene expression in the dengue vector mosquito, Aedes aegypti

    Directory of Open Access Journals (Sweden)

    James Anthony A

    2010-10-01

    Full Text Available Abstract Background Aedes aegypti is the principal vector of dengue and yellow fever viruses. The availability of the sequenced and annotated genome enables genome-wide analyses of gene expression in this mosquito. The large amount of data resulting from these analyses requires efficient cataloguing before it becomes useful as the basis for new insights into gene expression patterns and studies of the underlying molecular mechanisms for generating these patterns. Findings We provide a publicly-accessible database and data-mining tool, aeGEPUCI, that integrates 1 microarray analyses of sex- and stage-specific gene expression in Ae. aegypti, 2 functional gene annotation, 3 genomic sequence data, and 4 computational sequence analysis tools. The database can be used to identify genes expressed in particular stages and patterns of interest, and to analyze putative cis-regulatory elements (CREs that may play a role in coordinating these patterns. The database is accessible from the address http://www.aegep.bio.uci.edu. Conclusions The combination of gene expression, function and sequence data coupled with integrated sequence analysis tools allows for identification of expression patterns and streamlines the development of CRE predictions and experiments to assess how patterns of expression are coordinated at the molecular level.

  4. Lentiviral Vector-Mediated GFP/fluc gene introduction into primary mouse NK cells

    International Nuclear Information System (INIS)

    L, Thi Thanh Hoa; Tae, Seong Ho; Min, Jung Joon

    2007-01-01

    NK cell is a type of lymphocyte that has ability in defense against virus infection and some kinds of cancer diseases. Recently, using genetic engineering, studies about the roles and functions of NK cells have been developing. In this study, we used lentivirus-based vector encoding GFP/Fluc gene to transfer into primary mouse NK cells. This model is a tool in studying characteristics of NK cells. The lentivirus used in this study was a commercial one, named LentiM1.3-Fluc, encoding GFP and Flue reporter genes under the control of the murine cytomegalovirus (MCMV) promoter. LentiM1.3-Fluc was infected into freshly isolated mouse NK cells at 2 20 MOl by incubating or using spin infection. In the spin infection, we gently suspended NK cells in viral fluid, then centrifuged at 2000 rpm, 20 minutes at room temperature and incubated for 1 day. After 1 day, virus was discarded and NK cells were cultured in IL-2 with or without IL-12 supplemented media. Infected NK cells were monitored by using fluorescent microscope for GFP and IVIS machine for Fire-fly luciferase expression. The results showed that using spin infection had much effect on introducing lentiviral vector-mediated reporter gene into NK cells than the way without spin. Also, NK cells which were cultured in IL-2 and IL-12 added media expressed higher fluorescent and luminescent signals than those cultured in only IL-2 supplemented media. When these NK cells were injected subcutaneously in Balb/C mice, the imaging signal was observed transiently. Our study demonstrates that by using a simple method, mouse NK cells can be transfected by lentivirus. And this will be useful in studying biology and therapeutic potential of NK cells. However, we require developing alternative lentiviral vectors with different promoter for in vivo application

  5. Gene discovery for the bark beetle-vectored fungal tree pathogen Grosmannia clavigera

    Directory of Open Access Journals (Sweden)

    Robertson Gordon

    2010-10-01

    Full Text Available Abstract Background Grosmannia clavigera is a bark beetle-vectored fungal pathogen of pines that causes wood discoloration and may kill trees by disrupting nutrient and water transport. Trees respond to attacks from beetles and associated fungi by releasing terpenoid and phenolic defense compounds. It is unclear which genes are important for G. clavigera's ability to overcome antifungal pine terpenoids and phenolics. Results We constructed seven cDNA libraries from eight G. clavigera isolates grown under various culture conditions, and Sanger sequenced the 5' and 3' ends of 25,000 cDNA clones, resulting in 44,288 high quality ESTs. The assembled dataset of unique transcripts (unigenes consists of 6,265 contigs and 2,459 singletons that mapped to 6,467 locations on the G. clavigera reference genome, representing ~70% of the predicted G. clavigera genes. Although only 54% of the unigenes matched characterized proteins at the NCBI database, this dataset extensively covers major metabolic pathways, cellular processes, and genes necessary for response to environmental stimuli and genetic information processing. Furthermore, we identified genes expressed in spores prior to germination, and genes involved in response to treatment with lodgepole pine phloem extract (LPPE. Conclusions We provide a comprehensively annotated EST dataset for G. clavigera that represents a rich resource for gene characterization in this and other ophiostomatoid fungi. Genes expressed in response to LPPE treatment are indicative of fungal oxidative stress response. We identified two clusters of potentially functionally related genes responsive to LPPE treatment. Furthermore, we report a simple method for identifying contig misassemblies in de novo assembled EST collections caused by gene overlap on the genome.

  6. Genetic pathways to Neurodegeneration

    Indian Academy of Sciences (India)

    Renu

    The extensive resource on ataxia has led to the development of a clinico-genetic ... Keywords: Cerebellar ataxias, SCAs, ARCAs, NGS, Gene network, iPSCs, .... Besides, mutations in different regions of the same gene result in different ..... integration with population data can also allow focussed testing/screening in specific.

  7. Selective Inhibition of Histone Deacetylation in Melanoma Increases Targeted Gene Delivery by a Bacteriophage Viral Vector

    Directory of Open Access Journals (Sweden)

    Samuel Campbell

    2018-04-01

    Full Text Available The previously developed adeno-associated virus/phage (AAVP vector, a hybrid between M13 bacteriophage (phage viruses that infect bacteria only and human Adeno-Associated Virus (AAV, is a promising tool in targeted gene therapy against cancer. AAVP can be administered systemically and made tissue specific through the use of ligand-directed targeting. Cancer cells and tumor-associated blood vessels overexpress the αν integrin receptors, which are involved in tumor angiogenesis and tumor invasion. AAVP is targeted to these integrins via a double cyclic RGD4C ligand displayed on the phage capsid. Nevertheless, there remain significant host-defense hurdles to the use of AAVP in targeted gene delivery and subsequently in gene therapy. We previously reported that histone deacetylation in cancer constitutes a barrier to AAVP. Herein, to improve AAVP-mediated gene delivery to cancer cells, we combined the vector with selective adjuvant chemicals that inhibit specific histone deacetylases (HDAC. We examined the effects of the HDAC inhibitor C1A that mainly targets HDAC6 and compared this to sodium butyrate, a pan-HDAC inhibitor with broad spectrum HDAC inhibition. We tested the effects on melanoma, known for HDAC6 up-regulation, and compared this side by side with a normal human kidney HEK293 cell line. Varying concentrations were tested to determine cytotoxic levels as well as effects on AAVP gene delivery. We report that the HDAC inhibitor C1A increased AAVP-mediated transgene expression by up to ~9-fold. These findings indicate that selective HDAC inhibition is a promising adjuvant treatment for increasing the therapeutic value of AAVP.

  8. Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

    Science.gov (United States)

    Lv, Lulu; Zhao, Huiqing

    2014-01-01

    The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes. PMID:24392152

  9. Efficient gene transfer into lymphoma cells using adenoviral vectors combined with lipofection.

    Science.gov (United States)

    Buttgereit, P; Weineck, S; Röpke, G; Märten, A; Brand, K; Heinicke, T; Caselmann, W H; Huhn, D; Schmidt-Wolf, I G

    2000-08-01

    Tumor cells, such as lymphoma cells, are possible targets for gene therapy. In general, gene therapeutic approaches require efficient gene transfer to host cells and sufficient transgene expression. However, lymphoma cells previously have been demonstrated to be resistant to most of the currently available gene transfer methods. The aim of this study was to analyze various methods for transfection of lymphoma cells and to improve the efficiency of gene delivery. In accordance with previously published reports, lymphoma cells were demonstrated to be resistant to lipofection and electroporation. In contrast, we present an improved adenoviral protocol leading to highly efficient gene transfer to lymphoma cell lines derived from B cells as well as primary lymphoma cells being achieved with an adenoviral vector system encoding the beta-galactosidase protein. At a multiplicity of infection of 200, up to 100% of Daudi cells and Raji cells and 70% of OCI-Ly8-LAM53 cells could be transfected. Even at high adenoviral concentrations, no marked toxicity was observed, and the growth characteristics of the lymphoma cell lines were not impaired. The transfection rates in primary cells derived from six patients with non-Hodgkin's lymphoma were 30-65%, respectively. Transfection efficiency could be further increased by addition of cationic liposomes to adenoviral gene transfer. Furthermore, we examined the expression of the Coxsackie-adenoviral receptor (CAR) and the integrin receptors on the lymphoma cell surface. Flow cytometric analysis showed that 88% of Daudi cells, 69% of Raji cells, and 6% of OCI-Ly8-LAM53 cells expressed CAR on the cell surface. According to our data, adenoviral infection of lymphoma cells seems to be mediated by CAR. In contrast, integrin receptors are unlikely to play a major role, because lymphoma cells were negative for alphavbeta3-integrins and negative for alphavbeta5-integrins. In conclusion, this study demonstrates that B-lymphoma cell lines and

  10. [Construction and functional identification of eukaryotic expression vector carrying Sprague-Dawley rat MSX-2 gene].

    Science.gov (United States)

    Yang, Xian-Xian; Zhang, Mei; Yan, Zhao-Wen; Zhang, Ru-Hong; Mu, Xiong-Zheng

    2008-01-01

    To construct a high effective eukaryotic expressing plasmid PcDNA 3.1-MSX-2 encoding Sprague-Dawley rat MSX-2 gene for the further study of MSX-2 gene function. The full length SD rat MSX-2 gene was amplified by PCR, and the full length DNA was inserted in the PMD1 8-T vector. It was isolated by restriction enzyme digest with BamHI and Xhol, then ligated into the cloning site of the PcDNA3.1 expression plasmid. The positive recombinant was identified by PCR analysis, restriction endonudease analysis and sequence analysis. Expression of RNA and protein was detected by RT-PCR and Western blot analysis in PcDNA3.1-MSX-2 transfected HEK293 cells. Sequence analysis and restriction endonudease analysis of PcDNA3.1-MSX-2 demonstrated that the position and size of MSX-2 cDNA insertion were consistent with the design. RT-PCR and Western blot analysis showed specific expression of mRNA and protein of MSX-2 in the transfected HEK293 cells. The high effective eukaryotic expression plasmid PcDNA3.1-MSX-2 encoding Sprague-Dawley Rat MSX-2 gene which is related to craniofacial development can be successfully reconstructed. It may serve as the basis for the further study of MSX-2 gene function.

  11. Enhanced p53 gene transfer to human ovarian cancer cells using the cationic nonviral vector, DDC.

    Science.gov (United States)

    Kim, Chong-Kook; Choi, Eun-Jeong; Choi, Sung-Hee; Park, Jeong-Sook; Haider, Khawaja Hasnain; Ahn, Woong Shick

    2003-08-01

    Previously we have formulated a new cationic liposome, DDC, composed of dioleoyltrimethylamino propane (DOTAP), 1,2-dioeoyl-3-phosphophatidylethanolamine (DOPE), and cholesterol (Chol), and it efficiently delivered plasmid DNA into ovarian cancer cells. Mutations in the p53 tumor suppressor gene are the most common molecular genetic abnormalities to be described in ovarian cancer. However, there has been so far no report of nonviral vector-mediated p53 gene deliveries in ovarian cancer. In this study, wild-type p53 DNA was transfected into the ovarian cancer cells, using the DDC as a nonviral vector and the expression and activity of p53 gene were evaluated both in vitro and in vivo. DDC liposomes were prepared by mixing DOTAP:DOPE:Chol in a 1:0.7:0.3 molar ratio using the extrusion method. Plasmid DNA (pp53-EGFP) and DDC complexes were transfected into ovarian carcinoma cells (OVCAR-3 cells) and gene expression was determined by reverse transcription-polymerase chain reaction and Western blot analysis. The cellular growth inhibition and apoptosis of DDC-mediated p53 transfection were assessed by trypan blue exclusion assay and annexin-V staining, respectively. The OVCAR-3 cells treated with DDC/pp53-EGFP complexes were inoculated into female balb/c nude mice and tumor growth was observed. The transfection of liposome-complexed p53 gene resulted in a high level of wild-type p53 mRNA and protein expressions in OVCAR-3 cells. In vitro cell growth assay showed growth inhibition of cancer cells transfected with DDC/pp53-EGFP complexes compared with the control cells. The reestablishment of wild-type p53 function in ovarian cancer cells restored the apoptotic pathway. Following the inoculation of DDC/pp53-EGFP complexes, the volumes of tumors in nude mice were significantly reduced more than 60% compared to the control group. The DDC-mediated p53 DNA delivery may have the potential for clinical application as nonviral vector-mediated ovarian cancer therapy due to its

  12. Dual delivery systems based on polyamine analog BENSpm as prodrug and gene delivery vectors

    Science.gov (United States)

    Zhu, Yu

    Combination drug and gene therapy shows promise in cancer treatment. However, the success of such strategy requires careful selection of the therapeutic agents, as well as development of efficient delivery vectors. BENSpm (N 1, N11-bisethylnorspermine), a polyamine analogue targeting the intracellular polyamine pathway, draws our special attention because of the following reasons: (1) polyamine pathway is frequently dysregulated in cancer; (2) BENSpm exhibits multiple functions to interfere with the polyamine pathway, such as to up-regulate polyamine metabolism enzymes and down-regulate polyamine biosynthesis enzymes. Therefore BENSpm depletes all natural polyamines and leads to apoptosis and cell growth inhibition in a wide range of cancers; (3) preclinical studies proved that BENSpm can act synergistically with various chemotherapy agents, making it a promising candidate in combination therapy; (4) multiple positive charges in BENSpm enable it as a suitable building block for cationic polymers, which can be further applied to gene delivery. In this dissertation, our goal was to design dual-function delivery vector based on BENSpm that can function as a gene delivery vector and, after intracellular degradation, as an active anticancer agent targeting dysregulated polyamine metabolism. We first demonstrated strong synergism between BENSpm and a potential therapeutic gene product TRAIL. Strong synergism was obtained in both estrogen-dependent MCF-7 breast cancer cells and triple-negative MDA-MB-231 breast cancer cells. Significant dose reduction of TRAIL in combination with BENSpm in MDA-MB-231 cells, together with the fact that BENSpm rendered MCF-7 cells more sensitive to TRAIL treatment verified our rationale of designing BENSpm-based delivery platform. This was expected to be beneficial for overcoming drug resistance in chemotherapy, as well as boosting the therapeutic effect of therapeutic genes. We first designed a lipid-based BENSpm dual vector (Lipo

  13. Protection against California 2002 NDV strain afforded by adenovirus vectored vaccine expressing Fusion or Hemagglutination-neuraminidase genes

    Science.gov (United States)

    Vectored vaccines expressing the combination of the hemagglutinin-neuraminidase (HN) and fusion (F) genes generally have better clinical protection against Newcastle disease virus (NDV) than when either the F and HN genes are expressed alone. Interestingly, the protection induced by F is usually bet...

  14. Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector

    Directory of Open Access Journals (Sweden)

    Lauro Augusto de Oliveira

    2010-10-01

    Full Text Available PURPOSE: To evaluate the transfer of heterologous genes carrying a Green Fluorescent Protein (GFP reporter cassette to primary corneal epithelial cells ex vivo. METHODS: Freshly enucleated rabbit corneoscleral tissue was used to obtain corneal epithelial cell suspension via enzymatic digestion. Cells were plated at a density of 5×10³ cells/cm² and allowed to grow for 5 days (to 70-80% confluency prior to transduction. Gene transfer was monitored using fluorescence microscopy and fluorescence activated cell sorter (FACS. We evaluated the transduction efficiency (TE over time and the dose-response effect of different lentiviral particles. One set of cells were dual sorted by fluorescence activated cell sorter for green fluorescent protein expression as well as Hoechst dye exclusion to evaluate the transduction of potentially corneal epithelial stem cells (side-population phenotypic cells. RESULTS: Green fluorescent protein expressing lentiviral vectors were able to effectively transduce rabbit primary epithelial cells cultured ex vivo. Live cell imaging post-transduction demonstrated GFP-positive cells with normal epithelial cell morphology and growth. The transduction efficiency over time was higher at the 5th post-transduction day (14.1% and tended to stabilize after the 8th day. The number of transduced cells was dose-dependent, and at the highest lentivirus concentrations approached 7%. When double sorted by fluorescence activated cell sorter to isolate both green fluorescent protein positive and side population cells, transduced side population cells were identified. CONCLUSIONS: Lentiviral vectors can effectively transfer heterologous genes to primary corneal epithelial cells expanded ex vivo. Genes were stably expressed over time, transferred in a dose-dependence fashion, and could be transferred to mature corneal cells as well as presumable putative stem cells.

  15. Tropism-Modification Strategies for Targeted Gene Delivery Using Adenoviral Vectors

    Directory of Open Access Journals (Sweden)

    Andrew H. Baker

    2010-10-01

    Full Text Available Achieving high efficiency, targeted gene delivery with adenoviral vectors is a long-standing goal in the field of clinical gene therapy. To achieve this, platform vectors must combine efficient retargeting strategies with detargeting modifications to ablate native receptor binding (i.e. CAR/integrins/heparan sulfate proteoglycans and “bridging” interactions. “Bridging” interactions refer to coagulation factor binding, namely coagulation factor X (FX, which bridges hepatocyte transduction in vivo through engagement with surface expressed heparan sulfate proteoglycans (HSPGs. These interactions can contribute to the off-target sequestration of Ad5 in the liver and its characteristic dose-limiting hepatotoxicity, thereby significantly limiting the in vivo targeting efficiency and clinical potential of Ad5-based therapeutics. To date, various approaches to retargeting adenoviruses (Ad have been described. These include genetic modification strategies to incorporate peptide ligands (within fiber knob domain, fiber shaft, penton base, pIX or hexon, pseudotyping of capsid proteins to include whole fiber substitutions or fiber knob chimeras, pseudotyping with non-human Ad species or with capsid proteins derived from other viral families, hexon hypervariable region (HVR substitutions and adapter-based conjugation/crosslinking of scFv, growth factors or monoclonal antibodies directed against surface-expressed target antigens. In order to maximize retargeting, strategies which permit detargeting from undesirable interactions between the Ad capsid and components of the circulatory system (e.g. coagulation factors, erythrocytes, pre-existing neutralizing antibodies, can be employed simultaneously. Detargeting can be achieved by genetic ablation of native receptor-binding determinants, ablation of “bridging interactions” such as those which occur between the hexon of Ad5 and coagulation factor X (FX, or alternatively, through the use of polymer

  16. Generation of a non-transmissive Borna disease virus vector lacking both matrix and glycoprotein genes.

    Science.gov (United States)

    Fujino, Kan; Yamamoto, Yusuke; Daito, Takuji; Makino, Akiko; Honda, Tomoyuki; Tomonaga, Keizo

    2017-09-01

    Borna disease virus (BoDV), a prototype of mammalian bornavirus, is a non-segmented, negative strand RNA virus that often causes severe neurological disorders in infected animals, including horses and sheep. Unique among animal RNA viruses, BoDV transcribes and replicates non-cytopathically in the cell nucleus, leading to establishment of long-lasting persistent infection. This striking feature of BoDV indicates its potential as an RNA virus vector system. It has previously been demonstrated by our team that recombinant BoDV (rBoDV) lacking an envelope glycoprotein (G) gene develops persistent infections in transduced cells without loss of the viral genome. In this study, a novel non-transmissive rBoDV, rBoDV ΔMG, which lacks both matrix (M) and G genes in the genome, is reported. rBoDV-ΔMG expressing green fluorescence protein (GFP), rBoDV ΔMG-GFP, was efficiently generated in Vero/MG cells stably expressing both BoDV M and G proteins. Infection with rBoDV ΔMG-GFP was persistently maintained in the parent Vero cells without propagation within cell culture. The optimal ratio of M and G for efficient viral particle production by transient transfection of M and G expression plasmids into cells persistently infected with rBoDV ΔMG-GFP was also demonstrated. These findings indicate that the rBoDV ΔMG-based BoDV vector may provide an extremely safe virus vector system and could be a novel strategy for investigating the function of M and G proteins and the host range of bornaviruses. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  17. TALEN-based gene disruption in the dengue vector Aedes aegypti.

    Science.gov (United States)

    Aryan, Azadeh; Anderson, Michelle A E; Myles, Kevin M; Adelman, Zach N

    2013-01-01

    In addition to its role as the primary vector for dengue viruses, Aedes aegypti has a long history as a genetic model organism for other bloodfeeding mosquitoes, due to its ease of colonization, maintenance and reproductive productivity. Though its genome has been sequenced, functional characterization of many Ae. aegypti genes, pathways and behaviors has been slow. TALE nucleases (TALENs) have been used with great success in a number of organisms to generate site-specific DNA lesions. We evaluated the ability of a TALEN pair to target the Ae. aegypti kmo gene, whose protein product is essential in the production of eye pigmentation. Following injection into pre-blastoderm embryos, 20-40% of fertile survivors produced kmo alleles that failed to complement an existing kh(w) mutation. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. Mutant alleles were associated with lesions of 1-7 bp specifically at the selected target site. White-eyed individuals could also be recovered following a blind intercross of G1 progeny, yielding several new white-eyed strains in the genetic background of the sequenced Liverpool strain. We conclude that TALENs are highly active in the Ae. aegypti germline, and have the potential to transform how reverse genetic experiments are performed in this important disease vector.

  18. TALEN-Based Gene Disruption in the Dengue Vector Aedes aegypti

    Science.gov (United States)

    Aryan, Azadeh; Anderson, Michelle A. E.; Myles, Kevin M.; Adelman, Zach N.

    2013-01-01

    In addition to its role as the primary vector for dengue viruses, Aedes aegypti has a long history as a genetic model organism for other bloodfeeding mosquitoes, due to its ease of colonization, maintenance and reproductive productivity. Though its genome has been sequenced, functional characterization of many Ae. aegypti genes, pathways and behaviors has been slow. TALE nucleases (TALENs) have been used with great success in a number of organisms to generate site-specific DNA lesions. We evaluated the ability of a TALEN pair to target the Ae. aegypti kmo gene, whose protein product is essential in the production of eye pigmentation. Following injection into pre-blastoderm embryos, 20–40% of fertile survivors produced kmo alleles that failed to complement an existing khw mutation. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. Mutant alleles were associated with lesions of 1–7 bp specifically at the selected target site. White-eyed individuals could also be recovered following a blind intercross of G1 progeny, yielding several new white-eyed strains in the genetic background of the sequenced Liverpool strain. We conclude that TALENs are highly active in the Ae. aegypti germline, and have the potential to transform how reverse genetic experiments are performed in this important disease vector. PMID:23555893

  19. TALEN-based gene disruption in the dengue vector Aedes aegypti.

    Directory of Open Access Journals (Sweden)

    Azadeh Aryan

    Full Text Available In addition to its role as the primary vector for dengue viruses, Aedes aegypti has a long history as a genetic model organism for other bloodfeeding mosquitoes, due to its ease of colonization, maintenance and reproductive productivity. Though its genome has been sequenced, functional characterization of many Ae. aegypti genes, pathways and behaviors has been slow. TALE nucleases (TALENs have been used with great success in a number of organisms to generate site-specific DNA lesions. We evaluated the ability of a TALEN pair to target the Ae. aegypti kmo gene, whose protein product is essential in the production of eye pigmentation. Following injection into pre-blastoderm embryos, 20-40% of fertile survivors produced kmo alleles that failed to complement an existing kh(w mutation. Most of these individuals produced more than 20% white-eyed progeny, with some producing up to 75%. Mutant alleles were associated with lesions of 1-7 bp specifically at the selected target site. White-eyed individuals could also be recovered following a blind intercross of G1 progeny, yielding several new white-eyed strains in the genetic background of the sequenced Liverpool strain. We conclude that TALENs are highly active in the Ae. aegypti germline, and have the potential to transform how reverse genetic experiments are performed in this important disease vector.

  20. Neurodegeneration in the diabetic eye

    DEFF Research Database (Denmark)

    Simó, Rafael; Hernández, Cristina; Bandello, F

    2014-01-01

    Diabetic retinopathy (DR), one of the leading causes of preventable blindness, has been considered a microcirculatory disease of the retina. However, there is emerging evidence to suggest that retinal neurodegeneration is an early event in the pathogenesis of DR, which participates in the develop...

  1. Initiation and propagation of neurodegeneration.

    Science.gov (United States)

    Haass, Christian

    2010-11-01

    Although substantial progress has been made in understanding the molecular and pathological bases of neurodegeneration, there have been few successes in the clinic and a number of fundamental questions remain unanswered. Is this skepticism misplaced, or do the words of Sir Isaac Newton hold true, that "what we know is a drop, what we don't know is an ocean"?

  2. CNS-restricted Transduction and CRISPR/Cas9-mediated Gene Deletion with an Engineered AAV Vector

    Directory of Open Access Journals (Sweden)

    Giridhar Murlidharan

    2016-01-01

    Full Text Available Gene therapy using recombinant adeno-associated viral (AAV vectors is emerging as a promising approach to treat central nervous system disorders such as Spinal muscular atrophy, Batten, Parkinson and Alzheimer disease amongst others. A critical remaining challenge for central nervous system-targeted gene therapy, silencing or gene editing is to limit potential vector dose-related toxicity in off-target cells and organs. Here, we characterize a lab-derived AAV chimeric (AAV2g9, which displays favorable central nervous system attributes derived from both parental counterparts, AAV2 and AAV9. This synthetic AAV strain displays preferential, robust, and widespread neuronal transduction within the brain and decreased glial tropism. Importantly, we observed minimal systemic leakage, decreased sequestration and gene transfer in off-target organs with AAV2g9, when administered into the cerebrospinal fluid. A single intracranial injection of AAV2g9 vectors encoding guide RNAs targeting the schizophrenia risk gene MIR137 (encoding MIR137 in CRISPR/Cas9 knockin mice resulted in brain-specific gene deletion with no detectable events in the liver. This engineered AAV vector is a promising platform for treating neurological disorders through gene therapy, silencing or editing modalities.

  3. Parameters selection in gene selection using Gaussian kernel support vector machines by genetic algorithm

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    In microarray-based cancer classification, gene selection is an important issue owing to the large number of variables and small number of samples as well as its non-linearity. It is difficult to get satisfying results by using conventional linear statistical methods. Recursive feature elimination based on support vector machine (SVM RFE) is an effective algorithm for gene selection and cancer classification, which are integrated into a consistent framework. In this paper, we propose a new method to select parameters of the aforementioned algorithm implemented with Gaussian kernel SVMs as better alternatives to the common practice of selecting the apparently best parameters by using a genetic algorithm to search for a couple of optimal parameter. Fast implementation issues for this method are also discussed for pragmatic reasons. The proposed method was tested on two representative hereditary breast cancer and acute leukaemia datasets. The experimental results indicate that the proposed method performs well in selecting genes and achieves high classification accuracies with these genes.

  4. [Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System].

    Science.gov (United States)

    Li, Lili; Wang, Zhan; Zhou, Yubai; Zhang, Fang; Shen, Sisi; Li, Zelin; Zeng, Yi

    2015-09-01

    For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.

  5. Construction and identification of double-gene co-expression vector with radiation-inducible human TRAIL and endostatin

    International Nuclear Information System (INIS)

    Li Yanbo; Guo Caixia; Gong Pingsheng; Liu Yang; Liangshuo; Wang Hongfang; Wang Jianfeng; Gong Shouliang

    2010-01-01

    Objective: To construct a recombinant plasmid pshuttle-Egr1-shTRAIL-shES containing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and endostatin double genes. Methods: The secretary endostatin gene (shES) fragment was amplified from the pMD19T-endostatin vector by PCR. The shES gene was ligated to pMD19Tand sequenced. Finally, using the gene recombinant technique, the recombinant plasmid pshuttle-Egr1- shTRAIL-shES with radiation-inducible Egr1 promoter, secretary TRAIL and endostatin double-gene was constructed. Results: The sequence of the shES gene was in concordance with that anticipated indicating shES gene was acquired successfully.Moreover, the results acquired by PCR and restrictive digestion identification of the recombinant plasmid pshuttle-Egr1-shTRAIL-shES and all the vectors refered to its construction confirmed that pshuttle-Egr1-shTRAIL-shES was constructed correctly. Conclusion: The radiation-inducible double-gene co-expression vector pshuttle-Egr1-shTRAIL-shES is constructed successfully, which would set the experimental foundation for further study on the anti-tumor effect of TRAIL and endostatin double-gene-radiotherapy and its related mechanisms. (authors)

  6. Preliminary studies on gene therapy with TGF β1 antisense gene/liposome complexes and adenovirus transfer vector in RPF rats

    International Nuclear Information System (INIS)

    Liu Chunjie; Wang Dewen; Zhang Zhaoshan; Gao Yabing; Xiong Chengqi; Long Jianyin; Wang Huixin; Peng Ruiyun; Cui Xuemei

    2001-01-01

    Objective: To observed the efficiency of gene therapy with TGF β1 antisense gene/liposome complexes and adenovirus transfer vector in RPF rats. Methods: TGFβ1 sense and antisense gene expression vectors and adenovirus transfer vector were introduced into rat bronchus by way of intratracheal instillation. Results: At day 1.5 after TGFβ1 sense and antisense gene transfer, PCR amplification using neo gene-specific primer from lung tissue DNA was all positive. After day 5.5, 67% (2/3) of lung tissue DNA was positive. RNA dot blot hybridization indicated that TGFβ1 mRNA content of lung tissue transfected with pMAMneo-antiTGFβ1 gene decreased. Detection of lung hydroxyproline (Hyp) content after day 35 of gene transfer showed that even in lung of rats received pMAMneo-AntiTGFβ1 lipid complexes it raised remarkably (P 9 pfu/ml were instilled into bronchus at 0.5 ml per rat. After day 2 day 6, the lung tissues of all six rats (three per each group )expressed the transfected luciferase gene by luminometer. Conclusion: Cationic lipid-mediated TGFβ1 antisense gene therapy was a simple and easy method. It can slow down the course of pathogenesis of lung fibrosis. Replication-deficient recombinant adenovirus-mediated gene therapy of lung diseases is a good and efficient method

  7. A set of vectors for introduction of antibiotic resistance genes by in vitro Cre-mediated recombination

    Directory of Open Access Journals (Sweden)

    Vassetzky Yegor S

    2008-12-01

    Full Text Available Abstract Background Introduction of new antibiotic resistance genes in the plasmids of interest is a frequent task in molecular cloning practice. Classical approaches involving digestion with restriction endonucleases and ligation are time-consuming. Findings We have created a set of insertion vectors (pINS carrying genes that provide resistance to various antibiotics (puromycin, blasticidin and G418 and containing a loxP site. Each vector (pINS-Puro, pINS-Blast or pINS-Neo contains either a chloramphenicol or a kanamycin resistance gene and is unable to replicate in most E. coli strains as it contains a conditional R6Kγ replication origin. Introduction of the antibiotic resistance genes into the vector of interest is achieved by Cre-mediated recombination between the replication-incompetent pINS and a replication-competent target vector. The recombination mix is then transformed into E. coli and selected by the resistance marker (kanamycin or chloramphenicol present in pINS, which allows to recover the recombinant plasmids with 100% efficiency. Conclusion Here we propose a simple strategy that allows to introduce various antibiotic-resistance genes into any plasmid containing a replication origin, an ampicillin resistance gene and a loxP site.

  8. Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector.

    Science.gov (United States)

    Paiboonsukwong, Kittiphong; Ohbayashi, Fumi; Shiiba, Haruka; Aizawa, Emi; Yamashita, Takayuki; Mitani, Kohnosuke

    2009-11-01

    Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR. We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells. We subsequently constructed an AAV vector encoding the normal sequences from the Fanconi anemia group A (FANCA) locus to correct a mutation in the gene in BCL derived from a FANCA patient. Under optimal conditions, approximately 50% of BCL cells were transduced with an AAV serotype 2 (AAV-2) vector. In FANCA BCL cells, up to 0.016% of infected cells were gene-corrected by HR. AAV-mediated restoration of normal genotypic and phenotypic characteristics in FANCA-mutant cells was confirmed at the DNA, protein and functional levels. The results obtained in the present study indicate that AAV vectors may be applicable for gene correction therapy of inherited hematopoietic disorders.

  9. Construction of a recombinant viral vector containing part of the nucleocapsid protein gene of newcastle disease virus

    Energy Technology Data Exchange (ETDEWEB)

    Bader, D.E.

    1995-09-01

    This report describes the procedures used to clone a 673 base pair gene fragment of the major nucleocapsid protein gene of Newcastle disease virus into a viral vector molecule for the purpose of maintaining a stable, long-term, renewable source of this target sequence for gene probe studies. The gene fragment was prepared by reverse-transcription polymerase chain reaction of Newcastle disease virus RNA and was cloned into the viral DNA vector Ml3mp18 RF to produce a recombinant DNA molecule. The cloned fragment was shown to be present in the recombinant clones based on (i) clonal selection on indicator plates; (ii) restriction enzyme analysis; (iii) gene probe analysis and (iv) nested PCR amplification.

  10. A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo

    Science.gov (United States)

    Lehto, Taavi; Simonson, Oscar E; Mäger, Imre; Ezzat, Kariem; Sork, Helena; Copolovici, Dana-Maria; Viola, Joana R; Zaghloul, Eman M; Lundin, Per; Moreno, Pedro MD; Mäe, Maarja; Oskolkov, Nikita; Suhorutšenko, Julia; Smith, CI Edvard; Andaloussi, Samir EL

    2011-01-01

    Finding suitable nonviral delivery vehicles for nucleic acid–based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice. PMID:21343913

  11. Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo

    Science.gov (United States)

    White, JD; Thesier, DM; Swain, JBD; Katz, MG; Tomasulo, C; Henderson, A; Wang, L; Yarnall, C; Fargnoli, A; Sumaroka, M; Isidro, A; Petrov, M; Holt, D; Nolen-Walston, R; Koch, WJ; Stedman, HH; Rabinowitz, J; Bridges, CR

    2013-01-01

    We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy. PMID:21228882

  12. Zn(II)-dipicolylamine-based metallo-lipids as novel non-viral gene vectors.

    Science.gov (United States)

    Su, Rong-Chuan; Liu, Qiang; Yi, Wen-Jing; Zhao, Zhi-Gang

    2017-08-01

    In this study, a series of Zn(II)-dipicolylamine (Zn-DPA) based cationic lipids bearing different hydrophobic tails (long chains, α-tocopherol, cholesterol or diosgenin) were synthesized. Structure-activity relationship (SAR) of these lipids was studied in detail by investigating the effects of several structural aspects including the type of hydrophobic tails, the chain length and saturation degree. In addition, several assays were used to study their interactions with plasmid DNA, and results reveal that these lipids could condense DNA into nanosized particles with appropriate size and zeta-potentials. MTT-based cell viability assays showed that lipoplexes 5 had low cytotoxicity. The in vitro gene transfection studies showed the hydrophobic tails clearly affected the TE, and hexadecanol-containing lipid 5b gives the best TE, which was 2.2 times higher than bPEI 25k in the presence of 10% serum. The results not only demonstrate that these lipids might be promising non-viral gene vectors, but also afford us clues for further optimization of lipidic gene delivery materials.

  13. Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy.

    Science.gov (United States)

    Capowski, Elizabeth E; Schneider, Bernard L; Ebert, Allison D; Seehus, Corey R; Szulc, Jolanta; Zufferey, Romain; Aebischer, Patrick; Svendsen, Clive N

    2007-07-30

    Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.

  14. Synthesis and characterization of PEG-conjugated quaternized chitosan and its application as a gene vector.

    Science.gov (United States)

    Zhang, Xi; Yao, Juan; Zhang, Lihong; Fang, Jianguo; Bian, Fengling

    2014-03-15

    Poly(ethylene glycol)-conjugated N-(2-hydroxy) propyl-3-trimethyl ammonium chitosan chloride (PHTAC) derivatives were prepared by incorporating PEG molecules onto quaternized chitosan backbone. The copolymers were characterized by FTIR, (1)H NMR and XRD. Agarose gel retardation assay indicated that PHTAC had good plasmid DNA (pDNA) binding capability and the particle sizes of PHTAC/pDNA complexes determined by DLS were about 200 nm. Cytotoxicity assays in HeLa and 293T cells showed that PHTAC had low cytotoxicity. In vitro luciferase assay showed that PHTAC with PEGylation degree of 9% (PHTAC-1) had good transfection efficiency about 5.3-fold higher than quaternized chitosan, which was comparable with PEI (25 kDa). These results suggest that PHTAC-1 is a promising candidate as an efficient nonviral gene vector. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins: new building blocks for cell marking and multi-gene analysis.

    Science.gov (United States)

    Weber, K; Mock, U; Petrowitz, B; Bartsch, U; Fehse, B

    2010-04-01

    Vector-encoded fluorescent proteins (FPs) facilitate unambiguous identification or sorting of gene-modified cells by fluorescence-activated cell sorting (FACS). Exploiting this feature, we have recently developed lentiviral gene ontology (LeGO) vectors (www.LentiGO-Vectors.de) for multi-gene analysis in different target cells. In this study, we extend the LeGO principle by introducing 10 different drug-selectable FPs created by fusing one of the five selection marker (protecting against blasticidin, hygromycin, neomycin, puromycin and zeocin) and one of the five FP genes (Cerulean, eGFP, Venus, dTomato and mCherry). All tested fusion proteins allowed both fluorescence-mediated detection and drug-mediated selection of LeGO-transduced cells. Newly generated codon-optimized hygromycin- and neomycin-resistance genes showed improved expression as compared with their ancestors. New LeGO constructs were produced at titers >10(6) per ml (for non-concentrated supernatants). We show efficient combinatorial marking and selection of various cells, including mesenchymal stem cells, simultaneously transduced with different LeGO constructs. Inclusion of the cytomegalovirus early enhancer/chicken beta-actin promoter into LeGO vectors facilitated robust transgene expression in and selection of neural stem cells and their differentiated progeny. We suppose that the new drug-selectable markers combining advantages of FACS and drug selection are well suited for numerous applications and vector systems. Their inclusion into LeGO vectors opens new possibilities for (stem) cell tracking and functional multi-gene analysis.

  16. Tissue-specific expression of silkmoth chorion genes in vivo using Bombyx mori nuclear polyhedrosis virus as a transducing vector.

    Science.gov (United States)

    Iatrou, K; Meidinger, R G

    1990-01-01

    A pair of silkmoth chorion chromosomal genes, HcA.12-HcB.12, was inserted into a baculovirus transfer vector, pBmp2, derived from the nuclear polyhedrosis virus of Bombyx mori. This vector, which permits the insertion of foreign genetic material in the vicinity of a mutationally inactivated polyhedrin gene, was used to acquire the corresponding recombinant virus. Injection of mutant silkmoth pupae that lack all Hc chorion genes with the recombinant virus resulted in the infection of all internal organs including follicular tissue. Analysis of RNA from infected tissues has demonstrated that the two chorion genes present in the viral genome are correctly transcribed under the control of their own promoter in follicular cells, the tissue in which chorion genes are normally expressed. The chorion primary transcripts are also correctly processed in the infected follicular cells and yield mature mRNAs indistinguishable from authentic chorion mRNAs present in wild-type follicles. These results demonstrate that recombinant nuclear polyhedrosis viruses can be used as transducing vectors for introducing genetic material of host origin into the cells of the organism and that the transduced genes are transiently expressed in a tissue-specific manner under the control of their resident regulatory sequences. Thus we show the in vivo expression of cloned genes under cellular promoter control in an insect other than Drosophila melanogaster. The approach should be applicable to all insect systems that are subject to nuclear polyhedrosis virus infection. Images PMID:2187186

  17. miRNA genes of an invasive vector mosquito, Aedes albopictus.

    Directory of Open Access Journals (Sweden)

    Jinbao Gu

    Full Text Available Aedes albopictus, a vector of Dengue and Chikungunya viruses, is a robust invasive species in both tropical and temperate environments. MicroRNAs (miRNAs regulate gene expression and biological processes including embryonic development, innate immunity and infection. While a number of miRNAs have been discovered in some mosquitoes, no comprehensive effort has been made to characterize them from different developmental stages from a single species. Systematic analysis of miRNAs in Ae. albopictus will improve our understanding of its basic biology and inform novel strategies to prevent virus transmission. Between 10-14 million Illumina sequencing reads per sample were obtained from embryos, larvae, pupae, adult males, sugar-fed and blood-fed adult females. A total of 119 miRNA genes represented by 215 miRNA or miRNA star (miRNA* sequences were identified, 15 of which are novel. Eleven, two, and two of the newly-discovered miRNA genes appear specific to Aedes, Culicinae, and Culicidae, respectively. A number of miRNAs accumulate predominantly in one or two developmental stages and the large number that showed differences in abundance following a blood meal likely are important in blood-induced mosquito biology. Gene Ontology (GO analysis of the targets of all Ae. albopictus miRNAs provides a useful starting point for the study of their functions in mosquitoes. This study is the first systematic analysis of miRNAs based on deep-sequencing of small RNA samples of all developmental stages of a mosquito species. A number of miRNAs are related to specific physiological states, most notably, pre- and post-blood feeding. The distribution of lineage-specific miRNAs is consistent with mosquito phylogeny and the presence of a number of Aedes-specific miRNAs likely reflects the divergence between the Aedes and Culex genera.

  18. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems.

    Directory of Open Access Journals (Sweden)

    Tamer Z Salem

    Full Text Available The simian virus 40 polyadenylation signal (SV40 polyA has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS. In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV, the polyhedrin promoter (very late promoter transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt, and gp37. In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications.

  19. The Influence of SV40 polyA on Gene Expression of Baculovirus Expression Vector Systems

    Science.gov (United States)

    Salem, Tamer Z.; Seaborn, Craig P.; Turney, Colin M.; Xue, Jianli; Shang, Hui; Cheng, Xiao-Wen

    2015-01-01

    The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome. In order to test the significance of adding the SV40 polyA sequence on gene expression, the expression of the enhanced green fluorescent protein (egfp) was evaluated with and without the presence of SV40 polyA under the control of the polyhedrin promoter at different genomic loci (polyherin, ecdysteroid UDP-glucosyltransferase (egt), and gp37). In this study, spectrofluorometry and western blot showed reduction of EGFP protein for all recombinant viruses with SV40 polyA, whereas qPCR showed an increase in the egfp mRNA levels. Therefore, we conclude that SV40 polyA increases mRNA levels but decreases protein production in the BEVS when the polyhedrin promoter is used at different loci. This work suggests that SV40 polyA in BEVSs should be replaced by an AcMNPV late gene polyA for optimal protein production or left untouched for optimal RNA production (RNA interference applications). PMID:26659470

  20. Efficient gene delivery to primary human retinal pigment epithelial cells: The innate and acquired properties of vectors.

    Science.gov (United States)

    Tasharrofi, Nooshin; Kouhkan, Fatemeh; Soleimani, Masoud; Soheili, Zahra-Soheila; Atyabi, Fatemeh; Akbari Javar, Hamid; Abedin Dorkoosh, Farid

    2017-02-25

    The purpose of this study is designing non-viral gene delivery vectors for transfection of the primary human retinal pigment epithelial cells (RPE). In the design process of gene delivery vectors, considering physicochemical properties of vectors alone does not seem to be enough since they interact with constituents of the surrounding environment and hence gain new characteristics. Moreover, due to these interactions, their cargo can be released untimely or undergo degradation before reaching to the target cells. Further, the characteristics of cells itself can also influence the transfection efficacy. For example, the non-dividing property of RPE cells can impede the transfection efficiency which in most studies was ignored by using immortal cell lines. In this study, vectors with different characteristics differing in mixing orders of pDNA, PEI polymer, and PLGA/PEI or PLGA nanoparticles were prepared and characterized. Then, their characteristics and efficacy in gene delivery to RPE cells in the presence of vitreous or fetal bovine serum (FBS) were evaluated. All formulations showed no cytotoxicity and were able to protect pDNA from premature release and degradation in extracellular media. Also, the adsorption of vitreous or serum proteins onto the surface of vectors changed their properties and hence cellular uptake and transfection efficacy. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs.

    Science.gov (United States)

    Levine, Timothy P; Daniels, Rachel D; Gatta, Alberto T; Wong, Louise H; Hayes, Matthew J

    2013-02-15

    Fronto-temporal dementia (FTD) and amyotrophic lateral sclerosis (ALS, also called motor neuron disease, MND) are severe neurodegenerative diseases that show considerable overlap at the clinical and cellular level. The most common single mutation in families with FTD or ALS has recently been mapped to a non-coding repeat expansion in the uncharacterized gene C9ORF72. Although a plausible mechanism for disease is that aberrant C9ORF72 mRNA poisons splicing, it is important to determine the cellular function of C9ORF72, about which nothing is known. Sensitive homology searches showed that C9ORF72 is a full-length distant homologue of proteins related to Differentially Expressed in Normal and Neoplasia (DENN), which is a GDP/GTP exchange factor (GEF) that activates Rab-GTPases. Our results suggest that C9ORF72 is likely to regulate membrane traffic in conjunction with Rab-GTPase switches, and we propose to name the gene and its product DENN-like 72 (DENNL72).

  2. Construction Of An Optimized Lentiviral Vector Containing Pdx-1 Gene For Transduction Of Stem Cells Towards Gene Therapy Diabetes Type 1

    Directory of Open Access Journals (Sweden)

    S Rahmati

    2013-02-01

    Full Text Available Abstract Background & aim: Nowadays, most of gene therapy protocols are performed by lentiviral vectors. One of the most important factors which is involved in pancreas development and transcription of insulin gene is pancreatic & duodenal homeobox 1 (PDX-1 transcription factor. The goal of this study was to optimize a lentiviral construct, containing pdx-1 gene, to transfect stem cells towards gene therapy of type-1 diabetes. Methods: In this experimental study, first, the pdx-1 gene was multiplied by PCR from pcDNA3.1-pdx-1 and cloned into pTG19-T vector. Then, pdx-1 was subcloned on upstream of IRES-EGFP gene into IRES2-EGFP vector. At the next step, the cloned parts of IRES-EGFP and pdx-1 were isolated and cloned into the lentiviral expression vector pSINTREM in upstream of TRE-CMV gene. After sequencing, final construct was transfected into HEK 293 cells and gene expression of pdx-1 was evaluated using flow cytometry analysis and reverse fluorescent microscopy. Results: Flow cytometry results and inverted fluorescent microscopy observing showed that pdx-1 and GFP genes are expressed in cells transfected with final recombinant construct. Conclusion: Regarding the design of this construct, to ensure long time expression with higher in vivo and in vitro expression efficiency for stem cells and also use of Tet on induced optimized system, it seems that the current construct can be among the best ones to transfect stem cells. Key words: Gene therapy, Diabetes, Stem cells

  3. Multicistronic lentiviral vectors containing the FMDV 2A cleavage factor demonstrate robust expression of encoded genes at limiting MOI

    Directory of Open Access Journals (Sweden)

    Margison Geoffrey P

    2006-03-01

    Full Text Available Abstract Background A number of gene therapy applications would benefit from vectors capable of expressing multiple genes. In this study we explored the feasibility and efficiency of expressing two or three transgenes in HIV-1 based lentiviral vector. Bicistronic and tricistronic self-inactivating lentiviral vectors were constructed employing the internal ribosomal entry site (IRES sequence of encephalomyocarditis virus (EMCV and/or foot-and-mouth disease virus (FMDV cleavage factor 2A. We employed enhanced green fluorescent protein (eGFP, O6-methylguanine-DNA-methyltransferase (MGMT, and homeobox transcription factor HOXB4 as model genes and their expression was detected by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. Results All the multigene vectors produced high titer virus and were able to simultaneously express two or three transgenes in transduced cells. However, the level of expression of individual transgenes varied depending on: the transgene itself; its position within the construct; the total number of transgenes expressed; the strategy used for multigene expression and the average copy number of pro-viral insertions. Notably, at limiting MOI, the expression of eGFP in a bicistronic vector based on 2A was ~4 times greater than that of an IRES based vector. Conclusion The small and efficient 2A sequence can be used alone or in combination with an IRES for the construction of multicistronic lentiviral vectors which can express encoded transgenes at functionally relevant levels in cells containing an average of one pro-viral insert.

  4. Advances in Viral Vector-Based TRAIL Gene Therapy for Cancer

    International Nuclear Information System (INIS)

    Norian, Lyse A.; James, Britnie R.; Griffith, Thomas S.

    2011-01-01

    Numerous biologic approaches are being investigated as anti-cancer therapies in an attempt to induce tumor regression while circumventing the toxic side effects associated with standard chemo- or radiotherapies. Among these, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown particular promise in pre-clinical and early clinical trials, due to its preferential ability to induce apoptotic cell death in cancer cells and its minimal toxicity. One limitation of TRAIL use is the fact that many tumor types display an inherent resistance to TRAIL-induced apoptosis. To circumvent this problem, researchers have explored a number of strategies to optimize TRAIL delivery and to improve its efficacy via co-administration with other anti-cancer agents. In this review, we will focus on TRAIL-based gene therapy approaches for the treatment of malignancies. We will discuss the main viral vectors that are being used for TRAIL gene therapy and the strategies that are currently being attempted to improve the efficacy of TRAIL as an anti-cancer therapeutic

  5. Modeling horizontal gene transfer (HGT in the gut of the Chagas disease vector Rhodnius prolixus

    Directory of Open Access Journals (Sweden)

    Durvasula Ravi V

    2011-05-01

    Full Text Available Abstract Background Paratransgenesis is an approach to reducing arthropod vector competence using genetically modified symbionts. When applied to control of Chagas disease, the symbiont bacterium Rhodococcus rhodnii, resident in the gut lumen of the triatomine vector Rhodnius prolixus (Hemiptera: Reduviidae, is transformed to export cecropin A, an insect immune peptide. Cecropin A is active against Trypanosoma cruzi, the causative agent of Chagas disease. While proof of concept has been achieved in laboratory studies, a rigorous and comprehensive risk assessment is required prior to consideration of field release. An important part of this assessment involves estimating probability of transgene horizontal transfer to environmental organisms (HGT. This article presents a two-part risk assessment methodology: a theoretical model predicting HGT in the gut of R. prolixus from the genetically transformed symbiont R. rhodnii to a closely related non-target bacterium, Gordona rubropertinctus, in the absence of selection pressure, and a series of laboratory trials designed to test the model. Results The model predicted an HGT frequency of less than 1.14 × 10-16 per 100,000 generations at the 99% certainty level. The model was iterated twenty times, with the mean of the ten highest outputs evaluated at the 99% certainty level. Laboratory trials indicated no horizontal gene transfer, supporting the conclusions of the model. Conclusions The model treats HGT as a composite event, the probability of which is determined by the joint probability of three independent events: gene transfer through the modalities of transformation, transduction, and conjugation. Genes are represented in matrices and Monte Carlo method and Markov chain analysis are used to simulate and evaluate environmental conditions. The model is intended as a risk assessment instrument and predicts HGT frequency of less than 1.14 × 10-16 per 100,000 generations. With laboratory studies that

  6. Cloning vector

    Science.gov (United States)

    Guilfoyle, Richard A.; Smith, Lloyd M.

    1994-01-01

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site.

  7. Cloning vector

    Science.gov (United States)

    Guilfoyle, R.A.; Smith, L.M.

    1994-12-27

    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site. 2 figures.

  8. Suppression of leaky expression of adenovirus genes by insertion of microRNA-targeted sequences in the replication-incompetent adenovirus vector genome

    Directory of Open Access Journals (Sweden)

    Kahori Shimizu

    2014-01-01

    Full Text Available Leaky expression of adenovirus (Ad genes occurs following transduction with a conventional replication-incompetent Ad vector, leading to an induction of cellular immunity against Ad proteins and Ad protein-induced toxicity, especially in the late phase following administration. To suppress the leaky expression of Ad genes, we developed novel Ad vectors by incorporating four tandem copies of sequences with perfect complementarity to miR-122a or miR-142-3p into the 3′-untranslated region (UTR of the E2A, E4, or pIX gene, which were mainly expressed from the Ad vector genome after transduction. These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional 293 cells. The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold, compared with a conventional Ad vector, by insertion of the miRNA-targeted sequences. Notably, the Ad vector carrying the miR-122a–targeted sequences into the 3′-UTR of the E4 gene expressed higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver than a conventional Ad vector. miR-122a–mediated suppression of the E4 gene expression in the liver significantly reduced the hepatotoxicity which an Ad vector causes via both adaptive and non-adaptive immune responses.

  9. Gene therapy with adeno-associated virus vector 5-human factor IX in adults with hemophilia B

    DEFF Research Database (Denmark)

    Miesbach, Wolfgang; Meijer, Karina; Coppens, Michiel

    2018-01-01

    Hemophilia B gene therapy aims to ameliorate bleeding risk and provide endogenous factor IX (FIX) activity/synthesis through a single treatment, eliminating the requirement for FIX concentrate. AMT-060 combines an adeno-associated virus-5 (AAV5) vector with a liver-specific promoter driving expre...

  10. Transformation of Cowpea Vigna unguiculata Cells with an Antibiotic Resistance Gene Using a Ti-Plasmid-Derived Vector

    NARCIS (Netherlands)

    Hille, Jacques; Goldbach, Rob

    1986-01-01

    A chimaeric antibiotic resistance gene was transferred to cowpea (Vigna unguiculata), a member of the legume family. This transfer was established by inoculating cowpea leaf discs with an Agrobacterium tumefaciens strain harboring a Ti-plasmid-derived vector that contained two copies of a chimaeric

  11. A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets.

    Science.gov (United States)

    Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

    2014-12-05

    Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.

  12. Generation of a Vero-Based Packaging Cell Line to Produce SV40 Gene Delivery Vectors for Use in Clinical Gene Therapy Studies

    Directory of Open Access Journals (Sweden)

    Miguel G. Toscano

    2017-09-01

    Full Text Available Replication-defective (RD recombinant simian virus 40 (SV40-based gene delivery vectors hold a great potential for clinical applications because of their presumed non-immunogenicity and capacity to induce immune tolerance to the transgene products in humans. However, the clinical use of SV40 vectors has been hampered by the lack of a packaging cell line that produces replication-competent (RC free SV40 particles in the vector production process. To solve this problem, we have adapted the current SV40 vector genome used for the production of vector particles and generated a novel Vero-based packaging cell line named SuperVero that exclusively expresses the SV40 large T antigen. SuperVero cells produce similar numbers of SV40 vector particles compared to the currently used packaging cell lines, albeit in the absence of contaminating RC SV40 particles. Our unique SV40 vector platform named SVac paves the way to clinically test a whole new generation of SV40-based therapeutics for a broad range of important diseases.

  13. Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes

    International Nuclear Information System (INIS)

    Igarashi, Aki; Yamagata, Kousuke; Sugai, Tomokazu; Takahashi, Yukari; Sugawara, Emiko; Tamura, Akihiro; Yaegashi, Hajime; Yamagishi, Noriko; Takahashi, Tsubasa; Isogai, Masamichi; Takahashi, Hideki; Yoshikawa, Nobuyuki

    2009-01-01

    Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to silence a meristem gene, proliferating cell nuclear antigen and disease resistant N gene in tobacco and RCY1 gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad range of plant species.

  14. Expression of Separate Proteins in the Same Plant Leaves and Cells Using Two Independent Virus-Based Gene Vectors

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    Maria R. Mendoza

    2017-11-01

    Full Text Available Plant viral vectors enable the expression of proteins at high levels in a relatively short time. For many purposes (e.g., cell biological interaction studies it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two compatible and non-competitive viruses that can co-exist to each express a separate protein. Here, we report on the use of two agro-launchable coat-protein gene substitution GFP-expressing virus vector systems based on Tomato bushy stunt virus (TBSV referred to as TG, and Tobacco mosaic virus (TMV annotated as TRBO-G. TG expressed GFP in Nicotiana benthamiana, tomato, lettuce and cowpea, whereas expression from TRBO-G was detected only in the first two species. Upon co-infiltration of the two vectors co-expression was monitored by: molecular detection of the two slightly differently sized GFPs, suppressor-complementation assays, and using TG in combination with TRBO-RFP. All the results revealed that in N. benthamiana and tomato the TBSV and TMV vectors accumulated and expressed proteins in the same plants, the same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a platform for fast and high level expression of proteins to study their cell biology or other properties.

  15. Cloning of synthetic gene including antigens against Urinary Tract Infections in pET28a+ vector

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    Zohreh Haghri

    2017-12-01

    Full Text Available There are many different bacterial infections in the world that patients are suffering from and research teams are trying to find suitable ways to prevent and treat them. Urinary Tract Infections (UTIs are most important infections in the world , and they are more common among women because vaginal cavity is near to urethral opening. The aim of this study is cloning of synthetic gene include antigens against UTIs in pET28a+ vector. Antibiotic resistant has been increasing because of antibiotic overuse recently, so It shows the necessity of developing a vaccine against these infections. There for, it will be imperative to develop a vaccine instead of antibiotics. This infection causes by many organisms, most important of which are Uropathogenic Escherichia coli (UPEC, Proteus mirabilis and Klebsiella pneumoniae Uropathogenic Escherichia .coli is the most important microorganism that causes these infections more than other bacteria, so in developing a vaccine it is the most important one, that have to be considered. The synthetic Gene which was designed against these three bacteria including antigens which are important and common to cause these infections. This gene has involved 1293bp. It was ordered to Gene Ray Biotechnology. Primers were designed by Gene Runner. Gene and pET28a+ vector was checked by SnappGene. Synthetic gene was multiplied by PCR and cloned in pET28a+ vector. Construct was transformed into E. coli TOP10.The clone was confirmed by PCR, Digestion. This data indicates that this gene can be expressed and it might be a vaccine candidate to protect people from these infections in the future.

  16. Widespread and highly persistent gene transfer to the CNS by retrovirus vector in utero: implication for gene therapy to Krabbe disease.

    Science.gov (United States)

    Shen, Jin-Song; Meng, Xing-Li; Yokoo, Takashi; Sakurai, Ken; Watabe, Kazuhiko; Ohashi, Toya; Eto, Yoshikatsu

    2005-05-01

    Brain-directed prenatal gene therapy may benefit some lysosomal storage diseases that affect the central nervous system (CNS) before birth. Our previous study showed that intrauterine introduction of recombinant adenoviruses into cerebral ventricles results in efficient gene transfer to the CNS in the mouse. However, transgene expression decreased with time due to the non-integrative property of adenoviral vectors. In this study, in order to obtain permanent gene transduction, we investigated the feasibility of retrovirus-mediated in utero gene transduction. Concentrated retrovirus encoding the LacZ gene was injected into the cerebral ventricles of the embryos of normal and twitcher mice (a murine model of Krabbe disease) at embryonic day 12. The distribution and maintenance of the transgene expression in the recipient brain were analyzed histochemically, biochemically and by the quantitative polymerase chain reaction method pre- and postnatally. Efficient and highly persistent gene transduction to the brain was achieved both in normal and the twitcher mouse. Transduced neurons, astrocytes and oligodendrocytes were distributed throughout the brain. The transduced LacZ gene, its transcript and protein expression in the brain were maintained for 14 months without decrement. In addition, gene transduction to multiple tissues other than the brain was also detected at low levels. This study suggests that brain-directed in utero gene transfer using retrovirus vector may be beneficial to the treatment of lysosomal storage diseases with severe brain damage early in life, such as Krabbe disease. Copyright (c) 2005 John Wiley & Sons, Ltd.

  17. An efficient nonviral gene-delivery vector based on hyperbranched cationic glycogen derivatives

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    Liang X

    2014-01-01

    Full Text Available Xuan Liang,1,* Xianyue Ren,2,* Zhenzhen Liu,1 Yingliang Liu,1 Jue Wang,2 Jingnan Wang,2 Li-Ming Zhang,1 David YB Deng,2 Daping Quan,1 Liqun Yang1 1Institute of Polymer Science, School of Chemistry and Chemical Engineering, Key Laboratory of Designed Synthesis and Application of Polymer Material, Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, Sun Yat-Sen University, Guangzhou, People's Republic of China; 2Research Center of Translational Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China *Both these authors contributed equally to this work Background: The purpose of this study was to synthesize and evaluate hyperbranched cationic glycogen derivatives as an efficient nonviral gene-delivery vector. Methods: A series of hyperbranched cationic glycogen derivatives conjugated with 3-(dimethylamino-1-propylamine (DMAPA-Glyp and 1-(2-aminoethyl piperazine (AEPZ-Glyp residues were synthesized and characterized by Fourier-transform infrared and hydrogen-1 nuclear magnetic resonance spectroscopy. Their buffer capacity was assessed by acid–base titration in aqueous NaCl solution. Plasmid deoxyribonucleic acid (pDNA condensation ability and protection against DNase I degradation of the glycogen derivatives were assessed using agarose gel electrophoresis. The zeta potentials and particle sizes of the glycogen derivative/pDNA complexes were measured, and the images of the complexes were observed using atomic force microscopy. Blood compatibility and cytotoxicity were evaluated by hemolysis assay and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, respectively. pDNA transfection efficiency mediated by the cationic glycogen derivatives was evaluated by flow cytometry and fluorescence microscopy in the 293T (human embryonic kidney and the CNE2 (human nasopharyngeal carcinoma cell lines. In vivo delivery of pDNA in model animals (Sprague Dawley

  18. CompareSVM: supervised, Support Vector Machine (SVM) inference of gene regularity networks.

    Science.gov (United States)

    Gillani, Zeeshan; Akash, Muhammad Sajid Hamid; Rahaman, M D Matiur; Chen, Ming

    2014-11-30

    Predication of gene regularity network (GRN) from expression data is a challenging task. There are many methods that have been developed to address this challenge ranging from supervised to unsupervised methods. Most promising methods are based on support vector machine (SVM). There is a need for comprehensive analysis on prediction accuracy of supervised method SVM using different kernels on different biological experimental conditions and network size. We developed a tool (CompareSVM) based on SVM to compare different kernel methods for inference of GRN. Using CompareSVM, we investigated and evaluated different SVM kernel methods on simulated datasets of microarray of different sizes in detail. The results obtained from CompareSVM showed that accuracy of inference method depends upon the nature of experimental condition and size of the network. For network with nodes (SVM Gaussian kernel outperform on knockout, knockdown, and multifactorial datasets compared to all the other inference methods. For network with large number of nodes (~500), choice of inference method depend upon nature of experimental condition. CompareSVM is available at http://bis.zju.edu.cn/CompareSVM/ .

  19. Intercontinental genetic structure and gene flow in Dunlin (Calidris alpina), a potential vector of avian influenza

    Science.gov (United States)

    Miller, Mark P.; Haig, Susan M.; Mullins, Thomas D.; Ruan, Luzhang; Casler, Bruce; Dondua, Alexei; Gates, River H.; Johnson, J. Matthew; Kendall, Steven J.; Tomkovich, Pavel S.; Tracy, Diane; Valchuk, Olga P.; Lanctot, Richard B.

    2015-01-01

    Waterfowl (Anseriformes) and shorebirds (Charadriiformes) are the most common wild vectors of influenza A viruses. Due to their migratory behavior, some may transmit disease over long distances. Migratory connectivity studies can link breeding and nonbreeding grounds while illustrating potential interactions among populations that may spread diseases. We investigated Dunlin (Calidris alpina), a shorebird with a subspecies (C. a. arcticola) that migrates from nonbreeding areas endemic to avian influenza in eastern Asia to breeding grounds in northern Alaska. Using microsatellites and mitochondrial DNA, we illustrate genetic structure among six subspecies: C. a. arcticola, C. a. pacifica, C. a. hudsonia, C. a. sakhalina, C. a. kistchinski, and C. a. actites. We demonstrate that mitochondrial DNA can help distinguish C. a. arcticola on the Asian nonbreeding grounds with >70% accuracy depending on their relative abundance, indicating that genetics can help determine whether C. a. arcticola occurs where they may be exposed to highly pathogenic avian influenza (HPAI) during outbreaks. Our data reveal asymmetric intercontinental gene flow, with some C. a. arcticola short-stopping migration to breed with C. a. pacifica in western Alaska. Because C. a. pacifica migrates along the Pacific Coast of North America, interactions between these subspecies and other taxa provide route for transmission of HPAI into other parts of North America.

  20. Expression profile of genes during resistance reversal in a temephos selected strain of the dengue vector, Aedes aegypti.

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    Clare Strode

    Full Text Available BACKGROUND: The mosquito Aedes aegypti is one of the most important disease vectors because it transmits two major arboviruses, dengue and yellow fever, which cause significant global morbidity and mortality. Chemical insecticides form the cornerstone of vector control. The organophosphate temephos a larvicide recommended by WHO for controlling Ae. aegypti, however, resistance to this compound has been reported in many countries, including Brazil. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to identify genes implicated in metabolic resistance in an Ae. aegypti temephos resistant strain, named RecR, through microarray analysis. We utilized a custom 'Ae. aegypti detox chip' and validated microarray data through RT-PCR comparing susceptible and resistant individuals. In addition, we analyzed gene expression in 4(th instar larvae from a reversed susceptible strain (RecRev, exposed and unexposed to temephos. The results obtained revealed a set of 13 and 6 genes significantly over expressed in resistant adult mosquitoes and larvae, respectively. One of these genes, the cytochrome P450 CYP6N12, was up-regulated in both stages. RT-PCR confirmed the microarray results and, additionally, showed no difference in gene expression between temephos exposed and unexposed RecRev mosquitoes. This suggested that the differences in the transcript profiles among the strains are heritable due to a selection process and are not caused by immediate insecticide exposure. Reversal of temephos resistance was demonstrated and, importantly, there was a positive correlation between a decrease in the resistance ratio and an accompanying decrease in the expression levels of previously over expressed genes. Some of the genes identified here have also been implicated in metabolic resistance in other mosquito species and insecticide resistant populations of Ae. aegypti. CONCLUSIONS/SIGNIFICANCE: The identification of gene expression signatures associated to

  1. Clonal Dominance With Retroviral Vector Insertions Near the ANGPT1 and ANGPT2 Genes in a Human Xenotransplant Mouse Model

    Directory of Open Access Journals (Sweden)

    Reinhard Haemmerle

    2014-01-01

    Full Text Available Insertional leukemogenesis represents the major risk factor of hematopoietic stem cell (HSC based gene therapy utilizing integrating viral vectors. To develop a pre-clinical model for the evaluation of vector-related genotoxicity directly in the relevant human target cells, cord blood CD34+ HSCs were transplanted into immunodeficient NOD.SCID.IL2rg−/− (NSG mice after transduction with an LTR-driven gammaretroviral vector (GV. Furthermore, we specifically investigated the effect of prolonged in vitro culture in the presence of cytokines recently described to promote HSC expansion or maintenance. Clonality of human hematopoiesis in NSG mice was assessed by high throughput insertion site analyses and validated by insertion site-specific PCR depicting a GV typical integration profile with insertion sites resembling to 25% those of clinical studies. No overrepresentation of integrations in the vicinity of cancer-related genes was observed, however, several dominant clones were identified including two clones harboring integrations in the ANGPT1 and near the ANGPT2 genes associated with deregulated ANGPT1- and ANGPT2-mRNA levels. While these data underscore the potential value of the NSG model, our studies also identified short-comings such as overall low numbers of engrafted HSCs, limited in vivo observation time, and the challenges of in-depth insertion site analyses by low contribution of gene modified hematopoiesis.

  2. Vector for IS element entrapment and functional characterization based on turning on expression of distal promoterless genes.

    Science.gov (United States)

    Szeverényi, I; Hodel, A; Arber, W; Olasz, F

    1996-09-26

    We constructed and characterized a novel trap vector for rapid isolation of insertion sequences. The strategy used for the isolation of IS elements is based on the ability of many IS elements to turn on the expression of otherwise silent genes distal to some sites of insertion. The simple transposition of an IS element can sometimes cause the constitutive expression of promoterless antibiotic resistance genes resulting in selectable phenotypes. The trap vector pAW1326 is based on a pBR322 replicon, it carries ampicillin and streptomycin resistance genes, and also silenced genes that confer chloramphenicol and kanamycin resistance once activated. The trap vector pAW1326 proved to be efficient and 85 percent of all isolated mutations were insertions. The majority of IS elements resident in the studied Escherichia coli strains tested became trapped, namely IS2, IS3, IS5, IS150, IS186 and Tn1000. We also encountered an insertion sequence, called IS10L/R-2, which is a hybrid of the two IS variants IS10L and IS10R. IS10L/R-2 is absent from most E. coli strains, but it is detectable in some strains such as JM109 which had been submitted to Tn10 mutagenesis. The distribution of the insertion sequences within the trap region was not random. Rather, the integration of chromosomal mobile genetic elements into the offered target sequence occurred in element-specific clusters. This is explained both by the target specificity and by the specific requirements for the activation of gene transcription by the DNA rearrangement. The employed trap vector pAW1326 proved to be useful for the isolation of mobile genetic elements, for a demonstration of their transposition activity as well as for the further characterization of some of the functional parameters of transposition.

  3. Generation and characterization of a novel candidate gene therapy and vaccination vector based on human species D adenovirus type 56.

    Science.gov (United States)

    Duffy, Margaret R; Alonso-Padilla, Julio; John, Lijo; Chandra, Naresh; Khan, Selina; Ballmann, Monika Z; Lipiec, Agnieszka; Heemskerk, Evert; Custers, Jerome; Arnberg, Niklas; Havenga, Menzo; Baker, Andrew H; Lemckert, Angelique

    2018-01-01

    The vectorization of rare human adenovirus (HAdV) types will widen our knowledge of this family and their interaction with cells, tissues and organs. In this study we focus on HAdV-56, a member of human Ad species D, and create ease-of-use cloning systems to generate recombinant HAdV-56 vectors carrying foreign genes. We present in vitro transduction profiles for HAdV-56 in direct comparison to the most commonly used HAdV-5-based vector. In vivo characterizations demonstrate that when it is delivered intravenously (i.v.) HAdV-56 mainly targets the spleen and, to a lesser extent, the lungs, whilst largely bypassing liver transduction in mice. HAdV-56 triggered robust inflammatory and cellular immune responses, with higher induction of IFNγ, TNFα, IL5, IL6, IP10, MCP1 and MIG1 compared to HAdV-5 following i.v. administration. We also investigated its potential as a vaccine vector candidate by performing prime immunizations in mice with HAdV-56 encoding luciferase (HAdV-56-Luc). Direct comparisons were made to HAdV-26, a highly potent human vaccine vector currently in phase II clinical trials. HAdV-56-Luc induced luciferase 'antigen'-specific IFNγ-producing cells and anti-HAdV-56 neutralizing antibodies in Balb/c mice, demonstrating a near identical profile to that of HAdV-26. Taken together, the data presented provides further insight into human Ad receptor/co-receptor usage, and the first report on HAdV-56 vectors and their potential for gene therapy and vaccine applications.

  4. NYVAC vector modified by C7L viral gene insertion improves T cell immune responses and effectiveness against leishmaniasis.

    Science.gov (United States)

    Sánchez-Sampedro, L; Mejías-Pérez, E; S Sorzano, Carlos Óscar; Nájera, J L; Esteban, M

    2016-07-15

    The NYVAC poxvirus vector is used as vaccine candidate for HIV and other diseases, although there is only limited experimental information on its immunogenicity and effectiveness for use against human pathogens. Here we defined the selective advantage of NYVAC vectors in a mouse model by comparing the immune responses and protection induced by vectors that express the LACK (Leishmania-activated C-kinase antigen), alone or with insertion of the viral host range gene C7L that allows the virus to replicate in human cells. Using DNA prime/virus boost protocols, we show that replication-competent NYVAC-LACK that expresses C7L (NYVAC-LACK-C7L) induced higher-magnitude polyfunctional CD8(+) and CD4(+) primary adaptive and effector memory T cell responses (IFNγ, TNFα, IL-2, CD107a) to LACK antigen than non-replicating NYVAC-LACK. Compared to NYVAC-LACK, the NYVAC-LACK-C7L-induced CD8(+) T cell population also showed higher proliferation when stimulated with LACK antigen. After a challenge by subcutaneous Leishmania major metacyclic promastigotes, NYVAC-LACK-C7L-vaccinated mouse groups showed greater protection than the NYVAC-LACK-vaccinated group. Our results indicate that the type and potency of immune responses induced by LACK-expressing NYVAC vectors is improved by insertion of the C7L gene, and that a replication-competent vector as a vaccine renders greater protection against a human pathogen than a non-replicating vector. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Vectors for Inhaled Gene Therapy in Lung Cancer. Application for Nano Oncology and Safety of Bio Nanotechnology

    Science.gov (United States)

    Zarogouldis, Paul; Karamanos, Nikos K.; Porpodis, Konstantinos; Domvri, Kalliopi; Huang, Haidong; Hohenforst-Schimdt, Wolfgang; Goldberg, Eugene P.; Zarogoulidis, Konstantinos

    2012-01-01

    Novel aerosol therapeutic modalities have been investigated for lung cancer. Inhaled gene therapy has presented safety and effectiveness previously in cystic fibrosis. However, safety concerns have been raised regarding the safety of non-viral vectors for inhaled gene therapy in lung cancer, and therefore small steps have been made towards this multifunctional treatment modality. During the last decade, numerous new nanocomplexes have been created and investigated as a safe gene delivery nano-vehicle. These formulations are multifunctional; they can be used as either local therapy or carrier for an effective inhaled gene therapy for lung cancer. Herein, we present current and future perspectives of nanocomplexes for inhaled gene therapy treatment in lung cancer. PMID:23109824

  6. Construction and characterization in vitro of a bicistronic retroviral vector coding endostatin and interleukin-2 for use in gene therapy

    International Nuclear Information System (INIS)

    Calvo, Fernanda Bernardes

    2009-01-01

    Gene therapy has been used in preclinical studies and clinical trials in order to alleviate or cure a disease. Retroviral vectors are a tool for gene transfer is widely used. Bicistronic vectors are an attractive alternative for treatment of complex diseases. A variety of options exists to simultaneously express two genes in genetically modified cells. The most common approach relies on bicistronic vectors in which the genes are linked to each other by an internal ribosome entry site allowing co-translational expression of both cistrons. Endostatin, the C-terminal fragment of collagen XVIII, is a potent angiogenesis inhibitor. At present, ES has been widely used in anti-angiogenic in a variety of experimental tumor models, and clinical trials to test it as an anti-tumor agent are already under way. Immunotherapy has been used as adjuvant treatment for tumors and has been used in several preclinical studies and clinical trials. The objective of this project was to construct and characterize 'in vitro' an IRES-based bicistronic retroviral vector encoding endostatin and interleukin-2. The construction of the vector was performed in three stages, the final construction was analyzed by restriction analysis and sequencing. Packaging cells were prepared. The endostatin and interleukin-2 levels were determined by Dot blot. Monocistronic and bicistronic mRNA expression were analyzed by real time RT-PCR. Bicistronic vector showed high levels of virus trites, ranging from 4.20x10 5 to 1.53x10 6 UFC/ml. Secreted levels of endostatin and interleukin-2 ranged from 1.08 to 2.08μg/10 6 cells.24h and 0.66 - 0.89μg/10 6 cells.24h, respectively. The mRNA expression of ES in the NIH3T3 clone pLend-IRES-IL2SN was 2 times higher than the level presented by the NIH3T3 clone pLendSN. The endostatin promoted inhibition (40%) of endothelial cell proliferation. Interleukin-2 promoted a proliferation of 10.6% lymphocytes CD4 and 8.9% of CD8. We conclude that the IRES bicistronic vector

  7. RNA-seq analyses of blood-induced changes in gene expression in the mosquito vector species, Aedes aegypti

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    Olson Ken E

    2011-01-01

    Full Text Available Abstract Background Hematophagy is a common trait of insect vectors of disease. Extensive genome-wide transcriptional changes occur in mosquitoes after blood meals, and these are related to digestive and reproductive processes, among others. Studies of these changes are expected to reveal molecular targets for novel vector control and pathogen transmission-blocking strategies. The mosquito Aedes aegypti (Diptera, Culicidae, a vector of Dengue viruses, Yellow Fever Virus (YFV and Chikungunya virus (CV, is the subject of this study to look at genome-wide changes in gene expression following a blood meal. Results Transcriptional changes that follow a blood meal in Ae. aegypti females were explored using RNA-seq technology. Over 30% of more than 18,000 investigated transcripts accumulate differentially in mosquitoes at five hours after a blood meal when compared to those fed only on sugar. Forty transcripts accumulate only in blood-fed mosquitoes. The list of regulated transcripts correlates with an enhancement of digestive activity and a suppression of environmental stimuli perception and innate immunity. The alignment of more than 65 million high-quality short reads to the Ae. aegypti reference genome permitted the refinement of the current annotation of transcript boundaries, as well as the discovery of novel transcripts, exons and splicing variants. Cis-regulatory elements (CRE and cis-regulatory modules (CRM enriched significantly at the 5'end flanking sequences of blood meal-regulated genes were identified. Conclusions This study provides the first global view of the changes in transcript accumulation elicited by a blood meal in Ae. aegypti females. This information permitted the identification of classes of potentially co-regulated genes and a description of biochemical and physiological events that occur immediately after blood feeding. The data presented here serve as a basis for novel vector control and pathogen transmission

  8. An episomal vector-based CRISPR/Cas9 system for highly efficient gene knockout in human pluripotent stem cells.

    Science.gov (United States)

    Xie, Yifang; Wang, Daqi; Lan, Feng; Wei, Gang; Ni, Ting; Chai, Renjie; Liu, Dong; Hu, Shijun; Li, Mingqing; Li, Dajin; Wang, Hongyan; Wang, Yongming

    2017-05-24

    Human pluripotent stem cells (hPSCs) represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases. CRISPR/Cas9 is a powerful tool to introduce genetic mutations into the hPSCs for loss-of-function studies. Here, we developed an episomal vector-based CRISPR/Cas9 system, which we called epiCRISPR, for highly efficient gene knockout in hPSCs. The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates. In addition, the epiCRISPR system enables efficient double-gene knockout and genomic deletion. To minimize off-target cleavage, we combined the episomal vector technology with double-nicking strategy and recent developed high fidelity Cas9. Thus the epiCRISPR system offers a highly efficient platform for genetic analysis in hPSCs.

  9. A potyvirus-based gene vector allows producing active human S-COMT and animal GFP, but not human sorcin, in vector-infected plants.

    Science.gov (United States)

    Kelloniemi, Jani; Mäkinen, Kristiina; Valkonen, Jari P T

    2006-05-01

    Potato virus A (PVA), a potyvirus with a (+)ssRNA genome translated to a large polyprotein, was engineered and used as a gene vector for expression of heterologous proteins in plants. Foreign genes including jellyfish GFP (Aequorea victoria) encoding the green fluorescent protein (GFP, 27 kDa) and the genes of human origin (Homo sapiens) encoding a soluble resistance-related calcium-binding protein (sorcin, 22 kDa) and the catechol-O-methyltransferase (S-COMT; 25 kDa) were cloned between the cistrons for the viral replicase and coat protein (CP). The inserts caused no adverse effects on viral infectivity and virulence, and the inserted sequences remained intact in progeny viruses in the systemically infected leaves. The heterologous proteins were released from the viral polyprotein following cleavage by the main viral proteinase, NIa, at engineered proteolytic processing sites flanking the insert. Active GFP, as indicated by green fluorescence, and S-COMT with high levels of enzymatic activity were produced. In contrast, no sorcin was detected despite the expected equimolar amounts of the foreign and viral proteins being expressed as a polyprotein. These data reveal inherent differences between heterologous proteins in their suitability for production in plants.

  10. DNA repair deficiency in neurodegeneration

    DEFF Research Database (Denmark)

    Jeppesen, Dennis Kjølhede; Bohr, Vilhelm A; Stevnsner, Tinna V.

    2011-01-01

    Deficiency in repair of nuclear and mitochondrial DNA damage has been linked to several neurodegenerative disorders. Many recent experimental results indicate that the post-mitotic neurons are particularly prone to accumulation of unrepaired DNA lesions potentially leading to progressive...... neurodegeneration. Nucleotide excision repair is the cellular pathway responsible for removing helix-distorting DNA damage and deficiency in such repair is found in a number of diseases with neurodegenerative phenotypes, including Xeroderma Pigmentosum and Cockayne syndrome. The main pathway for repairing oxidative...... base lesions is base excision repair, and such repair is crucial for neurons given their high rates of oxygen metabolism. Mismatch repair corrects base mispairs generated during replication and evidence indicates that oxidative DNA damage can cause this pathway to expand trinucleotide repeats, thereby...

  11. Unconventional neurotransmitters, neurodegeneration and neuroprotection

    Directory of Open Access Journals (Sweden)

    M. Leonelli

    2009-01-01

    Full Text Available Neurotransmitters are also involved in functions other than conventional signal transfer between nerve cells, such as development, plasticity, neurodegeneration, and neuroprotection. For example, there is a considerable amount of data indicating developmental roles for the glutamatergic, cholinergic, dopaminergic, GABA-ergic, and ATP/adenosine systems. In this review, we discuss the existing literature on these "new" functions of neurotransmitters in relation to some unconventional neurotransmitters, such as the endocannabinoids and nitric oxide. Data indicating both transcriptional and post-transcriptional modulation of endocannabinoid and nitrinergic systems after neural lesions are discussed in relation to the non-conventional roles of these neurotransmitters. Knowledge of the roles of neurotransmitters in brain functions other than information transfer is critical for a more complete understanding of the functional organization of the brain and to provide more opportunities for the development of therapeutical tools aimed at minimizing neuronal death.

  12. Cloning of cellulase genes using pUC18 and lambda 2001 vectors

    International Nuclear Information System (INIS)

    Bashir, A.; Ashfaq, S.R.; Rajoka, M.I.; Malik, K.A.; Batt, C.A.

    1991-01-01

    Chromosomal DNA from cellulomonas biazotea NIAB 442 was used for isolation and cloning of cellulase genes. For this purpose plasmid pUC18 was used for cloning fragments in the range of 109 Kb and phase vector lambda 2001 for cloning fragments in the range of 15-20 Kb respectively. Three restriction enzymes BamHI, Sau3AI and SaII were used for partial restriction of chromosomal DNA to obtain fragment size in the range of 0.5 - 20 Kb. BamHI and SaII were used to linearize pUC18 to obtain compatible ends against the three enzymes used in chromosomal DNA restriction. Linearized pUC18 was then ligated to respective compatible chromosomal DNA fragments and transformed to JM109 competent cells. A total of 6781 recombinants were tested for the production of B-glucosidase and carboxy methyl cellulase (CMC-ase) production. Only one of the recombinants was found to be positive for B-glucosidase production in solid culture. One of the recombinants was found positive for CMC-ase production in solid culture and is being verified and characterized. Larger DNA fragments in the range of 15-20 Kilobase were obtained by partial restriction of chromosomal DNA with BamHI, SaII and Xhol. Lambda 2001 was double digested with BamHI/EcoRI and Xhol/EcoRI for removal of stuffer fragment. Ligation of respective compatible ends was performed between Lambda DNA and chromosomal DNA. Ligation mixture was used for packaging and infection of P2 lysogen. No plaques could be obtained on P2 lysogen due to inefficient packaging. (author)

  13. The feasibility of using a baculovirus vector to deliver the sodium-iodide symporter gene as a reporter

    Energy Technology Data Exchange (ETDEWEB)

    Zhou Xiang; Li Biao; Wang Jun; Yin Hongyan [Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China); Zhang Yifan [Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025 (China)], E-mail: zhangyifan1992@yahoo.com.cn

    2010-04-15

    Purpose: To evaluate the efficiency of baculovirus vectors in transducing FTC-133 cells and to examine the feasibility of using baculovirus vectors for the delivery of the sodium-iodide symporter (NIS) gene as a reporter through co-transduction to monitor the expression of the target gene. Method: Two recombinant baculoviruses were constructed to express NIS and green fluorescent protein (GFP) respectively. FTC-133, 8050C, SW1116, A549 cells, were infected with Bac-GFP. The infection efficiency of Bac-GFP and the intensity of fluorescence, in either the presence or absence of sodium butyrate, were monitored by flow cytometry. The iodine uptake by FTC-133 cells infected with Bac-NIS was measured using a {gamma} counter. FTC-133 cells were infected with a mixture of equal amounts of Bac-NIS and Bac-GFP at different setting of multiplicity of infection (MOI). The changes of GFP fluorescence intensity and iodine uptake were monitored 24 h after infection in the coinfected cells. Results: We have successfully constructed recombinant baculoviruses carrying NIS and GFP under the control of the cytomegalovirus IE-1 promoter. We found that transduced efficiency of baculovirus in 8505C, SW1116, A549 cells are low in absence of sodium butyrate. Yet Bac-GFP infects FTC-133 cells at a high efficiency, 77.67%, 85.57% and 93.23% with MOI of 100, 200 and 400, respectively. The fluorescence intensity of the Bac-GFP infected tumor cells correlated positively with the MOI of the virus. Sodium butyrate induction increased both the infection efficiency and the fluorescence intensity, but increase of infection efficiency was insignificant in FTC-133 cells. Reporter gene (GFP) expression in FTC-133 is stable within 7 days after infection. The radioactivity incorporated by the tumor cells infected with Bac-NIS correlated positively with the MOI of Bac-NIS as well. In tumor cells co-infected with Bac-NIS and Bac-GFP, the amount of radioactivity incorporated significantly correlated with

  14. UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

    Directory of Open Access Journals (Sweden)

    Emanuela Chiarella

    Full Text Available Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6 where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in

  15. UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

    Science.gov (United States)

    Chiarella, Emanuela; Carrà, Giovanna; Scicchitano, Stefania; Codispoti, Bruna; Mega, Tiziana; Lupia, Michela; Pelaggi, Daniela; Marafioti, Maria G; Aloisio, Annamaria; Giordano, Marco; Nappo, Giovanna; Spoleti, Cristina B; Grillone, Teresa; Giovannone, Emilia D; Spina, Raffaella; Bernaudo, Francesca; Moore, Malcolm A S; Bond, Heather M; Mesuraca, Maria; Morrone, Giovanni

    2014-01-01

    Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter. However, while the product of the gene located upstream of the IRES is generally abundantly expressed, the translation of the downstream cDNA (typically encoding the reporter protein) is often inconsistent, which hinders the detection and the isolation of transduced cells. To overcome these limitations, we developed novel lentiviral dual-promoter vectors (named UMG-LV5 and -LV6) where transgene expression is driven by the potent UBC promoter and that of the reporter protein, EGFP, by the minimal regulatory element of the WASP gene. These vectors, harboring two distinct transgenes, were tested in a variety of human haematopoietic cell lines as well as in primary human CD34+ cells in comparison with the FUIGW vector that contains the expression cassette UBC-transgene-IRES-EGFP. In these experiments both UMG-LV5 and UMG-LV6 yielded moderately lower transgene expression than FUIGW, but dramatically higher levels of EGFP, thereby allowing the easy distinction between transduced and non-transduced cells. An additional construct was produced, in which the cDNA encoding the reporter protein is upstream, and the transgene downstream of the IRES sequence. This vector, named UMG-LV11, proved able to promote abundant expression of both transgene product and EGFP in all cells tested. The UMG-LVs represent therefore useful vectors for gene transfer-based studies in hematopoietic stem and

  16. Use of endophytic diazotrophic bacteria as a vector to express the cry3A gene from Bacillus thuringiensis

    Directory of Open Access Journals (Sweden)

    Salles Joana Falcão

    2000-01-01

    Full Text Available The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vector to express a cry gene from Bacillus thuringiensis, envisaging the control of pests that attack sugarcane plants. The endophytic nitrogen-fixing bacteria Gluconacetobacter diazotrophicus strain BR11281 and Herbaspirillum seropedicae strain BR11335 were used as models. The cry3A gene was transferred by conjugation using a suicide plasmid and the recombinant strains were selected by their ability to fix nitrogen in semi-solid N-free medium. The presence of the cry gene was detected by Southern-blot using an internal fragment of 1.0 kb as a probe. The production of delta-endotoxin by the recombinant H. seropedicae strain was detected by dot blot while for G. diazotrophicus the Western-blot technique was used. In both cases, a specific antibody raised against the B. thuringiensis toxin was applied. The delta-endotoxin production showed by the G. diazotrophicus recombinant strain was dependent on the nitrogen fixing conditions since the cry3A gene was fused to a nif promoter. In the case of H. seropedicae the delta-endotoxin expression was not affected by the promoter (rhi used. These results suggest that endophytic diazotrophic bacteria can be used as vectors to express entomopathogenic genes envisaging control of sugarcane pests.

  17. Herpes simplex virus vectors overexpressing the glucose transporter gene protect against seizure-induced neuron loss.

    OpenAIRE

    Lawrence, M S; Ho, D Y; Dash, R; Sapolsky, R M

    1995-01-01

    We have generated herpes simplex virus (HSV) vectors vIE1GT and v alpha 4GT bearing the GLUT-1 isoform of the rat brain glucose transporter (GT) under the control of the human cytomegalovirus ie1 and HSV alpha 4 promoters, respectively. We previously reported that such vectors enhance glucose uptake in hippocampal cultures and the hippocampus. In this study we demonstrate that such vectors can maintain neuronal metabolism and reduce the extent of neuron loss in cultures after a period of hypo...

  18. Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts.

    Science.gov (United States)

    Ding, Xin Shun; Schneider, William L; Chaluvadi, Srinivasa Rao; Mian, M A Rouf; Nelson, Richard S

    2006-11-01

    Virus-induced gene silencing (VIGS) is used to analyze gene function in dicotyledonous plants but less so in monocotyledonous plants (particularly rice and corn), partially due to the limited number of virus expression vectors available. Here, we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb. (tall fescue) that caused systemic mosaic symptoms on barley, rice, and a specific cultivar of maize (Va35) under greenhouse conditions. Through sequencing, the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca), and genetic determinants that controlled the systemic infection of rice were mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Coinoculation of RNAs 1 and 2 from F-BMV and RNA 3 from R-BMV expressing a portion of a plant gene to leaves of barley, rice, and maize plants resulted in visual silencing-like phenotypes. The visual phenotypes were correlated with decreased target host transcript levels in the corresponding leaves. The VIGS visual phenotype varied from maintained during silencing of actin 1 transcript expression to transient with incomplete penetration through affected tissue during silencing of phytoene desaturase expression. F-BMV RNA 3 was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector C-BMV(A/G) (C for chimeric) was shown to be useful for VIGS. These BMV vectors will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.

  19. Three-dimensional tumor spheroids for in vitro analysis of bacteria as gene delivery vectors in tumor therapy.

    Science.gov (United States)

    Osswald, Annika; Sun, Zhongke; Grimm, Verena; Ampem, Grace; Riegel, Karin; Westendorf, Astrid M; Sommergruber, Wolfgang; Otte, Kerstin; Dürre, Peter; Riedel, Christian U

    2015-12-12

    Several studies in animal models demonstrated that obligate and facultative anaerobic bacteria of the genera Bifidobacterium, Salmonella, or Clostridium specifically colonize solid tumors. Consequently, these and other bacteria are discussed as live vectors to deliver therapeutic genes to inhibit tumor growth. Therapeutic approaches for cancer treatment using anaerobic bacteria have been investigated in different mouse models. In the present study, solid three-dimensional (3D) multicellular tumor spheroids (MCTS) of the colorectal adenocarcinoma cell line HT-29 were generated and tested for their potential to study prodrug-converting enzyme therapies using bacterial vectors in vitro. HT-29 MCTS resembled solid tumors displaying all relevant features with an outer zone of proliferating cells and hypoxic and apoptotic regions in the core. Upon incubation with HT-29 MCTS, Bifidobacterium bifidum S17 and Salmonella typhimurium YB1 selectively localized, survived and replicated in hypoxic areas inside MCTS. Furthermore, spores of the obligate anaerobe Clostridium sporogenes germinated in these hypoxic areas. To further evaluate the potential of MCTS to investigate therapeutic approaches using bacteria as gene delivery vectors, recombinant bifidobacteria expressing prodrug-converting enzymes were used. Expression of a secreted cytosine deaminase in combination with 5-fluorocytosine had no effect on growth of MCTS due to an intrinsic resistance of HT-29 cells to 5-fluorouracil, i.e. the converted drug. However, a combination of the prodrug CB1954 and a strain expressing a secreted chromate reductase effectively inhibited MCTS growth. Collectively, the presented results indicate that MCTS are a suitable and reliable model to investigate live bacteria as gene delivery vectors for cancer therapy in vitro.

  20. An efficient marker-free vector for clean gene transfer into plants

    African Journals Online (AJOL)

    Yomi

    2012-02-23

    Feb 23, 2012 ... vector sequences inserted into plant genomes. Transformation ..... Thymosin Alpha 1 (Tα1) in Marker-free Transgenic Lettuce (Lactuca sativa). ... obtaining marker-free plants of a cross-pollinating and vegetatively propagated ...

  1. Molecular pathways underpinning ethanol-induced neurodegeneration

    Directory of Open Access Journals (Sweden)

    Dan eGoldowitz*

    2014-07-01

    Full Text Available While genetics impacts the type and severity of damage following developmental ethanol exposure, little is currently known about the molecular pathways that mediate these effects. Traditionally, research in this area has used a candidate gene approach and evaluated effects on a gene-by-gene basis. Recent studies, however, have begun to use unbiased approaches and genetic reference populations to evaluate the roles of genotype and epigenetic modifications in phenotypic changes following developmental ethanol exposure, similar to studies that evaluated numerous alcohol-related phenotypes in adults. Here, we present work assessing the role of genetics and chromatin-based alterations in mediating ethanol-induced apoptosis in the developing nervous system. Utilizing the expanded family of BXD recombinant inbred mice, animals were exposed to ethanol at postnatal day 7 via subcutaneous injection (5.0 g/kg in 2 doses. Tissue was collected 7 hours after the initial ethanol treatment and analyzed by activated caspase-3 immunostaining to visualize dying cells in the cerebral cortex and hippocampus. In parallel, the levels of two histone modifications relevant to apoptosis, γH2AX and H3K14 acetylation, were examined in the cerebral cortex using protein blot analysis. Activated caspase-3 staining identified marked differences in cell death across brain regions between different mouse strains. Genetic analysis of ethanol susceptibility in the hippocampus led to the identification of a quantitative trait locus on chromosome 12, which mediates, at least in part, strain-specific differential vulnerability to ethanol-induced apoptosis. Furthermore, analysis of chromatin modifications in the cerebral cortex revealed a global increase in γH2AX levels following ethanol exposure, but did not show any change in H3K14 acetylation levels. Together, these findings provide new insights into the molecular mechanisms and genetic contributions underlying ethanol

  2. Methylenedioxymethamphetamine (MDMA, 'Ecstasy': Neurodegeneration versus Neuromodulation

    Directory of Open Access Journals (Sweden)

    Elena Puerta

    2011-07-01

    Full Text Available The amphetamine analogue 3,4-methylenedioxymethamphetamine (MDMA, ‘ecstasy’ is widely abused as a recreational drug due to its unique psychological effects. Of interest, MDMA causes long-lasting deficits in neurochemical and histological markers of the serotonergic neurons in the brain of different animal species. Such deficits include the decline in the activity of tryptophan hydroxylase in parallel with the loss of 5-HT and its main metabolite 5-hydoxyindoleacetic acid (5-HIAA along with a lower binding of specific ligands to the 5-HT transporters (SERT. Of concern, reduced 5-HIAA levels in the CSF and SERT density have also been reported in human ecstasy users, what has been interpreted to reflect the loss of serotonergic fibers and terminals. The neurotoxic potential of MDMA has been questioned in recent years based on studies that failed to show the loss of the SERT protein by western blot or the lack of reactive astrogliosis after MDMA exposure. In addition, MDMA produces a long-lasting down-regulation of SERT gene expression; which, on the whole, has been used to invoke neuromodulatory mechanisms as an explanation to MDMA-induced 5-HT deficits. While decreased protein levels do not necessarily reflect neurodegeneration, the opposite is also true, that is, neuroregulatory mechanisms do not preclude the existence of 5-HT terminal degeneration.

  3. Construction and expression of eukaryotic expression vectors of full-length, amino-terminus and carboxyl-terminus Raf gene

    Directory of Open Access Journals (Sweden)

    Zhuomin WANG

    2008-06-01

    Full Text Available Background and objective Raf is a key molecule in the Ras-Raf-MEK-ERK signal transduction pathway and is highly activated in different human carcinomas. However, its biological functions and regulation mechanisms are still unclear. The aims of this study were to construct eukaryotic expression vectors with Raf full encoding region, truncated amino-terminus and carboxyl-terminus, respectively. Methods Eukaryotic expression vectors of pCMV-Tag2b-Raf-1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-C-Raf were constructed by gene recombination technique and confirmed by restriction enzyme analysis and DNA sequencing. Furthermore, the expression of these fusion proteins was detected by western blot in transient transfected 293T cells. Results The sequences and open reading frames of these three vectors were completely consistent with experimental design. All target proteins can be detected in 293T cells. Conclusion Eukaryotic expression vectors of pCMV-Tag2b-Raf-1, pCMV-Tag2b-N-Raf and pCMV-Tag2b-C-Raf were successfully constructed and can be expressed in 293T cells.

  4. A MultiSite GatewayTM vector set for the functional analysis of genes in the model Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Nagels Durand Astrid

    2012-09-01

    Full Text Available Abstract Background Recombinatorial cloning using the GatewayTM technology has been the method of choice for high-throughput omics projects, resulting in the availability of entire ORFeomes in GatewayTM compatible vectors. The MultiSite GatewayTM system allows combining multiple genetic fragments such as promoter, ORF and epitope tag in one single reaction. To date, this technology has not been accessible in the yeast Saccharomyces cerevisiae, one of the most widely used experimental systems in molecular biology, due to the lack of appropriate destination vectors. Results Here, we present a set of three-fragment MultiSite GatewayTM destination vectors that have been developed for gene expression in S. cerevisiae and that allow the assembly of any promoter, open reading frame, epitope tag arrangement in combination with any of four auxotrophic markers and three distinct replication mechanisms. As an example of its applicability, we used yeast three-hybrid to provide evidence for the assembly of a ternary complex of plant proteins involved in jasmonate signalling and consisting of the JAZ, NINJA and TOPLESS proteins. Conclusion Our vectors make MultiSite GatewayTM cloning accessible in S. cerevisiae and implement a fast and versatile cloning method for the high-throughput functional analysis of (heterologous proteins in one of the most widely used model organisms for molecular biology research.

  5. [Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

    Science.gov (United States)

    Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

    2008-02-01

    To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

  6. Mechanism of Neurodegeneration Following Viral Infection

    National Research Council Canada - National Science Library

    Maheshwari, Radha

    2001-01-01

    The long term goal of this proposal is to delineate the mechanism(s) for neurodegeneration and neuropathogenesis following infection with a neurovirulent virus, Venezuelan equine encephalitis virus (VEE...

  7. BINARY VECTOR CONSTRUCTION OF KAPPA(κ-CARRAGEENASE GENE AND TRANSFORMATION TO Agrobacterium tumefaciens AS MEDIATOR FOR SEAWEED TRANSGENIC GENERATION

    Directory of Open Access Journals (Sweden)

    Muh Alias L. Rajamuddin

    2016-11-01

    Full Text Available Increasing of kappa (κ-carrageenan content in Kappaphycus alvarezii seaweed is potentially be achieved by applying transgenesis technology. This study was performed to obtain a construction of  κ-Carrageenase gene and Agrobacterium tumefaciens to carry those construction genes.  The κ-Carrageenase (κ-Car gene was involved in κ-carrageenan biosynthesis. The κ-Car gene sequence was ligated between the 35S CaMV promoter and tNos terminator sequences to generate pMSH/κ-Car expression vector. Transformation of pMSH/κ-Car plasmid to Escherichia coli was performed by heat-shock method, and to Agrobacterium tumefaciens by tri-parental mating method. The results showed that several colonies of E. coli and A. tumefaciens grew in the selective culture mediums containing antibiotic. PCR analysis using primers 35S-Forward and tNos-Reverse with DNA template from those bacterial colonies resulted DNA fragment of about 2,000 bp, the same as the total length of 35S CaMV promoter, κ-Car gene and tNos terminator sequences. Therefore, the construction of pMSH/κ-Car gene was succeeded and a colony of A. tumefaciens transformant carrying pMSH/κ-Car plasmid was successfully produced.                                                                                   Keywords:  Agrobacterium tumefaciens, kappa(κ-Carrageenase gene, transgenesis, vector

  8. A molecular toolbox for rapid generation of viral vectors to up- or down-regulate in vivo neuronal gene expression

    Directory of Open Access Journals (Sweden)

    Melanie D. White

    2011-07-01

    Full Text Available We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1 and Kir3.2 and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miR. We show that AAV assembled to express HCN1 miR produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miR with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience.

  9. Synthesis and optimization of cholesterol-based diquaternary ammonium Gemini Surfactant (Chol-GS) as a new gene delivery vector.

    Science.gov (United States)

    Kim, Bieong-Kil; Doh, Kyung-Oh; Bae, Yun-Ui; Seu, Young-Bae

    2011-01-01

    Amongst a number of potential nonviral vectors, cationic liposomes have been actively researched, with both gemini surfactants and bola amphiphiles reported as being in possession of good structures in terms of cell viability and in vitro transfection. In this study, a cholesterol-based diquaternary ammonium gemini surfactant (Chol-GS) was synthesized and assessed as a novel nonviral gene vector. Chol-GS was synthesized from cholesterol by way of four reaction steps. The optimal efficiency was found to be at a weight ratio of 1:4 of lipid:DOPE (1,2-dioleoyl-L-alpha- glycero-3-phosphatidylethanolamine), and at a ratio of between 10:1~15:1 of liposome:DNA. The transfection efficiency was compared with commercial liposomes and with Lipofectamine, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE-C), and N-[1-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTAP). The results indicate that the efficiency of Chol-GS is greater than that of all the tested commercial liposomes in COS7 and Huh7 cells, and higher than DOTAP and Lipofectamine in A549 cells. Confirmation of these findings was observed through the use of green fluorescent protein expression. Chol-GS exhibited a moderate level of cytotoxicity, at optimum concentrations for efficient transfection, indicating cell viability. Hence, the newly synthesized Chol-GS liposome has the potential of being an excellent nonviral vector for gene delivery.

  10. Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

    Science.gov (United States)

    White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

    2008-12-01

    Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

  11. Gene therapy for adenosine deaminase-deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans.

    Science.gov (United States)

    Candotti, Fabio; Shaw, Kit L; Muul, Linda; Carbonaro, Denise; Sokolic, Robert; Choi, Christopher; Schurman, Shepherd H; Garabedian, Elizabeth; Kesserwan, Chimene; Jagadeesh, G Jayashree; Fu, Pei-Yu; Gschweng, Eric; Cooper, Aaron; Tisdale, John F; Weinberg, Kenneth I; Crooks, Gay M; Kapoor, Neena; Shah, Ami; Abdel-Azim, Hisham; Yu, Xiao-Jin; Smogorzewska, Monika; Wayne, Alan S; Rosenblatt, Howard M; Davis, Carla M; Hanson, Celine; Rishi, Radha G; Wang, Xiaoyan; Gjertson, David; Yang, Otto O; Balamurugan, Arumugam; Bauer, Gerhard; Ireland, Joanna A; Engel, Barbara C; Podsakoff, Gregory M; Hershfield, Michael S; Blaese, R Michael; Parkman, Robertson; Kohn, Donald B

    2012-11-01

    We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34(+) cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m(2)). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

  12. Gene therapy for adenosine deaminase–deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans

    Science.gov (United States)

    Candotti, Fabio; Shaw, Kit L.; Muul, Linda; Carbonaro, Denise; Sokolic, Robert; Choi, Christopher; Schurman, Shepherd H.; Garabedian, Elizabeth; Kesserwan, Chimene; Jagadeesh, G. Jayashree; Fu, Pei-Yu; Gschweng, Eric; Cooper, Aaron; Tisdale, John F.; Weinberg, Kenneth I.; Crooks, Gay M.; Kapoor, Neena; Shah, Ami; Abdel-Azim, Hisham; Yu, Xiao-Jin; Smogorzewska, Monika; Wayne, Alan S.; Rosenblatt, Howard M.; Davis, Carla M.; Hanson, Celine; Rishi, Radha G.; Wang, Xiaoyan; Gjertson, David; Yang, Otto O.; Balamurugan, Arumugam; Bauer, Gerhard; Ireland, Joanna A.; Engel, Barbara C.; Podsakoff, Gregory M.; Hershfield, Michael S.; Blaese, R. Michael; Parkman, Robertson

    2012-01-01

    We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)–deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34+ cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m2). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency. PMID:22968453

  13. AAV vectors as gene delivery vehicles in the central nervous system

    NARCIS (Netherlands)

    Broekman, M.L.D.

    2006-01-01

    Recombinant gene delivery vehicles based on the replication-defective AAV have gained a preeminent position in the field of gene delivery to the brain. Efficient global gene delivery to the CNS is beneficial for the study of gene products is the entire CNS as well as for introducing and expressing

  14. Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters.

    Science.gov (United States)

    Ferreira, Joshua P; Peacock, Ryan W S; Lawhorn, Ingrid E B; Wang, Clifford L

    2011-12-01

    The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines. The online version of this article (doi:10.1007/s11693-011-9089-0) contains supplementary material, which is available to authorized users.

  15. Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector

    International Nuclear Information System (INIS)

    Watanabe, Daisuke; Brockman, Mark A.; Ndung'u, Thumbi; Mathews, Lydia; Lucas, William T.; Murphy, Cynthia G.; Felber, Barbara K.; Pavlakis, George N.; Deluca, Neal A.; Knipe, David M.

    2007-01-01

    Herpes simplex virus (HSV) recombinants induce durable immune responses in rhesus macaques and mice and have induced partial protection in rhesus macaques against mucosal challenge with virulent simian immunodeficiency virus (SIV). In this study, we evaluated the properties of a new generation HSV vaccine vector, an HSV-1 multiple immediate-early (IE) gene deletion mutant virus, d106, which contains deletions in the ICP4, ICP27, ICP22, and ICP47 genes. Because several of the HSV IE genes have been implicated in immune evasion, inactivation of the genes encoding these proteins was expected to result in enhanced immunogenicity. The d106 virus expresses few HSV gene products and shows minimal cytopathic effect in cultured cells. When d106 was inoculated into mice, viral DNA accumulated at high levels in draining lymph nodes, consistent with an ability to transduce dendritic cells and activate their maturation and movement to lymph nodes. A d106 recombinant expressing Escherichia coli β-galactosidase induced durable β-gal-specific IgG and CD8 + T cell responses in naive and HSV-immune mice. Finally, d106-based recombinants have been constructed that express simian immunodeficiency virus (SIV) gag, env, or a rev-tat-nef fusion protein for several days in cultured cells. Thus, d106 shows many of the properties desirable in a vaccine vector: limited expression of HSV gene products and cytopathogenicity, high level expression of transgenes, ability to induce durable immune responses, and an ability to transduce dendritic cells and induce their maturation and migration to lymph nodes

  16. Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application.

    Science.gov (United States)

    Merten, Otto-Wilhelm; Charrier, Sabine; Laroudie, Nicolas; Fauchille, Sylvain; Dugué, Céline; Jenny, Christine; Audit, Muriel; Zanta-Boussif, Maria-Antonietta; Chautard, Hélène; Radrizzani, Marina; Vallanti, Giuliana; Naldini, Luigi; Noguiez-Hellin, Patricia; Galy, Anne

    2011-03-01

    From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.

  17. Design and Construction of a Cloning Vector Containing the hspX Gene of Mycobacterium tuberculosis.

    Science.gov (United States)

    Yaghoubi, Atieh; Aryan, Ehsan; Zare, Hosna; Alami, Shadi; Teimourpour, Roghayeh; Meshkat, Zahra

    2016-10-01

    Tuberculosis (TB) is a major cause of death worldwide. Finding an effective vaccine against TB is the best way to control it. Several vaccines against this disease have been developed but none are completely protective. The aim of this study was to design and construct a cloning vector containing the Mycobacterium tuberculosis (M. tuberculosis) heat shock protein X (hspX) . First, an hspX fragment was amplified by PCR and cloned into plasmid pcDNA3.1(+) and recombinant vector was confirmed. A 435 bp hspX fragment was isolated. The fragment was 100% homologous with hspX of M. tuberculosis strain H37Rv in GenBank. In this study, the cloning vector pcDNA3.1(+), containing a 435-bp hspX fragment of M. tuberculosis , was constructed. This could be used as a DNA vaccine to induce immune responses in animal models in future studies.

  18. Lentiviral vectors in neurodegenrative disorders - Aspects in gene therapy and disease models

    DEFF Research Database (Denmark)

    Nielsen, Troels Tolstrup

    2009-01-01

    Neurodegenerative disorders remain a complex group of diseases (i.e. Huntington's disease, HD) that are characterized by progressive loss of neurons resulting in movement disorders, cognitive decline, dementia and death. There is no cure for these diseases and treatment relies on symptomatic relief...... expression and escape transgene silencing during differentiation of neural stem cell lines. However, insulator vectors appeared to be impaired in functionality, which has importance for the future use of insulators in viral vectors. Finally, cell based models of HD was constructed to elucidate...

  19. Construction and expression of secreting type human TRAIL gene vector mediated by hypoxia/radiation double sensitive promoter

    International Nuclear Information System (INIS)

    Yang Yanming; Jia Xiaojing; Qu Yaqin; Li Yanbo

    2009-01-01

    Objective: To construct secreting type human TRAIL (shTRAIL) gene vector pcDNA3.1-HRE/Egr1-shTRAIL mediated by hypoxia/radiation double sensitive promoter, and observe the effect of hypoxia and radiation on shTRAIL. Methods: HRE upper and lower strands were gotten by chemical synthesis, double strands HRE was gotten by PCR; pMD19T-Egr1 was digested by Sac I and Hind III, then Egr1 was obtained, pshuttle-shTRAIL was digested by Kpn I and BamH I, then shTRAIL was obtained; HRE/Egr1 double sensitive promoter mediated shTRAIL expression vector pcDNA3.1-HRE/Egr1-shTRAIL was constructed by gene recombination technique, it was identified correctly by enzyme digestion, PCR and sequencing. A549 cells were divided into normal, hypoxia (0.1%), irradiation (6 Gy) and hypoxia + irradiation groups. Results: After enzyme digestion by BamH I and Sma I, the fragments which lengths were 1284 bp and 4 998 bp, 2 292 bp and 3 990 bp were obtained; the vector was amplified by PCR with Egr1 and shTRAIL primer, the products which lengthens were 469 bp and 820 bp were obtained; pcDNA3.1-HRE/Egr1-shTRAIL was sequenced, the result was same to designed, this demonstrated that the construction was right. The vectors were transfected into A549 cells of adenocarcinoma of lung, the expression levels of shTRAIL mRNA and protein were increased after treated with hypoxia and radiation, it had statistically significant differences compared with normal group (P<0.05), and when they were combinated, the effect was more obvious. Conclusion: Secreting type human TRAIL gene vector pcDNA3.1-HRE/Egr1-shTRAIL mediated by hypoxia/radiation double sensitive promoter is constructed successfully, and hypoxia and radiation could increase the expression of TRAIL, and they have synergetic effect. (authors)

  20. Construction of a Shuttle Vector for Heterologous Expression of a Novel Fungal α-Amylase Gene in Aspergillus oryzae.

    Science.gov (United States)

    Yin, Yanchen; Mao, Youzhi; Yin, Xiaolie; Gao, Bei; Wei, Dongzhi

    2015-07-01

    The filamentous fungus Aspergillus oryzae is a well-known expression host used to express homologous and heterologous proteins in a number of industrial applications. To facilitate higher yields of proteins of interest, we constructed the pAsOP vector to express heterologous proteins in A. oryzae. pAsOP carries a selectable marker, pyrG, derived from Aspergillus nidulans, and a strong promoter and a terminator of the amyB gene derived from A. oryzae. pAsOP transformed A. oryzae efficiently via the PEG-CaCl2-mediated transformation method. As proof of concept, green fluorescent protein (GFP) was successfully expressed in A. oryzae transformed by pAsOP-GFP. Additionally, we identified a novel fungal α-amylase (PcAmy) gene from Penicillium sp. and cloned the gene into the vector. After transformation by pAsOPPcAmy, the α-amylase PcAmy from Penicillium sp. was successfully expressed in a heterologous host system for the first time. The α-amylase activity in the A. oryzae transformant was increased by 62.3% compared with the untransformed A. oryzae control. The PcAmy protein produced in the system had an optimum pH of 5.0 and optimum temperature of 30°C. As a cold-adapted enzyme, PcAmy shows potential value in industrial applications because of its high catalytic activity at low temperature. Furthermore, the expression vector reported in this study provides promising utility for further scientific research and biotechnological applications.

  1. Multimodality imaging of reporter gene expression using a novel fusion vector in living cells and animals

    Science.gov (United States)

    Gambhir, Sanjiv [Portola Valley, CA; Pritha, Ray [Mountain View, CA

    2011-06-07

    Novel double and triple fusion reporter gene constructs harboring distinct imagable reporter genes are provided, as well as applications for the use of such double and triple fusion constructs in living cells and in living animals using distinct imaging technologies.

  2. An efficient deletion mutant packaging system for defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology

    International Nuclear Information System (INIS)

    Geller, A.I.; Keyomarsi, K.; Bryan, J.; Pardee, A.B.

    1990-01-01

    The authors have previously described a defective herpes simplex virus (HSV-1) vector system that permits that introduction of virtually any gene into nonmitotic cells. pHSVlac, the prototype vector, stably expresses Escherichia coli β-galactosidase from a constitutive promoter in many human cell lines, in cultured rat neurons from throughout the nervous system, and in cells in the adult rat brain. HSV-1 vectors expressing other genes may prove useful for studying neuronal physiology or performing human gene therapy for neurological diseases, such as Parkinson disease or brain tumors. A HSV-1 temperature-sensitive (ts) mutant, ts K, has been used as helper virus; ts mutants revert to wild type. In contrast, HSV-1 deletion mutants essentially cannot revert to wild type; therefore, use of a deletion mutant as helper virus might permit human gene therapy with HSV-1 vectors. They now report an efficient packaging system for HSV-1 VECTORS USING A DELETION MUTANT, d30EBA, as helper virus; virus is grown on the complementing cell line M64A. pHSVlac virus prepared using the deletion mutant packaging system stably expresses β-galactosidase in cultured rat sympathetic neurons and glia. Both D30EBA and ts K contain a mutation in the IE3 gene of HSV-1 strain 17 and have the same phenotype; therefore, changing the helper virus from ts K to D30EBA does not alter the host range or other properties of the HSV-1 vector system

  3. CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors

    Directory of Open Access Journals (Sweden)

    Rasmus O. Bak

    2017-07-01

    Full Text Available The CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno-associated viral (AAV vectors to serve as donor template DNA during homologous recombination (HR. However, the maximum AAV packaging capacity of ∼4.5 kb limits the donor size. Here, we overcome this constraint by showing that two co-transduced AAV vectors can serve as donors during consecutive HR events for the integration of large transgenes. Importantly, the method involves a single-step procedure applicable to primary cells with relevance to therapeutic genome editing. We use the methodology in primary human T cells and CD34+ hematopoietic stem and progenitor cells to site-specifically integrate an expression cassette that, as a single donor vector, would otherwise amount to a total of 6.5 kb. This approach now provides an efficient way to integrate large transgene cassettes into the genomes of primary human cells using HR-mediated genome editing with AAV vectors.

  4. The sweet potato IbMYB1 gene as a potential visible marker for sweet potato intragenic vector system.

    Science.gov (United States)

    Kim, Cha Young; Ahn, Young Ock; Kim, Sun Ha; Kim, Yun-Hee; Lee, Haeng-Soon; Catanach, Andrew S; Jacobs, Jeanne M E; Conner, Anthony J; Kwak, Sang-Soo

    2010-07-01

    MYB transcription factors play important roles in transcriptional regulation of many secondary metabolites including anthocyanins. We cloned the R2R3-MYB type IbMYB1 complementary DNAs from the purple-fleshed sweet potato (Ipomoea batatas L. cv Sinzami) and investigated the expression patterns of IbMYB1 gene with IbMYB1a and IbMYB1b splice variants in leaf and root tissues of various sweet potato cultivars by reverse transcription-polymerase chain reaction. The transcripts of IbMYB1 were predominantly expressed in the purple-fleshed storage roots and they were also detectable in the leaf tissues accumulating anthocyanin pigments. In addition, transcript levels of IbMYB1 gene were up-regulated by treatment with methyl jasmonate or salicylic acid in leaf and root tissues of cv. White Star. To set up the intragenic vector system in sweet potato, we first evaluated the utilization of the IbMYB1 gene as a visible selectable marker. The IbMYB1a was transiently expressed in tobacco leaves under the control of a constitutive cauliflower mosaic virus 35S promoter, a root-specific and sucrose-inducible sporamin promoter, and an oxidative stress-inducible sweet potato anionic peroxidase2 promoter. We also showed that overexpression of IbMYB1a induced massive anthocyanin pigmentation in tobacco leaves and up-regulated the transcript levels of the structural genes in anthocyanin biosynthetic pathway. Furthermore, high-performance liquid chromatography analysis revealed that the expression of IbMYB1a led to production of cyanidin as a major core molecule of anthocyanidins in tobacco leaves. These results suggest that the IbMYB1 gene can be applicable to a visible marker for sweet potato transformation with intragenic vectors, as well as the production of anthocyanin as important nutritive value in other plant species.

  5. Phenotyping of VIGS-mediated gene silencing in rice using a vector derived from a DNA virus.

    Science.gov (United States)

    Kant, Ravi; Dasgupta, Indranil

    2017-07-01

    Target genes in rice can be optimally silenced if inserted in antisense or hairpin orientation in the RTBV-derived VIGS vector and plants grown at 28 °C and 80% humidity after inoculation. Virus induced gene silencing (VIGS) is a method used to transiently silence genes in dicot as well as monocot plants. For the important monocot species rice, the Rice tungro bacilliform virus (RTBV)-derived VIGS system (RTBV-VIGS), which uses agroinoculation to initiate silencing, has not been standardized for optimal use. Here, using RTBV-VIGS, three sets of conditions were tested to achieve optimal silencing of the rice marker gene phytoene desaturase (pds). The effect of orientation of the insert in the RTBV-VIGS plasmid (sense, antisense and hairpin) on the silencing of the target gene was then evaluated using rice magnesium chelatase subunit H (chlH). Finally, the rice Xa21 gene, conferring resistance against bacterial leaf blight disease (BLB) was silenced using RTBV-VIGS system. In each case, real-time PCR-based assessment indicated approximately 40-80% fall in the accumulation levels of the transcripts of pds, chlH and Xa21. In the case of pds, the appearance of white streaks in the emerging leaves, and for chlH, chlorophyll levels and F v /F m ratio were assessed as phenotypes for silencing. For Xa21, the resistance levels to BLB were assessed by measuring the lesion length and the percent diseased areas of leaves, following challenge inoculation with Xanthomonas oryzae. In each case, the RTBV-MVIGS system gave rise to a discernible phenotype indicating the silencing of the respective target gene using condition III (temperature 28 °C, humidity 80% and 1 mM MES and 20 µM acetosyringone in secondary agrobacterium culture), which revealed the robustness of this gene silencing system for rice.

  6. Novel adeno-associated viral vector delivering the utrophin gene regulator jazz counteracts dystrophic pathology in mdx mice.

    Science.gov (United States)

    Strimpakos, Georgios; Corbi, Nicoletta; Pisani, Cinzia; Di Certo, Maria Grazia; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Gabanella, Francesca; Monaco, Lucia; Mattei, Elisabetta; Passananti, Claudio

    2014-09-01

    Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor "Jazz" that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD. © 2014 Wiley Periodicals, Inc.

  7. The human ankyrin 1 promoter insulator sustains gene expression in a β-globin lentiviral vector in hematopoietic stem cells

    Directory of Open Access Journals (Sweden)

    Zulema Romero

    Full Text Available Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies.

  8. Targeted Gene Knock Out Using Nuclease-Assisted Vector Integration: Hemi- and Homozygous Deletion of JAG1.

    Science.gov (United States)

    Gapinske, Michael; Tague, Nathan; Winter, Jackson; Underhill, Gregory H; Perez-Pinera, Pablo

    2018-01-01

    Gene editing technologies are revolutionizing fields such as biomedicine and biotechnology by providing a simple means to manipulate the genetic makeup of essentially any organism. Gene editing tools function by introducing double-stranded breaks at targeted sites within the genome, which the host cells repair preferentially by Non-Homologous End Joining. While the technologies to introduce double-stranded breaks have been extensively optimized, this progress has not been matched by the development of methods to integrate heterologous DNA at the target sites or techniques to detect and isolate cells that harbor the desired modification. We present here a technique for rapid introduction of vectors at target sites in the genome that enables efficient isolation of successfully edited cells.

  9. Population structure of the malaria vector Anopheles sinensis (Diptera: Culicidae in China: two gene pools inferred by microsatellites.

    Directory of Open Access Journals (Sweden)

    Yajun Ma

    Full Text Available BACKGROUND: Anopheles sinensis is a competent malaria vector in China. An understanding of vector population structure is important to the vector-based malaria control programs. However, there is no adequate data of A. sinensis population genetics available yet. METHODOLOGY/PRINCIPAL FINDINGS: This study used 5 microsatellite loci to estimate population genetic diversity, genetic differentiation and demographic history of A. sinensis from 14 representative localities in China. All 5 microsatellite loci were highly polymorphic across populations, with high allelic richness and heterozygosity. Hardy-Weinberg disequilibrium was found in 12 populations associated with heterozygote deficits, which was likely caused by the presence of null allele and the Wahlund effect. Bayesian clustering analysis revealed two gene pools, grouping samples into two population clusters; one includes six and the other includes eight populations. Out of 14 samples, six samples were mixed with individuals from both gene pools, indicating the coexistence of two genetic units in the areas sampled. The overall differentiation between two genetic pools was moderate (F(ST = 0.156. Pairwise differentiation between populations were lower within clusters (F(ST = 0.008-0.028 in cluster I and F(ST = 0.004-0.048 in cluster II than between clusters (F(ST = 0.120-0.201. A reduced gene flow (Nm = 1-1.7 was detected between clusters. No evidence of isolation by distance was detected among populations neither within nor between the two clusters. There are differences in effective population size (Ne = 14.3-infinite across sampled populations. CONCLUSIONS/SIGNIFICANCE: Two genetic pools with moderate genetic differentiation were identified in the A. sinensis populations in China. The population divergence was not correlated with geographic distance or barrier in the range. Variable effective population size and other demographic effects of historical population

  10. Recombinant Newcastle disease virus (NDV) with inserted gene coding for GM-CSF as a new vector for cancer immunogene therapy

    NARCIS (Netherlands)

    Janke, M.; Peeters, B.P.H.; Leeuw, de O.S.; Moormann, R.J.M.; Arnold, A.; Fournier, P.; Schirrmacher, V.

    2007-01-01

    This is the first report describing recombinant (rec) Newcastle disease virus (NDV) as vector for gene therapy of cancer. The gene encoding granulocyte/macrophage colony-stimulating factor (GM-CSF) was inserted as an additional transcription unit at two different positions into the NDV genome. The

  11. Metallothionein prevents neurodegeneration and central nervous system cell death after treatment with gliotoxin 6-aminonicotinamide

    DEFF Research Database (Denmark)

    Penkowa, Milena; Quintana, Albert; Carrasco, Javier

    2004-01-01

    Transgenic expression of interleukin-6 (IL-6) in the CNS under the control of the glial fibrillary acidic protein (GFAP) gene promoter (GFAP-IL6 mice) induces significant inflammation and neurodegeneration but also affords neuroprotection against acute traumatic brain injury. This neuroprotection...

  12. Neuronal matrix metalloproteinase-9 is a determinant of selective neurodegeneration.

    Science.gov (United States)

    Kaplan, Artem; Spiller, Krista J; Towne, Christopher; Kanning, Kevin C; Choe, Ginn T; Geber, Adam; Akay, Turgay; Aebischer, Patrick; Henderson, Christopher E

    2014-01-22

    Selective neuronal loss is the hallmark of neurodegenerative diseases. In patients with amyotrophic lateral sclerosis (ALS), most motor neurons die but those innervating extraocular, pelvic sphincter, and slow limb muscles exhibit selective resistance. We identified 18 genes that show >10-fold differential expression between resistant and vulnerable motor neurons. One of these, matrix metalloproteinase-9 (MMP-9), is expressed only by fast motor neurons, which are selectively vulnerable. In ALS model mice expressing mutant superoxide dismutase (SOD1), reduction of MMP-9 function using gene ablation, viral gene therapy, or pharmacological inhibition significantly delayed muscle denervation. In the presence of mutant SOD1, MMP-9 expressed by fast motor neurons themselves enhances activation of ER stress and is sufficient to trigger axonal die-back. These findings define MMP-9 as a candidate therapeutic target for ALS. The molecular basis of neuronal diversity thus provides significant insights into mechanisms of selective vulnerability to neurodegeneration. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. The use of Listeria monocytogenes as a DNA delivery vector for cancer gene therapy.

    LENUS (Irish Health Repository)

    Tangney, Mark

    2012-01-31

    Listeria monocytogenes is an intracellular pathogen that lyses the phagosomal vacuole of infected cells, proliferates in the host cell cytoplasm and can actively enter adjacent cells. The pathogen is therefore well suited to exploitation as a vector for the delivery of DNA to target cells as the lifecycle favors cellular targeting with vector amplification and the potential for cell-to-cell spread. We have recently demonstrated DNA transfer by L. monocytogenes in growing tumors in murine models. Our approach exploited an ampicillin sensitive stain of L. monocytogenes which can be lysed through systemic administration of ampicillin to facilitate release of plasmid DNA for expression by infected mammalian cells. Here, we discuss the implications of this technology and the potential for future improvements of the system.

  14. Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters

    OpenAIRE

    Ferreira, Joshua P.; Peacock, Ryan W. S.; Lawhorn, Ingrid E. B.; Wang, Clifford L.

    2011-01-01

    The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evalua...

  15. A Patient with Beta-Propeller Protein-Associated Neurodegeneration: Treatment with Iron Chelation Therapy

    Directory of Open Access Journals (Sweden)

    Shen-Yang Lim

    2018-05-01

    Full Text Available We present a case of beta-propeller protein-associated neurodegeneration, a form of neurodegeneration with brain iron accumulation. The patient harbored a novel mutation in the WDR45 gene. A detailed video and description of her clinical condition are provided. Her movement disorder phenomenology was characterized primarily by limb stereotypies and gait dyspraxia. The patient’s disability was advanced by the time iron-chelating therapy with deferiprone was initiated, and no clinical response in terms of cognitive function, behavior, speech, or movements were observed after one year of treatment.

  16. The New Self-Inactivating Lentiviral Vector for Thalassemia Gene Therapy Combining Two HPFH Activating Elements Corrects Human Thalassemic Hematopoietic Stem Cells

    Science.gov (United States)

    Papanikolaou, Eleni; Georgomanoli, Maria; Stamateris, Evangelos; Panetsos, Fottes; Karagiorga, Markisia; Tsaftaridis, Panagiotis; Graphakos, Stelios

    2012-01-01

    Abstract To address how low titer, variable expression, and gene silencing affect gene therapy vectors for hemoglobinopathies, in a previous study we successfully used the HPFH (hereditary persistence of fetal hemoglobin)-2 enhancer in a series of oncoretroviral vectors. On the basis of these data, we generated a novel insulated self-inactivating (SIN) lentiviral vector, termed GGHI, carrying the Aγ-globin gene with the −117 HPFH point mutation and the HPFH-2 enhancer and exhibiting a pancellular pattern of Aγ-globin gene expression in MEL-585 clones. To assess the eventual clinical feasibility of this vector, GGHI was tested on CD34+ hematopoietic stem cells from nonmobilized peripheral blood or bone marrow from 20 patients with β-thalassemia. Our results show that GGHI increased the production of γ-globin by 32.9% as measured by high-performance liquid chromatography (p=0.001), with a mean vector copy number per cell of 1.1 and a mean transduction efficiency of 40.3%. Transduced populations also exhibited a lower rate of apoptosis and resulted in improvement of erythropoiesis with a higher percentage of orthochromatic erythroblasts. This is the first report of a locus control region (LCR)-free SIN insulated lentiviral vector that can be used to efficiently produce the anticipated therapeutic levels of γ-globin protein in the erythroid progeny of primary human thalassemic hematopoietic stem cells in vitro. PMID:21875313

  17. Characteristics of lentiviral vectors harboring the proximal promoter of the vav proto-oncogene: a weak and efficient promoter for gene therapy.

    Science.gov (United States)

    Almarza, Elena; Río, Paula; Meza, Nestor W; Aldea, Montserrat; Agirre, Xabier; Guenechea, Guillermo; Segovia, José C; Bueren, Juan A

    2007-08-01

    Recent published data have shown the efficacy of gene therapy treatments of certain monogenic diseases. Risks of insertional oncogenesis, however, indicate the necessity of developing new vectors with weaker or cell-restricted promoters to minimize the trans-activation activity of integrated proviruses. We have inserted the proximal promoter of the vav proto-oncogene into self-inactivating lentiviral vectors (vav-LVs) and investigated the expression pattern and therapeutic efficacy of these vectors. Compared with other LVs frequently used in gene therapy, vav-LVs mediated a weak, though homogeneous and stable, expression in in vitro-cultured cells. Transplantation experiments using transduced mouse bone marrow and human CD34(+) cells confirmed the stable activity of the promoter in vivo. To investigate whether the weak activity of this promoter was compatible with a therapeutic effect, a LV expressing the Fanconi anemia A (FANCA) gene was constructed (vav-FANCA LV). Although this vector induced a low expression of FANCA, compared to the expression induced by a LV harboring the spleen focus-forming virus (SFFV) promoter, the two vectors corrected the phenotype of cells from a patient with FA-A with the same efficacy. We propose that self-inactivating vectors harboring weak promoters, such as the vav promoter, will improve the safety of gene therapy and will be of particular interest for the treatment of diseases where a high expression of the transgene is not required.

  18. Design and Construction of a Cloning Vector Containing the hspX Gene of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Atieh Yaghoubi

    2016-10-01

    Full Text Available Background: Tuberculosis (TB is a major cause of death worldwide. Finding an effective vaccine against TB is the best way to control it. Several vaccines against this disease have been developed but none are completely protective. The aim of this study was to design and construct a cloning vector containing the Mycobacterium tuberculosis (M. tuberculosis heat shock protein X (hspX. Methods: First, an hspX fragment was amplified by PCR and cloned into plasmid pcDNA3.1(+ and recombinant vector was confirmed. Results: A 435 bp hspX fragment was isolated. The fragment was 100% homologous with hspX of M. tuberculosis strain H37Rv in GenBank. Conclusions: In this study, the cloning vector pcDNA3.1(+, containing a 435-bp hspX fragment of M. tuberculosis, was constructed. This could be used as a DNA vaccine to induce immune responses in animal models in future studies.

  19. Evaluation by fluorescence resonance energy transfer of the stability of nonviral gene delivery vectors under physiological conditions.

    Science.gov (United States)

    Itaka, Keiji; Harada, Atsushi; Nakamura, Kozo; Kawaguchi, Hiroshi; Kataoka, Kazunori

    2002-01-01

    The stability in physiological medium of polyplex- and lipoplex-type nonviral gene vectors was evaluated by detecting the conformational change of complexed plasmid DNA (pDNA) labeled simultaneously with fluorescein (energy donor) and X-rhodamine (energy acceptor) through fluorescence resonance energy transfer (FRET). Upon mixing with cationic components, such as LipofectAMINE, poly(L-lysine), and poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLys), the fluorescence spectrum of doubly labeled pDNA underwent a drastic change due to the occurrence of FRET between the donor-acceptor pair on pDNA taking a globular conformation (condensed state) through complexation. The measurement was carried out also in the presence of 20% serum, under which conditions FRET from condensed pDNA was clearly monitored without interference from coexisting components in the medium, allowing evaluation of the condensed state of pDNA in nonviral gene vectors under physiological conditions. Serum addition immediately induced a sharp decrease in FRET for the LipofectAMINE/pDNA (lipoplex) system, which was consistent with the sharp decrease in the transfection efficiency of the lipoplex system in serum-containing medium. In contrast, the PEG-PLys/pDNA polyplex (polyion complex micelle) system maintained appreciable transfection efficiency even in serum-containing medium, and FRET efficiency remained constant for up to 12 h, indicating the high stability of the polyion complex micelle under physiological conditions.

  20. A novel minicircle vector based system for inhibting the replication and gene expression of enterovirus 71 and coxsackievirus A16.

    Science.gov (United States)

    Yang, Zhuo; Li, Guodong; Zhang, Yingqiu; Liu, Xiaoman; Tien, Po

    2012-11-01

    Enterovirus 71 (EV 71) and Coxsackievirus A16 (CA 16) are two major causative agents of hand, foot and mouth disease (HFMD). They have been associated with severe neurological and cardiological complications worldwide, and have caused significant mortalities during large-scale outbreaks in China. Currently, there are no effective treatments against EV 71 and CA 16 infections. We now describe the development of a novel minicircle vector based RNA interference (RNAi) system as a therapeutic approach to inhibiting EV 71 and CA 16 replication. Small interfering RNA (siRNA) molecules targeting the conserved regions of the 3C(pro) and 3D(pol) function gene of the EV 71 and CA 16 China strains were designed based on their nucleotide sequences available in GenBank. This RNAi system was found to effectively block the replication and gene expression of these viruses in rhabdomyosarcoma (RD) cells and virus-infected mice model. The inhibitory effects were confirmed by a corresponding decrease in viral RNA, viral protein, and progeny virus production. In addition, no significant adverse off-target silencing or cytotoxic effects were observed. These results demonstrated the potential and feasibility of this novel minicircle vector based RNAi system for antiviral therapy against EV 71 and CA 16 infection. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Development of a Nature-Inspired Vector for Targeted Systemic Breast Cancer Gene Therapy

    National Research Council Canada - National Science Library

    Hatefi, Arash

    2008-01-01

    .... Using component integration approach, several subsystems of diverse biological origin were integrated onto a modular platform in order to carry diverse functions of an efficient gene delivery vehicle...

  2. Using the 2A Protein Coexpression System: Multicistronic 2A Vectors Expressing Gene(s) of Interest and Reporter Proteins.

    Science.gov (United States)

    Luke, Garry A; Ryan, Martin D

    2018-01-01

    To date, a huge range of different proteins-many with cotranslational and posttranslational subcellular localization signals-have been coexpressed together with various reporter proteins in vitro and in vivo using 2A peptides. The pros and cons of 2A co-expression technology are considered below, followed by a simple example of a "how to" protocol to concatenate multiple genes of interest, together with a reporter gene, into a single gene linked via 2As for easy identification or selection of transduced cells.

  3. Transcription regulation of sex-biased genes during ontogeny in the malaria vector Anopheles gambiae.

    Directory of Open Access Journals (Sweden)

    Kalle Magnusson

    Full Text Available In Anopheles gambiae, sex-regulated genes are responsible for controlling gender dimorphism and are therefore crucial in determining the ability of female mosquitoes to transmit human malaria. The identification and functional characterization of these genes will shed light on the sexual development and maturation of mosquitoes and provide useful targets for genetic control measures aimed at reducing mosquito fertility and/or distorting the sex ratio.We conducted a genome wide transcriptional analysis of sex-regulated genes from early developmental stages through adulthood combined with functional screening of novel gonadal genes. Our results demonstrate that the male-biased genes undergo a major transcription turnover starting from larval stages to adulthood. The male biased genes at the adult stage include a significant high number of unique sequences compared to the rest of the genome. This is in contrast to female-biased genes that are much more conserved and are mainly activated during late developmental stages.The high frequency of unique sequences would indicate that male-biased genes evolve more rapidly than the rest of the genome. This finding is particularly intriguing because A. gambiae is a strictly female monogamous species suggesting that driving forces in addition to sperm competition must account for the rapid evolution of male-biased genes. We have also identified and functionally characterized a number of previously unknown A. gambiae testis- and ovary-specific genes. Two of these genes, zero population growth and a suppressor of defective silencing 3 domain of the histone deacetylase co-repressor complex, were shown to play a key role in gonad development.

  4. Polyamidoamine-Decorated Nanodiamonds as a Hybrid Gene Delivery Vector and siRNA Structural Characterization at the Charged Interfaces.

    Science.gov (United States)

    Lim, Dae Gon; Rajasekaran, Nirmal; Lee, Dukhee; Kim, Nam Ah; Jung, Hun Soon; Hong, Sungyoul; Shin, Young Kee; Kang, Eunah; Jeong, Seong Hoon

    2017-09-20

    Nanodiamonds have been discovered as a new exogenous material source in biomedical applications. As a new potent form of nanodiamond (ND), polyamidoamine-decorated nanodiamonds (PAMAM-NDs) were prepared for E7 or E6 oncoprotein-suppressing siRNA gene delivery for high risk human papillomavirus-induced cervical cancer, such as types 16 and 18. It is critical to understand the physicochemical properties of siRNA complexes immobilized on cationic solid ND surfaces in the aspect of biomolecular structural and conformational changes, as the new inert carbon material can be extended into the application of a gene delivery vector. A spectral study of siRNA/PAMAM-ND complexes using differential scanning calorimetry and circular dichroism spectroscopy proved that the hydrogen bonding and electrostatic interactions between siRNA and PAMAM-NDs decreased endothermic heat capacity. Moreover, siRNA/PAMAM-ND complexes showed low cell cytotoxicity and significant suppressing effects for forward target E6 and E7 oncogenic genes, proving functional and therapeutic efficacy. The cellular uptake of siRNA/PAMAM-ND complexes at 8 h was visualized by macropinocytes and direct endosomal escape of the siRNA/PAMAM-ND complexes. It is presumed that PAMAM-NDs provided a buffering cushion to adjust the pH and hard mechanical stress to escape endosomes. siRNA/PAMAM-ND complexes provide a potential organic/inorganic hybrid material source for gene delivery carriers.

  5. Horizontal gene acquisition of Liberibacter plant pathogens from a bacteriome-confined endosymbiont of their psyllid vector.

    Directory of Open Access Journals (Sweden)

    Atsushi Nakabachi

    Full Text Available he Asian citrus psyllid Diaphorina citri is a notorious agricultural pest that transmits the phloem-inhabiting alphaproteobacterial 'Candidatus Liberibacter asiaticus' and allied plant pathogens, which cause the devastating citrus disease called Huanglongbing or greening disease. D. citri harbors two distinct bacterial mutualists in the symbiotic organ called bacteriome: the betaproteobacterium 'Candidatus Profftella armatura' in the syncytial cytoplasm at the center of the bacteriome, and the gammaproteobacterium 'Candidatus Carsonella ruddii' in uninucleate bacteriocytes. Here we report that a putative amino acid transporter LysE of Profftella forms a highly supported clade with proteins of L. asiaticus, L. americanus, and L. solanacearum. L. crescens, the most basal Liberibacter lineage currently known, lacked the corresponding gene. The Profftella-Liberibacter subclade of LysE formed a clade with proteins from betaproteobacteria of the order Burkholderiales, to which Profftella belongs. This phylogenetic pattern favors the hypothesis that the Liberibacter lineage acquired the gene from the Profftella lineage via horizontal gene transfer (HGT after L. crescens diverged from other Liberibacter lineages. K A/K S analyses further supported the hypothesis that the genes encoded in the Liberibacter genomes are functional. These findings highlight the possible evolutionary importance of HGT between plant pathogens and their insect vector's symbionts that are confined in the symbiotic organ and seemingly sequestered from external microbial populations.

  6. Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping.

    Science.gov (United States)

    Ni, W; Le Guiner, C; Gernoux, G; Penaud-Budloo, M; Moullier, P; Snyder, R O

    2011-07-01

    Legitimate uses of gene transfer technology can benefit from sensitive detection methods to determine vector biodistribution in pre-clinical studies and in human clinical trials, and similar methods can detect illegitimate gene transfer to provide sports-governing bodies with the ability to maintain fairness. Real-time PCR assays were developed to detect a performance-enhancing transgene (erythropoietin, EPO) and backbone sequences in the presence of endogenous cellular sequences. In addition to developing real-time PCR assays, the steps involved in DNA extraction, storage and transport were investigated. By real-time PCR, the vector transgene is distinguishable from the genomic DNA sequence because of the absence of introns, and the vector backbone can be identified by heterologous gene expression control elements. After performance of the assays was optimized, cynomolgus macaques received a single dose by intramuscular (IM) injection of plasmid DNA, a recombinant adeno-associated viral vector serotype 1 (rAAV1) or a rAAV8 vector expressing cynomolgus macaque EPO. Macaques received a high plasmid dose intended to achieve a significant, but not life-threatening, increase in hematocrit. rAAV vectors were used at low doses to achieve a small increase in hematocrit and to determine the limit of sensitivity for detecting rAAV sequences by single-step PCR. DNA extracted from white blood cells (WBCs) was tested to determine whether WBCs can be collaterally transfected by plasmid or transduced by rAAV vectors in this context, and can be used as a surrogate marker for gene doping. We demonstrate that IM injection of a conventional plasmid and rAAV vectors results in the presence of DNA that can be detected at high levels in blood before rapid elimination, and that rAAV genomes can persist for several months in WBCs.

  7. Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector

    OpenAIRE

    Peluffo, Hugo; Acarin, Laia; Arís, Anna; González, Pau; Villaverde, Antoni; Castellano, Bernardo; González, Berta

    2006-01-01

    Abstract Background Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD) after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn S...

  8. Therapeutic Efficacy of Vectored PGT121 Gene Delivery in HIV-1-Infected Humanized Mice.

    Science.gov (United States)

    Badamchi-Zadeh, Alexander; Tartaglia, Lawrence J; Abbink, Peter; Bricault, Christine A; Liu, Po-Ting; Boyd, Michael; Kirilova, Marinela; Mercado, Noe B; Nanayakkara, Ovini S; Vrbanac, Vladimir D; Tager, Andrew M; Larocca, Rafael A; Seaman, Michael S; Barouch, Dan H

    2018-04-01

    Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies. However, administration of purified bNAbs poses challenges in resource-poor settings, where the HIV-1 disease burden is greatest. In vivo vector-based production of bNAbs represents an alternative strategy. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121 in wild-type and immunocompromised C57BL/6 mice as well as in HIV-1-infected bone marrow-liver-thymus (BLT) humanized mice. Ad5.PGT121 and AAV1.PGT121 produced functional antibody in vivo Ad5.PGT121 produced PGT121 rapidly within 6 h, whereas AAV1.PGT121 produced detectable PGT121 in serum by 72 h. Serum PGT121 levels were rapidly reduced by the generation of anti-PGT121 antibodies in immunocompetent mice but were durably maintained in immunocompromised mice. In HIV-1-infected BLT humanized mice, Ad5.PGT121 resulted in a greater reduction of viral loads than did AAV1.PGT121. Ad5.PGT121 also led to more-sustained virologic control than purified PGT121 IgG. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Further evaluation of vector delivery of HIV-1 bNAbs is warranted, although approaches to prevent the generation of antiantibody responses may also be required. IMPORTANCE Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies, but delivery of purified antibodies may prove challenging. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Copyright © 2018 Badamchi-Zadeh et al.

  9. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA and HIV-1 nef Genes in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Siti Aisyah Mualif

    Full Text Available Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef, HIV-1 p24 (ca, and HIV-1 vif in NiCo21(DE3 E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes.

  10. Pantethine treatment is effective in recovering the disease phenotype induced by ketogenic diet in a pantothenate kinase-associated neurodegeneration mouse model

    NARCIS (Netherlands)

    Brunetti, Dario; Dusi, Sabrina; Giordano, Carla; Lamperti, Costanza; Morbin, Michela; Fugnanesi, Valeria; Marchet, Silvia; Fagiolari, Gigliola; Sibon, Ody; Moggio, Maurizio; d'Amati, Giulia; Tiranti, Valeria

    Pantothenate kinase-associated neurodegeneration, caused by mutations in the PANK2 gene, is an autosomal recessive disorder characterized by dystonia, dysarthria, rigidity, pigmentary retinal degeneration and brain iron accumulation. PANK2 encodes the mitochondrial enzyme pantothenate kinase type 2,

  11. Progress in developing cationic vectors for non-viral systemic gene therapy against cancer.

    OpenAIRE

    Morille , Marie; Passirani , Catherine; Vonarbourg , Arnaud; Clavreul , Anne; Benoit , Jean-Pierre

    2008-01-01

    International audience; Initially, gene therapy was viewed as an approach for treating hereditary diseases, but its potential role in the treatment of acquired diseases such as cancer is now widely recognized. The understanding of the molecular mechanisms involved in cancer and the development of nucleic acid delivery systems are two concepts that have led to this development. Systemic gene delivery systems are needed for therapeutic application to cells inaccessible by percutaneous injection...

  12. On the efficacy of malaria DNA vaccination with magnetic gene vectors.

    Science.gov (United States)

    Nawwab Al-Deen, Fatin; Ma, Charles; Xiang, Sue D; Selomulya, Cordelia; Plebanski, Magdalena; Coppel, Ross L

    2013-05-28

    We investigated the efficacy and types of immune responses from plasmid malaria DNA vaccine encoding VR1020-PyMSP119 condensed on the surface of polyethyleneimine (PEI)-coated SPIONs. In vivo mouse studies were done firstly to determine the optimum magnetic vector composition, and then to observe immune responses elicited when magnetic vectors were introduced via different administration routes. Higher serum antibody titers against PyMSP119 were observed with intraperitoneal and intramuscular injections than subcutaneous and intradermal injections. Robust IgG2a and IgG1 responses were observed for intraperitoneal administration, which could be due to the physiology of peritoneum as a major reservoir of macrophages and dendritic cells. Heterologous DNA prime followed by single protein boost vaccination regime also enhanced IgG2a, IgG1, and IgG2b responses, indicating the induction of appropriate memory immunity that can be elicited by protein on recall. These outcomes support the possibility to design superparamagnetic nanoparticle-based DNA vaccines to optimally evoke desired antibody responses, useful for a variety of diseases including malaria. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Implications of mitochondrial dynamics on neurodegeneration and on hypothalamic dysfunction

    Directory of Open Access Journals (Sweden)

    Antonio eZorzano

    2015-06-01

    Full Text Available Mitochondrial dynamics is a term that encompasses the movement of mitochondria along the cytoskeleton, regulation of their architecture, and connectivity mediated by tethering and fusion/fission. The importance of these events in cell physiology and pathology has been partially unraveled with the identification of the genes responsible for the catalysis of mitochondrial fusion and fission. Mutations in two mitochondrial fusion genes (MFN2 and OPA1 cause neurodegenerative diseases, namely Charcot-Marie Tooth type 2A and autosomal dominant optic atrophy. Alterations in mitochondrial dynamics may be involved in the pathophysiology of prevalent neurodegenerative conditions. Moreover, impairment of the activity of mitochondrial fusion proteins dysregulates the function of hypothalamic neurons, leading to alterations in food intake and in energy homeostasis. Here we review selected findings in the field of mitochondrial dynamics and their relevance for neurodegeneration and hypothalamic dysfunction.

  14. Effective in vivo and ex vivo gene transfer to intestinal mucosa by VSV-G-pseudotyped lentiviral vectors

    Directory of Open Access Journals (Sweden)

    Kasahara Noriyuki

    2010-05-01

    Full Text Available Abstract Background Gene transfer to the gastrointestinal (GI mucosa is a therapeutic strategy which could prove particularly advantageous for treatment of various hereditary and acquired intestinal disorders, including inflammatory bowel disease (IBD, GI infections, and cancer. Methods We evaluated vesicular stomatitis virus glycoprotein envelope (VSV-G-pseudotyped lentiviral vectors (LV for efficacy of gene transfer to both murine rectosigmoid colon in vivo and human colon explants ex vivo. LV encoding beta-galactosidase (LV-β-Gal or firefly-luciferase (LV-fLuc reporter genes were administered by intrarectal instillation in mice, or applied topically for ex vivo transduction of human colorectal explant tissues from normal individuals. Macroscopic and histological evaluations were performed to assess any tissue damage or inflammation. Transduction efficiency and systemic biodistribution were evaluated by real-time quantitative PCR. LV-fLuc expression was evaluated by ex vivo bioluminescence imaging. LV-β-Gal expression and identity of transduced cell types were examined by histochemical and immunofluorescence staining. Results Imaging studies showed positive fLuc signals in murine distal colon; β-Gal-positive cells were found in both murine and human intestinal tissue. In the murine model, β-Gal-positive epithelial and lamina propria cells were found to express cytokeratin, CD45, and CD4. LV-transduced β-Gal-positive cells were also seen in human colorectal explants, consisting mainly of CD45, CD4, and CD11c-positive cells confined to the LP. Conclusions We have demonstrated the feasibility of LV-mediated gene transfer into colonic mucosa. We also identified differential patterns of mucosal gene transfer dependent on whether murine or human tissue was used. Within the limitations of the study, the LV did not appear to induce mucosal damage and were not distributed beyond the distal colon.

  15. Lung cancer gene expression database analysis incorporating prior knowledge with support vector machine-based classification method

    Directory of Open Access Journals (Sweden)

    Huang Desheng

    2009-07-01

    Full Text Available Abstract Background A reliable and precise classification is essential for successful diagnosis and treatment of cancer. Gene expression microarrays have provided the high-throughput platform to discover genomic biomarkers for cancer diagnosis and prognosis. Rational use of the available bioinformation can not only effectively remove or suppress noise in gene chips, but also avoid one-sided results of separate experiment. However, only some studies have been aware of the importance of prior information in cancer classification. Methods Together with the application of support vector machine as the discriminant approach, we proposed one modified method that incorporated prior knowledge into cancer classification based on gene expression data to improve accuracy. A public well-known dataset, Malignant pleural mesothelioma and lung adenocarcinoma gene expression database, was used in this study. Prior knowledge is viewed here as a means of directing the classifier using known lung adenocarcinoma related genes. The procedures were performed by software R 2.80. Results The modified method performed better after incorporating prior knowledge. Accuracy of the modified method improved from 98.86% to 100% in training set and from 98.51% to 99.06% in test set. The standard deviations of the modified method decreased from 0.26% to 0 in training set and from 3.04% to 2.10% in test set. Conclusion The method that incorporates prior knowledge into discriminant analysis could effectively improve the capacity and reduce the impact of noise. This idea may have good future not only in practice but also in methodology.

  16. Huntingtin interacting proteins are genetic modifiers of neurodegeneration.

    Directory of Open Access Journals (Sweden)

    Linda S Kaltenbach

    2007-05-01

    Full Text Available Huntington's disease (HD is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%-4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to

  17. Integrase-Deficient Lentiviral Vector as an All-in-One Platform for Highly Efficient CRISPR/Cas9-Mediated Gene Editing

    Directory of Open Access Journals (Sweden)

    Pavel I. Ortinski

    2017-06-01

    Full Text Available The CRISPR/Cas9 systems have revolutionized the field of genome editing by providing unprecedented control over gene sequences and gene expression in many species, including humans. Lentiviral vectors (LVs are one of the primary delivery platforms for the CRISPR/Cas9 system due to their ability to accommodate large DNA payloads and sustain robust expression in a wide range of dividing and non-dividing cells. However, long-term expression of LV-delivered Cas9/guide RNA may lead to undesirable off-target effects characterized by non-specific RNA-DNA interactions and off-target DNA cleavages. Integrase-deficient lentiviral vectors (IDLVs present an attractive means for delivery of CRISPR/Cas9 components because: (1 they are capable of transducing a broad range of cells and tissues, (2 have superior packaging capacity compared to other vectors (e.g., adeno-associated viral vectors, and (3 they are expressed transiently and demonstrate very weak integration capability. In this manuscript, we aimed to establish IDLVs as a means for safe and efficient delivery of CRISPR/Cas9. To this end, we developed an all-in-one vector cassette with increased production efficacy and demonstrated that CRISPR/Cas9 delivered by the improved IDLV vectors can mediate rapid and robust gene editing in human embryonic kidney (HEK293T cells and post-mitotic brain neurons in vivo, via transient expression and with higher gene-targeting specificity than the corresponding integrase-competent vectors.

  18. New vectors in fission yeast: application for cloning the his2 gene

    DEFF Research Database (Denmark)

    Weilguny, D; Praetorius, M; Carr, Alan

    1991-01-01

    of transforming Sc. pombe ura4 strains, as well as ura 3 strains of the distantly related budding yeast Saccharomyces cerevisiae. We have used pON163 for the construction of two fission yeast genomic libraries. From these gene banks clones were isolated that were able to complement fission yeast his2 mutants...

  19. Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

    Science.gov (United States)

    Joseph, Joan; Fernández-Lloris, Raquel; Pezzat, Elías; Saubi, Narcís; Cardona, Pere-Joan; Mothe, Beatriz; Gatell, Josep Maria

    2010-01-01

    Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261) and Mycobacteria spp. α-antigen promoter (in plasmid pJH222). Among 14 rBCG:HIV-1gp120 (pMV261) colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222) colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors. PMID:20617151

  20. Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

    Directory of Open Access Journals (Sweden)

    Joan Joseph

    2010-01-01

    Full Text Available Mycobacterium bovis Bacillus Calmette-Guérin (BCG as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261 and Mycobacteria spp. α-antigen promoter (in plasmid pJH222. Among 14 rBCG:HIV-1gp120 (pMV261 colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222 colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors.

  1. A novel system for simultaneous or sequential integration of multiple gene-loading vectors into a defined site of a human artificial chromosome.

    Science.gov (United States)

    Suzuki, Teruhiko; Kazuki, Yasuhiro; Oshimura, Mitsuo; Hara, Takahiko

    2014-01-01

    Human artificial chromosomes (HACs) are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV) into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system) via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming.

  2. A novel system for simultaneous or sequential integration of multiple gene-loading vectors into a defined site of a human artificial chromosome.

    Directory of Open Access Journals (Sweden)

    Teruhiko Suzuki

    Full Text Available Human artificial chromosomes (HACs are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming.

  3. Hypoxia-inducible bidirectional shRNA expression vector delivery using PEI/chitosan-TBA copolymers for colorectal Cancer gene therapy.

    Science.gov (United States)

    Javan, Bita; Atyabi, Fatemeh; Shahbazi, Majid

    2018-04-12

    This investigation was conducted to construct a hypoxia/colorectal dual-specific bidirectional short hairpin RNA (shRNA) expression vector and to transfect it into the colon cancer cell line HT-29 with PEI/chitosan-TBA nanoparticles for the simultaneous knock down of β-catenin and Bcl-2 under hypoxia. To construct a pRNA-bipHRE-CEA vector, the carcinoma embryonic antigen (CEA) promoter designed in two directions and the vascular endothelial growth factor (VEGF) enhancer were inserted between two promoters for hypoxic cancer specific gene expression. To confirm the therapeutic effect of the dual-specific vector, β-catenin and Bcl-2 shRNAs were inserted downstream of each promoter. The physicochemical properties, the cytotoxicity, and the transfection efficiency of these PEI/chitosan-TBA nanoparticles were investigated. In addition, the antitumor effects of the designed vector on the expression of β-catenin and Bcl-2, cell cycle distribution, and apoptosis were investigated in vitro. The silencing effect of the hypoxia-response shRNA expression vector was relatively low (18%-25%) under normoxia, whereas it was significantly increased to approximately 50%-60% in the HT-29 cell line. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis due to gene silencing under hypoxia. Furthermore, MTS assay, fluorescence microscopy images, and flow cytometry analyses confirmed that the PEI/chitosan-TBA blend system provided effective transfection with low cytotoxicity. This novel hypoxia-responsive shRNA expression vector may be useful for RNA interference (RNAi)-based cancer gene therapy in hypoxic colorectal tumors. Moreover, the PEI/chitosan-TBA copolymer might be a promising gene carrier for use in gene transfer in vivo. Copyright © 2017. Published by Elsevier Inc.

  4. An efficient viral vector for functional genomic studies of Prunus fruit trees and its induced resistance to Plum pox virus via silencing of a host factor gene.

    Science.gov (United States)

    Cui, Hongguang; Wang, Aiming

    2017-03-01

    RNA silencing is a powerful technology for molecular characterization of gene functions in plants. A commonly used approach to the induction of RNA silencing is through genetic transformation. A potent alternative is to use a modified viral vector for virus-induced gene silencing (VIGS) to degrade RNA molecules sharing similar nucleotide sequence. Unfortunately, genomic studies in many allogamous woody perennials such as peach are severely hindered because they have a long juvenile period and are recalcitrant to genetic transformation. Here, we report the development of a viral vector derived from Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus that is endemic in all Prunus fruit production countries and regions in the world. We show that the modified PNRSV vector, harbouring the sense-orientated target gene sequence of 100-200 bp in length in genomic RNA3, could efficiently trigger the silencing of a transgene or an endogenous gene in the model plant Nicotiana benthamiana. We further demonstrate that the PNRSV-based vector could be manipulated to silence endogenous genes in peach such as eukaryotic translation initiation factor 4E isoform (eIF(iso)4E), a host factor of many potyviruses including Plum pox virus (PPV). Moreover, the eIF(iso)4E-knocked down peach plants were resistant to PPV. This work opens a potential avenue for the control of virus diseases in perennial trees via viral vector-mediated silencing of host factors, and the PNRSV vector may serve as a powerful molecular tool for functional genomic studies of Prunus fruit trees. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  5. Mouse Mammary Tumor Virus Promoter-Containing Retroviral Promoter Conversion Vectors for Gene-Directed Enzyme Prodrug Therapy are Functional in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Reinhard Klein

    2008-01-01

    Full Text Available Gene directed-enzyme prodrug therapy (GDEPT is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1 metabolizes the prodrugs cyclophosphamide (CPA and ifosfamide (IFA to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC50 values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model.

  6. Citrus tristeza virus-based RNAi in citrus plants induces gene silencing in Diaphorina citri, a phloem-sap sucking insect vector of citrus greening disease (Huanglongbing).

    Science.gov (United States)

    Hajeri, Subhas; Killiny, Nabil; El-Mohtar, Choaa; Dawson, William O; Gowda, Siddarame

    2014-04-20

    A transient expression vector based on Citrus tristeza virus (CTV) is unusually stable. Because of its stability it is being considered for use in the field to control Huanglongbing (HLB), which is caused by Candidatus Liberibacter asiaticus (CLas) and vectored by Asian citrus psyllid, Diaphorina citri. In the absence of effective control strategies for CLas, emphasis has been on control of D. citri. Coincident cohabitation in phloem tissue by CLas, D. citri and CTV was exploited to develop a novel method to mitigate HLB through RNA interference (RNAi). Since CTV has three RNA silencing suppressors, it was not known if CTV-based vector could induce RNAi in citrus. Yet, expression of sequences targeting citrus phytoene desaturase gene by CTV-RNAi resulted in photo-bleaching phenotype. CTV-RNAi vector, engineered with truncated abnormal wing disc (Awd) gene of D. citri, induced altered Awd expression when silencing triggers ingested by feeding D. citri nymphs. Decreased Awd in nymphs resulted in malformed-wing phenotype in adults and increased adult mortality. This impaired ability of D. citri to fly would potentially limit the successful vectoring of CLas bacteria between citrus trees in the grove. CTV-RNAi vector would be relevant for fast-track screening of candidate sequences for RNAi-mediated pest control. Copyright © 2014. Published by Elsevier B.V.

  7. Multifunctional non-viral gene vectors with enhanced stability, improved cellular and nuclear uptake capability, and increased transfection efficiency

    Science.gov (United States)

    Yang, Zhe; Jiang, Zhaozhong; Cao, Zhong; Zhang, Chao; Gao, Di; Luo, Xingen; Zhang, Xiaofang; Luo, Huiyan; Jiang, Qing; Liu, Jie

    2014-08-01

    We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell for nanoparticle stabilization, poly(γ-glutamic acid) (γ-PGA) and mTAT (a cell-penetrating peptide) for accelerated cellular uptake, and a nuclear localization signal peptide (NLS) for enhanced intracellular transport of DNA to the nucleus. In vitro study showed that coating of the binary PPMS/DNA polyplex with γ-PGA promotes cellular uptake of the polyplex particles, particularly by γ-glutamyl transpeptidase (GGT)-positive cells through the GGT-mediated endocytosis pathway. Conjugating PEG to the γ-PGA led to the formation of a ternary PPMS/DNA/PGA-g-PEG polyplex with decreased positive charges on the surface of the polyplex particles and substantially higher stability in serum-containing aqueous medium. The cellular uptake rate was further improved by incorporating mTAT into the ternary polyplex system. Addition of the NLS peptide was designed to facilitate intracellular delivery of the plasmid to the nucleus--a rate-limiting step in the gene transfection process. As a result, compared with the binary PPMS/LucDNA polyplex, the new mTAT-quaternary PPMS/LucDNA/NLS/PGA-g-PEG-mTAT system exhibited reduced cytotoxicity, remarkably faster cellular uptake rate, and enhanced transport of DNA to the nucleus. All these advantageous functionalities contribute to the remarkable gene transfection efficiency of the mTAT-quaternary polyplex both in vitro and in vivo, which exceeds that of the binary polyplex and commercial Lipofectamine™ 2000/DNA lipoplex. The multifunctional mTAT-quaternary polyplex system with improved efficiency and reduced cytotoxicity represents a new type of promising non-viral vectors for the delivery of therapeutic genes to treat tumors.We have developed a new multifunctional, non-viral gene delivery platform consisting of cationic poly(amine-co-ester) (PPMS) for DNA condensation, PEG shell

  8. Development of a multiple-gene-loading method by combining multi-integration system-equipped mouse artificial chromosome vector and CRISPR-Cas9.

    Directory of Open Access Journals (Sweden)

    Kazuhisa Honma

    Full Text Available Mouse artificial chromosome (MAC vectors have several advantages as gene delivery vectors, such as stable and independent maintenance in host cells without integration, transferability from donor cells to recipient cells via microcell-mediated chromosome transfer (MMCT, and the potential for loading a megabase-sized DNA fragment. Previously, a MAC containing a multi-integrase platform (MI-MAC was developed to facilitate the transfer of multiple genes into desired cells. Although the MI system can theoretically hold five gene-loading vectors (GLVs, there are a limited number of drugs available for the selection of multiple-GLV integration. To overcome this issue, we attempted to knock out and reuse drug resistance genes (DRGs using the CRISPR-Cas9 system. In this study, we developed new methods for multiple-GLV integration. As a proof of concept, we introduced five GLVs in the MI-MAC by these methods, in which each GLV contained a gene encoding a fluorescent or luminescent protein (EGFP, mCherry, BFP, Eluc, and Cluc. Genes of interest (GOI on the MI-MAC were expressed stably and functionally without silencing in the host cells. Furthermore, the MI-MAC carrying five GLVs was transferred to other cells by MMCT, and the resultant recipient cells exhibited all five fluorescence/luminescence signals. Thus, the MI-MAC was successfully used as a multiple-GLV integration vector using the CRISPR-Cas9 system. The MI-MAC employing these methods may resolve bottlenecks in developing multiple-gene humanized models, multiple-gene monitoring models, disease models, reprogramming, and inducible gene expression systems.

  9. A novel dendrimer based on poly (L-glutamic acid) derivatives as an efficient and biocompatible gene delivery vector

    International Nuclear Information System (INIS)

    Zeng Xin; Pan Shirong; Wang Chi; Wen Yuting; Wu Hongmei; Wang Cuifeng; Wu Chuanbin; Feng Min; Li Jie

    2011-01-01

    Non-viral gene delivery systems based on cationic polymers have faced limitations related to their relative low gene transfer efficiency, cytotoxicity and system instability in vivo. In this paper, a flexible and pompon-like dendrimer composed of poly (amidoamine) (PAMAM) G4.0 as the inner core and poly (L-glutamic acid) grafted low-molecular-weight polyethylenimine (PLGE) as the surrounding multiple arms was synthesized (MGI dendrimer). The novel MGI dendrimer was designed to combine the merits of size-controlled PAMAM G4.0 and the low toxicity and flexible chains of PLGE. In phosphate-buffered saline dispersions the well-defined DNA/MGI complex above a N/P ratio of 30 showed good stability with particle sizes of approximately 200 nm and a comparatively low polydispersity index. However, the particle size of the DNA/25 kDa polyethylenimine (DNA/PEI 25K) complex was larger than 700 nm under the same salt conditions. The shielding of the compact amino groups at the periphery of flexible PAMAM and biocompatible PLGE chains in MGI resulted in a dramatic decrease of the cytotoxicity compared to native PAMAM G4.0 dendrimer. The in vitro transfection efficiency of DNA/MGI dendrimer complex was higher than that of PAMAM G4.0 dendrimer. Importantly, in serum-containing medium, DNA/MGI complexes at their optimal N/P ratio maintained the same high levels of transfection efficiency as in serum-free medium, while the transfection efficiency of native PAMAM G4.0, PEI 25K and Lipofectamine 2000 were sharply decreased. In vivo gene delivery of pVEGF165/MGI complex into balloon-injured rabbit carotid arteries resulted in significant inhibition of restenosis by increasing VEGF165 expression in local vessels. Therefore, the pompon-like MGI dendrimer may be a promising vector candidate for efficient gene delivery in vivo.

  10. A multi-layered mechanistic modelling approach to understand how effector genes extend beyond phytoplasma to modulate plant hosts, insect vectors and the environment.

    Science.gov (United States)

    Tomkins, Melissa; Kliot, Adi; Marée, Athanasius Fm; Hogenhout, Saskia A

    2018-03-13

    Members of the Candidatus genus Phytoplasma are small bacterial pathogens that hijack their plant hosts via the secretion of virulence proteins (effectors) leading to a fascinating array of plant phenotypes, such as witch's brooms (stem proliferations) and phyllody (retrograde development of flowers into vegetative tissues). Phytoplasma depend on insect vectors for transmission, and interestingly, these insect vectors were found to be (in)directly attracted to plants with these phenotypes. Therefore, phytoplasma effectors appear to reprogram plant development and defence to lure insect vectors, similarly to social engineering malware, which employs tricks to lure people to infected computers and webpages. A multi-layered mechanistic modelling approach will enable a better understanding of how phytoplasma effector-mediated modulations of plant host development and insect vector behaviour contribute to phytoplasma spread, and ultimately to predict the long reach of phytoplasma effector genes. Copyright © 2018. Published by Elsevier Ltd.

  11. rAAV Vectors as Safe and Efficient Tools for the Stable Delivery of Genes to Primary Human Chondrosarcoma Cells In Vitro and In Situ

    Directory of Open Access Journals (Sweden)

    Henning Madry

    2012-01-01

    Full Text Available Treatment of chondrosarcoma remains a major challenge in orthopaedic oncology. Gene transfer strategies based on recombinant adenoassociated viral (rAAV vectors may provide powerful tools to develop new, efficient therapeutic options against these tumors. In the present study, we tested the hypothesis that rAAV is adapted for a stable and safe delivery of foreign sequences in human chondrosarcoma tissue by transducing primary human chondrosarcoma cells in vitro and in situ with different reporter genes (E. coli lacZ, firefly luc, Discosoma sp. RFP. The effects of rAAV administration upon cell survival and metabolic activities were also evaluated to monitor possibly detrimental effects of the gene transfer method. Remarkably, we provide evidence that efficient and prolonged expression of transgene sequences via rAAV can be safely achieved in all the systems investigated, demonstrating the potential of the approach of direct application of therapeutic gene vectors as a means to treat chondrosarcoma.

  12. Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector-mediated RNAi.

    Science.gov (United States)

    Patel, Utsav A; Patel, Amrutlal K; Joshi, Chaitanya G

    2015-01-01

    Myostatin (MSTN) is a secreted growth factor that negatively regulates skeletal muscle mass, and therefore, strategies to block myostatin-signaling pathway have been extensively pursued to increase the muscle mass in livestock. Here, we report a lentiviral vector-based delivery of shRNA to disrupt myostatin expression into goat fetal fibroblasts (GFFs) that were commonly used as karyoplast donors in somatic-cell nuclear transfer (SCNT) studies. Sh-RNA positive cells were screened by puromycin selection. Using real-time polymerase chain reaction (PCR), we demonstrated efficient knockdown of endogenous myostatin mRNA with 64% down-regulation in sh2 shRNA-treated GFF cells compared to GFF cells treated by control lentivirus without shRNA. Moreover, we have also demonstrated both the induction of interferon response and the expression of genes regulating myogenesis in GFF cells. The results indicate that myostatin-targeting siRNA produced endogenously could efficiently down-regulate myostatin expression. Therefore, targeted knockdown of the MSTN gene using lentivirus-mediated shRNA transgenics would facilitate customized cell engineering, allowing potential use in the establishment of stable cell lines to produce genetically engineered animals. © 2014 American Institute of Chemical Engineers.

  13. A convenient method of preparing gene vector for real time monitoring transfection process based on the quantum dots

    International Nuclear Information System (INIS)

    Zhang, Hai-Li; Zhang, Ming-Zhen; Li, Xiang-Yong; Wan, Min; Li, Yong-Qiang; Zhang, Rong-Ying; Zhao, Yuan-Di

    2012-01-01

    Highlights: ► An easy and direct way to prepare QDs–DNA complexes was developed. ► Surface charge of QDs was tuned with different ratio of amino and glycolate. ► Transfection efficiency was dependent on the surface zeta potentials of QDs. ► Cellular toxicity of this gene vectors is much lower than commercial liposome. ► Whole intracellular behavior of QDs–DNA complexes can be monitored in real time. -- Abstract: Nanoparticle carrier has been developed by combining water-soluble quantum dots and plasmid DNA expressed enhanced green fluorescent protein (EGFP) in a convenient and direct way. First the QDs with different surface charges were obtained by coating with amino and carboxyl terminals at different ratios. Then plasmid DNA was conjugated to QDs via electrostatic interaction. The resultant QDs–DNA complexes showed enhanced resistance to DNase I digestion. The following transfection experiments demonstrated that the transfection efficiency was dependent on the surface charges on QDs. The real time imaging of the transfection process showed that the nanoparticles experienced binding, penetrating the cell membrane and entering cytoplasm in the first 6 h of transfection. The green fluorescence of EGFP began to appear after 18 h transfection and plasmid DNA was fully expressed in the following 6 h. This new QDs–DNA platform showed great potential as new gene delivery carrier.

  14. A convenient method of preparing gene vector for real time monitoring transfection process based on the quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hai-Li; Zhang, Ming-Zhen; Li, Xiang-Yong [Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Wan, Min [Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Li, Yong-Qiang [Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Zhang, Rong-Ying [Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Zhao, Yuan-Di, E-mail: zydi@mail.hust.edu.cn [Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China); Key Laboratory of Biomedical Photonics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Department of Biomedical Engineering, Wuhan 430074 (China)

    2012-11-15

    Highlights: ► An easy and direct way to prepare QDs–DNA complexes was developed. ► Surface charge of QDs was tuned with different ratio of amino and glycolate. ► Transfection efficiency was dependent on the surface zeta potentials of QDs. ► Cellular toxicity of this gene vectors is much lower than commercial liposome. ► Whole intracellular behavior of QDs–DNA complexes can be monitored in real time. -- Abstract: Nanoparticle carrier has been developed by combining water-soluble quantum dots and plasmid DNA expressed enhanced green fluorescent protein (EGFP) in a convenient and direct way. First the QDs with different surface charges were obtained by coating with amino and carboxyl terminals at different ratios. Then plasmid DNA was conjugated to QDs via electrostatic interaction. The resultant QDs–DNA complexes showed enhanced resistance to DNase I digestion. The following transfection experiments demonstrated that the transfection efficiency was dependent on the surface charges on QDs. The real time imaging of the transfection process showed that the nanoparticles experienced binding, penetrating the cell membrane and entering cytoplasm in the first 6 h of transfection. The green fluorescence of EGFP began to appear after 18 h transfection and plasmid DNA was fully expressed in the following 6 h. This new QDs–DNA platform showed great potential as new gene delivery carrier.

  15. Cancer classification through filtering progressive transductive support vector machine based on gene expression data

    Science.gov (United States)

    Lu, Xinguo; Chen, Dan

    2017-08-01

    Traditional supervised classifiers neglect a large amount of data which not have sufficient follow-up information, only work with labeled data. Consequently, the small sample size limits the advancement of design appropriate classifier. In this paper, a transductive learning method which combined with the filtering strategy in transductive framework and progressive labeling strategy is addressed. The progressive labeling strategy does not need to consider the distribution of labeled samples to evaluate the distribution of unlabeled samples, can effective solve the problem of evaluate the proportion of positive and negative samples in work set. Our experiment result demonstrate that the proposed technique have great potential in cancer prediction based on gene expression.

  16. Parkinson’s disease managing reversible neurodegeneration

    Science.gov (United States)

    Hinz, Marty; Stein, Alvin; Cole, Ted; McDougall, Beth; Westaway, Mark

    2016-01-01

    Traditionally, the Parkinson’s disease (PD) symptom course has been classified as an irreversible progressive neurodegenerative disease. This paper documents 29 PD and treatment-induced systemic depletion etiologies which cause and/or exacerbate the seven novel primary relative nutritional deficiencies associated with PD. These reversible relative nutritional deficiencies (RNDs) may facilitate and accelerate irreversible progressive neurodegeneration, while other reversible RNDs may induce previously undocumented reversible pseudo-neurodegeneration that is hiding in plain sight since the symptoms are identical to the symptoms being experienced by the PD patient. Documented herein is a novel nutritional approach for reversible processes management which may slow or halt irreversible progressive neurodegenerative disease and correct reversible RNDs whose symptoms are identical to the patient’s PD symptoms. PMID:27103805

  17. Multi-parametric MRI at 14T for muscular dystrophy mice treated with AAV vector-mediated gene therapy.

    Directory of Open Access Journals (Sweden)

    Joshua Park

    Full Text Available The objective of this study was to investigate the efficacy of using quantitative magnetic resonance imaging (MRI as a non-invasive tool for the monitoring of gene therapy for muscular dystrophy. The clinical investigations for this family of diseases often involve surgical biopsy which limits the amount of information that can be obtained due to the invasive nature of the procedure. Thus, other non-invasive tools may provide more opportunities for disease assessment and treatment responses. In order to explore this, dystrophic mdx4cv mice were systemically treated with a recombinant adeno-associated viral (AAV vector containing a codon-optimized micro-dystrophin gene. Multi-parametric MRI of T2, magnetization transfer, and diffusion effects alongside 3-D volume measurements were then utilized to monitor disease/treatment progression. Mice were imaged at 10 weeks of age for pre-treatment, then again post-treatment at 8, 16, and 24 week time points. The efficacy of treatment was assessed by physiological assays for improvements in function and quantification of expression. Tissues from the hindlimbs were collected for histological analysis after the final time point for comparison with MRI results. We found that introduction of the micro-dystrophin gene restored some aspects of normal muscle histology and pathology such as decreased necrosis and resistance to contraction-induced injury. T2 relaxation values showed percentage decreases across all muscle types measured (tibialis anterior, gastrocnemius, and soleus when treated groups were compared to untreated groups. Additionally, the differences between groups were statistically significant for the tibialis anterior as well. The diffusion measurements showed a wider range of percentage changes and less statistical significance while the magnetization transfer effect measurements showed minimal change. MR images displayed hyper-intense regions of muscle that correlated with muscle pathology in

  18. Novel Cocaine Vaccine Linked to a Disrupted Adenovirus Gene Transfer Vector Blocks Cocaine Psychostimulant and Reinforcing Effects

    Science.gov (United States)

    Wee, Sunmee; Hicks, Martin J; De, Bishnu P; Rosenberg, Jonathan B; Moreno, Amira Y; Kaminsky, Stephen M; Janda, Kim D; Crystal, Ronald G; Koob, George F

    2012-01-01

    Immunotherapy is a promising treatment for drug addiction. However, insufficient immune responses to vaccines in most subjects pose a challenge. In this study, we tested the efficacy of a new cocaine vaccine (dAd5GNE) in antagonizing cocaine addiction-related behaviors in rats. This vaccine used a disrupted serotype 5 adenovirus (Ad) gene transfer vector coupled to a third-generation cocaine hapten, termed GNE (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido-hexanoic acid). Three groups of rats were immunized with dAd5GNE. One group was injected with 3H-cocaine, and radioactivity in the blood and brain was determined. A second group was tested for cocaine-induced locomotor sensitization. A third group was examined for cocaine self-administration, extinction, and reinstatement of responding for cocaine. Antibody titers were determined at various time-points. In each experiment, we added a control group that was immunized with dAd5 without a hapten. The vaccination with dAd5GNE produced long-lasting high titers (>105) of anti-cocaine antibodies in all of the rats. The vaccination inhibited cocaine-induced hyperlocomotor activity and sensitization. Vaccinated rats acquired cocaine self-administration, but they showed less motivation to self-administer cocaine under a progressive-ratio schedule than control rats. When cocaine was not available in a session, control rats exhibited ‘extinction burst' responding, whereas vaccinated rats did not. Moreover, when primed with cocaine, vaccinated rats did not reinstate responding, suggesting a blockade of cocaine-seeking behavior. These data strongly suggest that our dAd5GNE vector-based vaccine may be effective in treating cocaine abuse and addiction. PMID:21918504

  19. Metals and Neurodegeneration [version 1; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Pan Chen

    2016-03-01

    Full Text Available Metals play important roles in the human body, maintaining cell structure and regulating gene expression, neurotransmission, and antioxidant response, to name a few. However, excessive metal accumulation in the nervous system may be toxic, inducing oxidative stress, disrupting mitochondrial function, and impairing the activity of numerous enzymes. Damage caused by metal accumulation may result in permanent injuries, including severe neurological disorders. Epidemiological and clinical studies have shown a strong correlation between aberrant metal exposure and a number of neurological diseases, including Alzheimer’s disease, amyotrophic lateral sclerosis, autism spectrum disorders, Guillain–Barré disease, Gulf War syndrome, Huntington’s disease, multiple sclerosis, Parkinson’s disease, and Wilson’s disease. Here, we briefly survey the literature relating to the role of metals in neurodegeneration.

  20. Strategies for clinical approach to neurodegeneration in Amyotrophic lateral sclerosis.

    Science.gov (United States)

    Carlesi, Cecilia; Pasquali, Livia; Piazza, Selina; Lo Gerfo, Annalisa; Caldarazzo Ienco, Elena; Alessi, Rosaria; Fornai, Francesco; Siciliano, Gabriele

    2011-03-01

    Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and ultimately fatal neurodegenerative disorder of unknown aetiology that involves the loss of upper and lower motor neurons in the cerebral cortex, brainstem and spinal cord. Significant progress in understanding the cellular mechanisms of motor neuron degeneration in ALS has not been matched with the development of therapeutic strategies to prevent disease progression, and riluzole remains the only available therapy, with only marginal effects on disease survival. More recently alterations of mRNA processing in genetically defined forms of ALS, as those related to TDP-43 and FUS-TLS gene mutations have provided important insights into the molecular networks implicated in the disease pathogenesis. Here we review some of the recent progress in promoting therapeutic strategies for neurodegeneration.

  1. Octaarginine-modified chitosan as a nonviral gene delivery vector: properties and in vitro transfection efficiency

    International Nuclear Information System (INIS)

    Zhao Xiaoli; Li Zhaoyang; Liu Wenguang; Lam, Wingmoon; Sun Peng; Kao, Richard Y. T.; Luk, Keith D. K.; Lu, William W.

    2011-01-01

    Protein transduction domains (PTD) have been identified to have the capacity to facilitate molecular cargo to translocate through cell membrane. This study aims to utilize the cell membrane penetrating ability of octaarginine oligopeptide, a simplified prototype of the PTD, to enhance the transfection efficiency of chitosan. Octaarginine-modified chitosan (R 8 -CS) was synthesized as a gene transfer carrier by carbodiimide chemistry. The structure and composition of R 8 -CSs were characterized using FTIR and 1 H NMR. Agarose gel electrophoresis assay showed that R 8 -CS could efficiently condense the DNA. The particle size of R 8 -CS/DNA complexes were determined to be around 100–200 nm. The nanoparticle complexes exhibited a spherical and compact morphology. R 8 -CS demonstrated higher transfection activity and lower cytotoxicity as compared to the unmodified chitosan and also showed good serum resistance.

  2. Modelling Gene Flow between Fields of White Clover with Honeybees as Pollen Vectors

    DEFF Research Database (Denmark)

    Løjtnant, Christina; Boelt, Birte; Clausen, Sabine Karin

    2012-01-01

    The portion-dilution model is a parametric restatement of the conventional view of animal pollination; it predicts the level of pollinator-mediated gene dispersal. In this study, the model was applied to white clover (Trifolium repens) and its most frequent pollinator, the honeybee (Apis mellifera......). One of the three parameters in the portion-dilution model is the mean number of flowers a pollinator visits in one foraging bout. An alternative method to estimate this parameter was developed that was not depending on pollinator hive-seeking behaviour. The new estimation method, based on nectar......% with an estimated 95% percentile of 70%. The results show that the European Union threshold limit of 0.9% GM admixture for food and feed will likely be exceeded at times and especially organic farmers that do not accept GM admixture and often have clover and clover–grass fields might face challenges with admixture...

  3. Parkinson’s disease managing reversible neurodegeneration

    Directory of Open Access Journals (Sweden)

    Hinz M

    2016-04-01

    Full Text Available Marty Hinz,1 Alvin Stein,2 Ted Cole,3 Beth McDougall,4 Mark Westaway5 1Clinical Research, NeuroResearch Clinics, Inc., Cape Coral, FL, 2Stein Orthopedic Associates, Plantation, FL, 3Cole Center for Healing, Cincinnati, OH, 4CLEARCenter of Health, Mill Valley, CA, USA; 5Four Pillars Health, Brendale, QLD, Australia Abstract: Traditionally, the Parkinson’s disease (PD symptom course has been classified as an irreversible progressive neurodegenerative disease. This paper documents 29 PD and treatment-induced systemic depletion etiologies which cause and/or exacerbate the seven novel primary relative nutritional deficiencies associated with PD. These reversible relative nutritional deficiencies (RNDs may facilitate and accelerate irreversible progressive neurodegeneration, while other reversible RNDs may induce previously undocumented reversible pseudo-neurodegeneration that is hiding in plain sight since the symptoms are identical to the symptoms being experienced by the PD patient. Documented herein is a novel nutritional approach for reversible processes management which may slow or halt irreversible progressive neurodegenerative disease and correct reversible RNDs whose symptoms are identical to the patient’s PD symptoms. Keywords: Parkinson’s disease, L-dopa, carbidopa, B6, neurodegeneration

  4. Intracellular Cholesterol Trafficking and Impact in Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Fabian Arenas

    2017-11-01

    Full Text Available Cholesterol is a critical component of membrane bilayers where it plays key structural and functional roles by regulating the activity of diverse signaling platforms and pathways. Particularly enriched in brain, cholesterol homeostasis in this organ is singular with respect to other tissues and exhibits a heterogeneous regulation in distinct brain cell populations. Due to the key role of cholesterol in brain physiology and function, alterations in cholesterol homeostasis and levels have been linked to brain diseases and neurodegeneration. In the case of Alzheimer disease (AD, however, this association remains unclear with evidence indicating that either increased or decreased total brain cholesterol levels contribute to this major neurodegenerative disease. Here, rather than analyzing the role of total cholesterol levels in neurodegeneration, we focus on the contribution of intracellular cholesterol pools, particularly in endolysosomes and mitochondria through its trafficking via specialized membrane domains delineated by the contacts between endoplasmic reticulum and mitochondria, in the onset of prevalent neurodegenerative diseases such as AD, Parkinson disease, and Huntington disease as well as in lysosomal disorders like Niemann-Pick type C disease. We dissect molecular events associated with intracellular cholesterol accumulation, especially in mitochondria, an event that results in impaired mitochondrial antioxidant defense and function. A better understanding of the mechanisms involved in the distribution of cholesterol in intracellular compartments may shed light on the role of cholesterol homeostasis disruption in neurodegeneration and may pave the way for specific intervention opportunities.

  5. [Selection and construction of cell line stably expressing survivin gene in lower level through eukaryotic plasmid vector of shRNA].

    Science.gov (United States)

    Wang, Wen-Xia; Sun, Shan-Zhen; Song, Ying

    2008-06-01

    To construct a short hairpin RNA(shRNA) interference expression plasmid vector of survivin gene, transfect tongue squamous cell carcinoma line Tca8113 which expressed survivin gene in a high level, and choose the cells whose survivin gene were suppressed significantly. Two pairs of oligonucleotide sequences specific for survivin gene were designed and synthesized, and cloned into pSilencer-2.1U6-neo plasmid. The recombinant plasmids (named PS1 and PS2) were amplified in Ecoli. DH5alpha was identified by restriction digestion, PCR and sequencing. The vectors were transfected into Tca8113 cells with lipofectamine 2000. After selection with G418, the stable cell clones were attained. Survivn expression was assayed with real-time quantitative PCR and Western blotting. SAS8.0 software package was used for Student t test. Two vectors were constructed successfully and stable cell clones with PS1 or PS2 plasmid were obtained. As compared with those of control, survivin expression of transfected cell with PS1 or PS2 in mRNA level was significantly suppressed (P<0.05). In protein level, only those of transfected cell with PS2 was significantly suppressed (P<0.01). The shRNA interference expression plasmid vectors of survivin gene are successfully constructed, and Tca8113 cells which express survivin gene in a stable lower level are attained, which enable us to carry out further research on gene therapy of oral squamous cell carcinoma. Supported by National Natural Science Foundation of China (Grant No.30572056).

  6. Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

    Directory of Open Access Journals (Sweden)

    Zhao W

    2016-11-01

    Full Text Available Wen Zhao, Yifan Zhang, Xueyun Jiang, Chunying Cui School of Chemical Biology and Pharmaceutical Sciences, Capital Medical University, Beijing, China Abstract: Small interfering RNA (siRNA delivery is a prospective method in gene therapy, but it has application limitations such as negative charge, water solubility and high molecular weight. In this study, a safe and efficient nano-vector, CRS, was designed and synthesized to facilitate siRNA delivery. Physical and chemical properties of VEGF-siRNA/CRS were characterized by methods including scanning electron microscopy (SEM, transmission electron microscopy, zeta potential (ζ measurement, drug-releasing rate measurement, gel electrophoresis and confocal microscopy. The biological activities were evaluated using cell viability assay, gene-silencing efficacy assay in vitro, real-time polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA and antitumor tests in vivo. The mean nanoparticle size of VEGF-siRNA/CRS was 121.4±0.3 nm with positive ζ potential of 7.69±4.47 mV. The release rate of VEGF-siRNA from VEGF-siRNA/CRS was 82.50% sustained for 48 h in Tris-ethylenediaminetetraacetic acid buffer (pH 8.0. Real-time polymerase chain reaction was used to analyze the efficiency of the transfection, and the result showed that VEGF mRNA expression had been knocked down by 82.36%. The expression of VEGF protein was also recorded to be downregulated to 14.83% using ELISA. The results of cytotoxicity measured by Cell Counting Kit-8 assay showed that VEGF-siRNA/CRS had significant inhibitory effect on HeLa cells. The results of antitumor assays indicated that VEGF-siRNA/CRS exhibited tumor cell growth inhibition in vivo. The results demonstrated that VEGF-siRNA could be delivered and transported by the designed carrier, while siRNA could be released constantly and led to an increasing gene-silencing effect against VEGF gene. In conclusion, VEGF-siRNA/CRS is a promising carrier for si

  7. Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector

    Science.gov (United States)

    Peluffo, Hugo; Acarin, Laia; Arís, Anna; González, Pau; Villaverde, Antoni; Castellano, Bernardo; González, Berta

    2006-01-01

    Background Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD) after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn SOD or the control GFP transgenes 2 hours after intracortical N-methyl-D-aspartate (NMDA) administration, and daily functional evaluation was performed. Moreover, 3 days after, lesion volume, neuronal degeneration and nitrotyrosine immunoreactivity were evaluated. Results Overexpression of Cu/Zn SOD transgene after NMDA administration showed improved functional outcome and a reduced lesion volume at 3 days post lesion. In secondary degenerative areas, increased neuronal survival as well as decreased numbers of degenerating neurons and nitrotyrosine immunoreactivity was seen. Interestingly, injection of the NLSCt vector carrying the control GFP transgene also displayed a significant neuroprotective effect but less pronounced. Conclusion When the appropriate levels of Cu/Zn SOD are expressed transiently after injury using the non-viral modular protein vector NLSCt a neuroprotective effect is seen. Thus recombinant modular protein vectors may be suitable for in vivo gene therapy, and Cu/Zn SOD should be considered as an interesting therapeutic transgene. PMID:16638118

  8. Neuroprotection from NMDA excitotoxic lesion by Cu/Zn superoxide dismutase gene delivery to the postnatal rat brain by a modular protein vector

    Directory of Open Access Journals (Sweden)

    Castellano Bernardo

    2006-04-01

    Full Text Available Abstract Background Superoxide mediated oxidative stress is a key neuropathologic mechanism in acute central nervous system injuries. We have analyzed the neuroprotective efficacy of the transient overexpression of antioxidant enzyme Cu/Zn Superoxide dismutase (SOD after excitotoxic injury to the immature rat brain by using a recently constructed modular protein vector for non-viral gene delivery termed NLSCt. For this purpose, animals were injected with the NLSCt vector carrying the Cu/Zn SOD or the control GFP transgenes 2 hours after intracortical N-methyl-D-aspartate (NMDA administration, and daily functional evaluation was performed. Moreover, 3 days after, lesion volume, neuronal degeneration and nitrotyrosine immunoreactivity were evaluated. Results Overexpression of Cu/Zn SOD transgene after NMDA administration showed improved functional outcome and a reduced lesion volume at 3 days post lesion. In secondary degenerative areas, increased neuronal survival as well as decreased numbers of degenerating neurons and nitrotyrosine immunoreactivity was seen. Interestingly, injection of the NLSCt vector carrying the control GFP transgene also displayed a significant neuroprotective effect but less pronounced. Conclusion When the appropriate levels of Cu/Zn SOD are expressed transiently after injury using the non-viral modular protein vector NLSCt a neuroprotective effect is seen. Thus recombinant modular protein vectors may be suitable for in vivo gene therapy, and Cu/Zn SOD should be considered as an interesting therapeutic transgene.

  9. Clinical Heterogeneity of Atypical Pantothenate Kinase-Associated Neurodegeneration in Koreans

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    Jae-Hyeok Lee

    2016-01-01

    Full Text Available Objective Neurodegeneration with brain iron accumulation (NBIA represents a group of inherited movement disorders characterized by iron accumulation in the basal ganglia. Recent advances have included the identification of new causative genes and highlighted the wide phenotypic variation between and within the specific NBIA subtypes. This study aimed to investigate the current status of NBIA in Korea. Methods We collected genetically confirmed NBIA patients from twelve nationwide referral hospitals and from a review of the literature. We conducted a study to describe the phenotypic and genotypic characteristics of Korean adults with atypical pantothenate kinase-associated neurodegeneration (PKAN. Results Four subtypes of NBIA including PKAN (n = 30, PLA2G6-related neurodegeneration (n = 2, beta-propeller protein-associated neurodegeneration (n = 1, and aceruloplasminemia (n = 1 have been identified in the Korean population. The clinical features of fifteen adults with atypical PKAN included early focal limb dystonia, parkinsonism-predominant feature, oromandibular dystonia, and isolated freezing of gait (FOG. Patients with a higher age of onset tended to present with parkinsonism and FOG. The p.R440P and p.D378G mutations are two major mutations that represent approximately 50% of the mutated alleles. Although there were no specific genotype-phenotype correlations, most patients carrying the p.D378G mutation had a late-onset, atypical form of PKAN. Conclusions We found considerable phenotypic heterogeneity in Korean adults with atypical PKAN. The age of onset may influence the presentation of extrapyramidal symptoms.

  10. [Construction of eukaryotic recombinant vector and expression in COS7 cell of LipL32-HlyX fusion gene from Leptospira serovar Lai].

    Science.gov (United States)

    Huang, Bi; Bao, Lang; Zhong, Qi; Zhang, Huidong; Zhang, Ying

    2009-04-01

    This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.

  11. Construction of expression vector containing glnA gene and detection of NPT II activity in the transgenic rice calli using 32P-labelled compound

    International Nuclear Information System (INIS)

    Su Jin; Zhang Xueqin; Yan Qiusheng; Chen Zhangliang; You Chongbiao

    1993-08-01

    The glnA gene encoding glutamine synthetase (GS) was amplified from Azospirillum brasilense Sp7 by PCR technique. the amplified 1.4 kb DNA fragment was cloned at the EcoRV site of Bluescript-SK. Both sequencing and restriction digestion data showed that the 1.4 kb DNA fragment flanked with BamHI site at each end was really the glnA gene of A. brasilense Sp7. The glnA gene was ligated with Bg1 II site of pCo24. As a result, an expression vector pGSC35 with CaMV35S promoter was obtained. Using colony in situ hybridization with α- 32 P-dATP labelled probes to screen the positive clones, another glnA gene expression vector pAGNB92 with rice actin 1 promoter was constructed after three rounds of ligation and transformation. Protoplasts isolated from rice cell suspension line cv. T986 were transformed with glnA expression vectors pGSC35 and pAGNB92 containing neomycin phosphotransferase II (NPTII) gene by using PEG fusion and electroporation. Transformed microcalli were selected on media containing G418 disulfate salt. NPT II activity was detected in 37% of G418 resistant calli by using dot blot hybridization with γ- 32 P-ATP and kanamycin as substrate

  12. Construction of Double Right-Border Binary Vector Carrying Non-Host Gene Rxol Resistant to Bacterial Leaf Streak of Rice

    Institute of Scientific and Technical Information of China (English)

    Xu Mei-rong; XIA Zhi-hui; ZHAI Wen-xue; XU Jian-long; ZHOU Yong-li; LI Zhi-kang

    2008-01-01

    Rxol cloned from maize is a non-host gene resistant to bacterial leaf streak of rice. pCAMBIA1305-1 with Rxol was digested with Sca Ⅰ and NgoM Ⅳ and the double right-border binary vector pMNDRBBin6 was digested with Hpa Ⅰ and Xma Ⅰ.pMNDRBBin6 carrying the gene Rxol was acquired by ligation of blunt-end and cohesive end. The results of PCR, restriction enzyme analysis and sequencing indicated that the Rxol gene had been cloned into pMNDRBBin6. This double right-border binary vector,named as pMNDRBBin6-Rxol, will play a role in breeding marker-free plants resistant to bacterial leaf streak of rice by genetic transformation.

  13. The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae.

    Science.gov (United States)

    Nguyen, Khuyen Thi; Ho, Quynh Ngoc; Pham, Thu Ha; Phan, Tuan-Nghia; Tran, Van-Tuan

    2016-12-01

    Aspergillus oryzae is a safe mold widely used in food industry. It is also considered as a microbial cell factory for production of recombinant proteins and enzymes. Currently, genetic manipulation of filamentous fungi is achieved via Agrobacterium tumefaciens-mediated transformation methods usually employing antibiotic resistance markers. These methods are hardly usable for A. oryzae due to its strong resistance to the common antifungal compounds used for fungal transformation. In this study, we have constructed two binary vectors carrying the pyrG gene from A. oryzae as a biochemical marker than an antibiotic resistance marker, and an expression cassette for GFP or DsRed reporter gene under control of the constitutive gpdA promoter from Aspergillus nidulans. All components of these vectors are changeable to generate new versions for specific research purposes. The developed vectors are fully functional for heterologous expression of the GFP and DsRed fluorescent proteins in the uridine/uracil auxotrophic A. oryzae strain. Our study provides a new approach for A. oryzae transformation using pyrG as the selectable auxotrophic marker, A. tumefaciens as the DNA transfer tool and fungal spores as the transformation material. The binary vectors constructed can be used for gene expression studies in this industrially important filamentous fungus.

  14. A gene delivery system with a human artificial chromosome vector based on migration of mesenchymal stem cells towards human glioblastoma HTB14 cells.

    Science.gov (United States)

    Kinoshita, Yusuke; Kamitani, Hideki; Mamun, Mahabub Hasan; Wasita, Brian; Kazuki, Yasuhiro; Hiratsuka, Masaharu; Oshimura, Mitsuo; Watanabe, Takashi

    2010-05-01

    Mesenchymal stem cells (MSCs) have been expected to become useful gene delivery vehicles against human malignant gliomas when coupled with an appropriate vector system, because they migrate towards the lesion. Human artificial chromosomes (HACs) are non-integrating vectors with several advantages for gene therapy, namely, no limitations on the size and number of genes that can be inserted. We investigated the migration of human immortalized MSCs bearing a HAC vector containing the herpes simplex virus thymidine kinase gene (HAC-tk-hiMSCs) towards malignant gliomas in vivo. Red fluorescence protein-labeled human glioblastoma HTB14 cells were implanted into a subcortical region in nude mice. Four days later, green fluorescence protein-labeled HAC-tk-hiMSCs were injected into a contralateral subcortical region (the HTB14/HAC-tk-hiMSC injection model). Tropism to the glioma mass and the route of migration were visualized by fluorescence microscopy and immunohistochemical staining. HAC-tk-hiMSCs began to migrate toward the HTB14 glioma area via the corpus callosum on day 4, and gathered around the HTB14 glioma mass on day 7. To test whether the delivered gene could effectively treat glioblastoma in vivo, HTB14/HAC-tk-hiMSC injected mice were treated with ganciclovir (GCV) or PBS. The HTB14 glioma mass was significantly reduced by GCV treatment in mice injected with HAC-tk-hiMSCs. It was confirmed that gene delivery by our HAC-hiMSC system was effective after migration of MSCs to the glioma mass in vivo. Therefore, MSCs containing HACs carrying an anticancer gene or genes may provide a new tool for the treatment of malignant gliomas and possibly of other tumor types.

  15. Paraquat and maneb co-exposure induces noradrenergic locus coeruleus neurodegeneration through NADPH oxidase-mediated microglial activation

    International Nuclear Information System (INIS)

    Hou, Liyan; Zhang, Cong; Wang, Ke; Liu, Xiaofang; Wang, Hongwei; Che, Yuning; Sun, Fuqiang; Zhou, Xueying; Zhao, Xiulan; Wang, Qingshan

    2017-01-01

    Highlights: • Microglial activation induced by paraquat and maneb precedes noradrenergic neurodegeneration in locus coeruleus. • NADPH oxidase activation contributes to microglia-mediated neuroinflammation and related noradrenergic neurodegeneration. • Inhibition of NADPH oxidase by apocynin protects noradrenergic neurons against paraquat and maneb-induced toxicity. - Abstract: Co-exposure to paraquat (PQ) and maneb (Mb) has been shown to increase the risk of Parkinson’s disease (PD) and dopaminergic (DA) neurodegeneration in the substantia nigra pars compacta (SNpc) is observed in PQ and Mb-treated experimental animals. The loss of noradrenergic locus coeruleus (LC/NE) neurons in brainstem is a common feature shared by multiple neurodegenerative diseases, including PD. However, whether PQ and Mb is able to damage LC/NE neurons remains undefined. In this study, mice treated with combined PQ and Mb displayed progressive LC/NE neurodegeneration. Time course studies revealed that the activation of microglia preceded LC/NE neurodegeneration. Mechanistically, the activation of NADPH oxidase contributed to microglial activation and subsequent LC/NE neurodegeneration. We found that PQ and Mb co-exposure induced activation of NADPH oxidase as shown by increased superoxide production and membrane translocation of p47 phox , a cytosolic subunit of NADPH oxidase. Inhibition of NADPH oxidase by apocynin, a widely used NADPH oxidase inhibitor, suppressed microglial activation and gene expressions of proinflammatory factors. Furthermore, reduced activation of nuclear factor-κB (NF-κB) pathway was observed in apocynin-treated mice. More importantly, inhibition of NADPH oxidase by apocynin afforded LC/NE neuroprotection against PQ and Mb-induced neurotoxicity. Thus, our findings revealed the critical role NADPH oxidase-mediated microglial activation in driving LC/NE neurodegeneration induced by PQ and Mb, providing new insights into the pathogenesis of environmental

  16. Stable replication of the EBNA1/OriP-mediated baculovirus vector and its application to anti-HCV gene therapy

    Directory of Open Access Journals (Sweden)

    Chang Myint OO

    2009-10-01

    Full Text Available Abstract Background Hepatitis C virus (HCV is one of the main causes of liver-related morbidity and mortality. Although combined interferon-α-ribavirin therapy is effective for about 50% of the patients with HCV, better therapies are needed and preventative vaccines have yet to be developed. Short-hairpin RNAs (shRNAs inhibit gene expression by RNA interference. The application of transient shRNA expression is limited, however, due to the inability of the shRNA to replicate in mammalian cells and its inefficient transduction. The duration of transgene (shRNA expression in mammalian cells can be significantly extended using baculovirus-based shRNA-expressing vectors that contain the latent viral protein Epstein-Barr nuclear antigen 1 (EBNA1 and the origin of latent viral DNA replication (OriP sequences. These recombinant vectors contain compatible promoters and are highly effective for infecting primary hepatocyte and hepatoma cell lines, making them very useful tools for studies of hepatitis B and hepatitis C viruses. Here, we report the use of these baculovirus-based vector-derived shRNAs to inhibit core-protein expression in full-length hepatitis C virus (HCV replicon cells. Results We constructed a long-term transgene shRNA expression vector that contains the EBV EBNA1 and OriP sequences. We also designed baculovirus vector-mediated shRNAs against the highly conserved core-protein region of HCV. HCV core protein expression was inhibited by the EBNA1/OriP baculovirus vector for at least 14 days, which was considerably longer than the 3 days of inhibition produced by the wild-type baculovirus vector. Conclusion These findings indicate that we successfully constructed a long-term transgene (shRNA expression vector (Ac-EP-shRNA452 using the EBNA1/OriP system, which was propagated in Escherichia coli and converted into mammalian cells. The potential anti-HCV activity of the long-term transgene (shRNA expression vector was evaluated with the view

  17. Homology Requirements for Efficient, Footprintless Gene Editing at the CFTR Locus in Human iPSCs with Helper-dependent Adenoviral Vectors

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    Donna J Palmer

    2016-01-01

    Full Text Available Helper-dependent adenoviral vectors mediate high efficiency gene editing in induced pluripotent stem cells without needing a designer nuclease thereby avoiding off-target cleavage. Because of their large cloning capacity of 37 kb, helper-dependent adenoviral vectors with long homology arms are used for gene editing. However, this makes vector construction and recombinant analysis difficult. Conversely, insufficient homology may compromise targeting efficiency. Thus, we investigated the effect of homology length on helper-dependent adenoviral vector targeting efficiency at the cystic fibrosis transmembrane conductance regulator locus in induced pluripotent stem cells and found a positive correlation. With 23.8 and 21.4 kb of homology, the frequencies of targeted recombinants were 50–64.6% after positive selection for vector integration, and 97.4–100% after negative selection against random integrations. With 14.8 kb, the frequencies were 26.9–57.1% after positive selection and 87.5–100% after negative selection. With 9.6 kb, the frequencies were 21.4 and 75% after positive and negative selection, respectively. With only 5.6 kb, the frequencies were 5.6–16.7% after positive selection and 50% after negative selection, but these were more than high enough for efficient identification and isolation of targeted clones. Furthermore, we demonstrate helper-dependent adenoviral vector-mediated footprintless correction of cystic fibrosis transmembrane conductance regulator mutations through piggyBac excision of the selectable marker. However, low frequencies (≤ 1 × 10−3 necessitated negative selection for piggyBac-excision product isolation.

  18. Vascular Changes and Neurodegeneration in the Early Stages of Diabetic Retinopathy

    DEFF Research Database (Denmark)

    Jonsson, Karoline Boegeberg; Frydkjaer-Olsen, Ulrik; Grauslund, Jakob

    2016-01-01

    INTRODUCTION: Neurodegeneration is an early component of diabetic retinopathy (DR). It is unclear whether neurodegeneration is an independent factor or a consequence of damaged retinal vasculature. The aims of this study were to review the literature concerning neurodegeneration in diabetic...

  19. Food-grade host/vector expression system for Lactobacillus casei based on complementation of plasmid-associated phospho-beta-galactosidase gene lacG.

    Science.gov (United States)

    Takala, T M; Saris, P E J; Tynkkynen, S S H

    2003-01-01

    A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.

  20. Molecular Mechanisms of Neurodegeneration in Spinal Muscular Atrophy

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    Saif Ahmad

    2016-01-01

    Full Text Available Spinal muscular atrophy (SMA is an autosomal recessive motor neuron disease with a high incidence and is the most common genetic cause of infant mortality. SMA is primarily characterized by degeneration of the spinal motor neurons that leads to skeletal muscle atrophy followed by symmetric limb paralysis, respiratory failure, and death. In humans, mutation of the Survival Motor Neuron 1 (SMN1 gene shifts the load of expression of SMN protein to the SMN2 gene that produces low levels of full-length SMN protein because of alternative splicing, which are sufficient for embryonic development and survival but result in SMA. The molecular mechanisms of the (a regulation of SMN gene expression and (b degeneration of motor neurons caused by low levels of SMN are unclear. However, some progress has been made in recent years that have provided new insights into understanding of the cellular and molecular basis of SMA pathogenesis. In this review, we have briefly summarized recent advances toward understanding of the molecular mechanisms of regulation of SMN levels and signaling mechanisms that mediate neurodegeneration in SMA.

  1. Transcriptional Response of Human Neurospheres to Helper-Dependent CAV-2 Vectors Involves the Modulation of DNA Damage Response, Microtubule and Centromere Gene Groups.

    Directory of Open Access Journals (Sweden)

    Stefania Piersanti

    Full Text Available Brain gene transfer using viral vectors will likely become a therapeutic option for several disorders. Helper-dependent (HD canine adenovirus type 2 vectors (CAV-2 are well suited for this goal. These vectors are poorly immunogenic, efficiently transduce neurons, are retrogradely transported to afferent structures in the brain and lead to long-term transgene expression. CAV-2 vectors are being exploited to unravel behavior, cognition, neural networks, axonal transport and therapy for orphan diseases. With the goal of better understanding and characterizing HD-CAV-2 for brain therapy, we analyzed the transcriptomic modulation induced by HD-CAV-2 in human differentiated neurospheres derived from midbrain progenitors. This 3D model system mimics several aspects of the dynamic nature of human brain. We found that differentiated neurospheres are readily transduced by HD-CAV-2 and that transduction generates two main transcriptional responses: a DNA damage response and alteration of centromeric and microtubule probes. Future investigations on the biochemistry of processes highlighted by probe modulations will help defining the implication of HD-CAV-2 and CAR receptor binding in enchaining these functional pathways. We suggest here that the modulation of DNA damage genes is related to viral DNA, while the alteration of centromeric and microtubule probes is possibly enchained by the interaction of the HD-CAV-2 fibre with CAR.

  2. Site-specific integration of CAR gene into Jurkat T cells with a linear close-ended AAV-based DNA vector for CAR-T engineering.

    Science.gov (United States)

    Zhang, Yun; Liu, Xiaomei; Zhang, Jinju; Zhang, Chun

    2016-09-01

    To develop a site-specific integration strategy for CAR-T engineering by using a non-viral vector dependent on adeno-associated viral (AAV) genome, which tends to be integrated into AAVS1 site with the help of its Rep proteins. AAV-dependent vectors were produced in Sf9 cells. Structural analyses revealed the vector as covalently close-ended, linear duplex molecules, which was termed "CELiD" DNA. A plasmid CMV-Rep was constructed to express the integrases Rep78 and Rep68. Jurkat cells were co-electroporated with "CELiD" DNA and plasmid CMV-Rep in order to specifically integrate CAR gene into AAVS1 site. We examined 71 stably transfected Jurkat clones by nested PCR, sequencing and southern blotting, of which 30 clones bore CAR gene within AAVS1 site. The site-specific integration efficiency was nearly 42.2 %. The AAV-dependent vector preferentially integrated CAR into AAVS1 site, which could be further used in human T cell modification and enhance the security of CAR-T therapy.

  3. A small and efficient dimerization/packaging signal of rat VL30 RNA and its use in murine leukemia virus-VL30-derived vectors for gene transfer.

    Science.gov (United States)

    Torrent, C; Gabus, C; Darlix, J L

    1994-02-01

    Retroviral genomes consist of two identical RNA molecules associated at their 5' ends by the dimer linkage structure located in the packaging element (Psi or E) necessary for RNA dimerization in vitro and packaging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi + sequence of 600 nucleotides directs the packaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and inserted into an MLV-derived vector lacking MLV Psi +, it directed the efficient encapsidation of recombinant RNAs into MLV virions. Because this VL30 packaging signal is smaller and more efficient in packaging recombinant RNAs than the MLV Psi + and does not contain gag or glyco-gag coding sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent viruses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer.

  4. Expression of heterologous genes from an IRES translational cassette in replication-competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Jespersen, T.; Duch, M.; Carrasco, M.L.

    1999-01-01

    of spliced env mRNA for the SL3-3 derived vector relative to the Akv derived vectors, seemingly contributing to its low replication capacity. The EGFP expressing Akv-MLV was genetically stable for multiple rounds of infection; marker-cassette deletion revertants appeared after several replication rounds...

  5. Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells.

    Science.gov (United States)

    Huo, Wenying; Zhao, Guannan; Yin, Jinggang; Ouyang, Xuan; Wang, Yinan; Yang, Chuanhe; Wang, Baojing; Dong, Peixin; Wang, Zhixiang; Watari, Hidemichi; Chaum, Edward; Pfeffer, Lawrence M; Yue, Junming

    2017-01-01

    CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells.

  6. A sensitive, support-vector-machine method for the detection of horizontal gene transfers in viral, archaeal and bacterial genomes.

    Science.gov (United States)

    Tsirigos, Aristotelis; Rigoutsos, Isidore

    2005-01-01

    In earlier work, we introduced and discussed a generalized computational framework for identifying horizontal transfers. This framework relied on a gene's nucleotide composition, obviated the need for knowledge of codon boundaries and database searches, and was shown to perform very well across a wide range of archaeal and bacterial genomes when compared with previously published approaches, such as Codon Adaptation Index and C + G content. Nonetheless, two considerations remained outstanding: we wanted to further increase the sensitivity of detecting horizontal transfers and also to be able to apply the method to increasingly smaller genomes. In the discussion that follows, we present such a method, Wn-SVM, and show that it exhibits a very significant improvement in sensitivity compared with earlier approaches. Wn-SVM uses a one-class support-vector machine and can learn using rather small training sets. This property makes Wn-SVM particularly suitable for studying small-size genomes, similar to those of viruses, as well as the typically larger archaeal and bacterial genomes. We show experimentally that the new method results in a superior performance across a wide range of organisms and that it improves even upon our own earlier method by an average of 10% across all examined genomes. As a small-genome case study, we analyze the genome of the human cytomegalovirus and demonstrate that Wn-SVM correctly identifies regions that are known to be conserved and prototypical of all beta-herpesvirinae, regions that are known to have been acquired horizontally from the human host and, finally, regions that had not up to now been suspected to be horizontally transferred. Atypical region predictions for many eukaryotic viruses, including the alpha-, beta- and gamma-herpesvirinae, and 123 archaeal and bacterial genomes, have been made available online at http://cbcsrv.watson.ibm.com/HGT_SVM/.

  7. Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector.

    Science.gov (United States)

    Herzog, R W; Yang, E Y; Couto, L B; Hagstrom, J N; Elwell, D; Fields, P A; Burton, M; Bellinger, D A; Read, M S; Brinkhous, K M; Podsakoff, G M; Nichols, T C; Kurtzman, G J; High, K A

    1999-01-01

    Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5x10(12) vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

  8. Construction of Expression Vector for Anti-Alpha-Fetoprotein Gene and Its Inhibition Effects on Alpha-Fetoprotein Positive Hepg2 Cells

    Science.gov (United States)

    Wang, Ze; Zhang, Hui

    As research previously demonstrated, suppression of AFP expression or its biological activities might inhibit the proliferation of AFP positive human hepatocellular carcinoma cells. In this study, we constructed an anti-AFP gene vector and transfected it to HepG2 cells. RT-PCR showed AFP gene expression in the transfected cells was reduced. MTT assay suggested the proliferation of the transfected cells was also inhibited comparing with the untransfected cells. This result provides a new insight into AFP as the target for preventing and treating hepatocellular carcinoma.

  9. Mutations in the Heme Exporter FLVCR1 Cause Sensory Neurodegeneration with Loss of Pain Perception.

    Science.gov (United States)

    Chiabrando, Deborah; Castori, Marco; di Rocco, Maja; Ungelenk, Martin; Gießelmann, Sebastian; Di Capua, Matteo; Madeo, Annalisa; Grammatico, Paola; Bartsch, Sophie; Hübner, Christian A; Altruda, Fiorella; Silengo, Lorenzo; Tolosano, Emanuela; Kurth, Ingo

    2016-12-01

    Pain is necessary to alert us to actual or potential tissue damage. Specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. Pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (HSANs). Although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. Using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the FLVCR1 (Feline Leukemia Virus subgroup C Receptor 1) gene, which encodes a broadly expressed heme exporter. Different FLVCR1 isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. Mutations in FLVCR1 have previously been linked to vision impairment and posterior column ataxia in humans, but not to HSAN. Using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the FLVCR1-mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. Our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans.

  10. The Drosophila carbonyl reductase sniffer prevents oxidative stress-induced neurodegeneration.

    Science.gov (United States)

    Botella, Jose A; Ulschmid, Julia K; Gruenewald, Christoph; Moehle, Christoph; Kretzschmar, Doris; Becker, Katja; Schneuwly, Stephan

    2004-05-04

    A growing body of evidence suggests that oxidative stress is a common underlying mechanism in the pathogenesis of neurodegenerative disorders such as Alzheimer's, Huntington's, Creutzfeld-Jakob and Parkinson's diseases. Despite the increasing number of reports finding a causal relation between oxidative stress and neurodegeneration, little is known about the genetic elements that confer protection against the deleterious effects of oxidation in neurons. We have isolated and characterized the Drosophila melanogaster gene sniffer, whose function is essential for preventing age-related neurodegeneration. In addition, we demonstrate that oxidative stress is a direct cause of neurodegeneration in the Drosophila central nervous system and that reduction of sniffer activity leads to neuronal cell death. The overexpression of the gene confers neuronal protection against oxygen-induced apoptosis, increases resistance of flies to experimental normobaric hyperoxia, and improves general locomotor fitness. Sniffer belongs to the family of short-chain dehydrogenase/reductase (SDR) enzymes and exhibits carbonyl reductase activity. This is the first in vivo evidence of the direct and important implication of this enzyme as a neuroprotective agent in the cellular defense mechanisms against oxidative stress.

  11. Mutations in the Heme Exporter FLVCR1 Cause Sensory Neurodegeneration with Loss of Pain Perception.

    Directory of Open Access Journals (Sweden)

    Deborah Chiabrando

    2016-12-01

    Full Text Available Pain is necessary to alert us to actual or potential tissue damage. Specialized nerve cells in the body periphery, so called nociceptors, are fundamental to mediate pain perception and humans without pain perception are at permanent risk for injuries, burns and mutilations. Pain insensitivity can be caused by sensory neurodegeneration which is a hallmark of hereditary sensory and autonomic neuropathies (HSANs. Although mutations in several genes were previously associated with sensory neurodegeneration, the etiology of many cases remains unknown. Using next generation sequencing in patients with congenital loss of pain perception, we here identify bi-allelic mutations in the FLVCR1 (Feline Leukemia Virus subgroup C Receptor 1 gene, which encodes a broadly expressed heme exporter. Different FLVCR1 isoforms control the size of the cytosolic heme pool required to sustain metabolic activity of different cell types. Mutations in FLVCR1 have previously been linked to vision impairment and posterior column ataxia in humans, but not to HSAN. Using fibroblasts and lymphoblastoid cell lines from patients with sensory neurodegeneration, we here show that the FLVCR1-mutations reduce heme export activity, enhance oxidative stress and increase sensitivity to programmed cell death. Our data link heme metabolism to sensory neuron maintenance and suggest that intracellular heme overload causes early-onset degeneration of pain-sensing neurons in humans.

  12. Dynamic properties of the Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when challenged with vector-borne viral and plasmid genes and protospacers

    DEFF Research Database (Denmark)

    Guðbergsdóttir, Sóley Ruth; Deng, Ling; Chen, Zhengjun

    2011-01-01

    The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching...... individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different...... protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers...

  13. Development of a duplex real-time RT-qPCR assay to monitor genome replication, gene expression and gene insert stability during in vivo replication of a prototype live attenuated canine distemper virus vector encoding SIV gag.

    Science.gov (United States)

    Coleman, John W; Wright, Kevin J; Wallace, Olivia L; Sharma, Palka; Arendt, Heather; Martinez, Jennifer; DeStefano, Joanne; Zamb, Timothy P; Zhang, Xinsheng; Parks, Christopher L

    2015-03-01

    Advancement of new vaccines based on live viral vectors requires sensitive assays to analyze in vivo replication, gene expression and genetic stability. In this study, attenuated canine distemper virus (CDV) was used as a vaccine delivery vector and duplex 2-step quantitative real-time RT-PCR (RT-qPCR) assays specific for genomic RNA (gRNA) or mRNA have been developed that concurrently quantify coding sequences for the CDV nucleocapsid protein (N) and a foreign vaccine antigen (SIV Gag). These amplicons, which had detection limits of about 10 copies per PCR reaction, were used to show that abdominal cavity lymphoid tissues were a primary site of CDV vector replication in infected ferrets, and importantly, CDV gRNA or mRNA was undetectable in brain tissue. In addition, the gRNA duplex assay was adapted for monitoring foreign gene insert genetic stability during in vivo replication by analyzing the ratio of CDV N and SIV gag genomic RNA copies over the course of vector infection. This measurement was found to be a sensitive probe for assessing the in vivo genetic stability of the foreign gene insert. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep

    Science.gov (United States)

    Davey, Marcus G.; Riley, John S.; Andrews, Abigail; Tyminski, Alec; Limberis, Maria; Pogoriler, Jennifer E.; Partridge, Emily; Olive, Aliza; Hedrick, Holly L.; Flake, Alan W.; Peranteau, William H.

    2017-01-01

    A major limitation to adeno-associated virus (AAV) gene therapy is the generation of host immune responses to viral vector antigens and the transgene product. The ability to induce immune tolerance to foreign protein has the potential to overcome this host immunity. Acquisition and maintenance of tolerance to viral vector antigens and transgene products may also permit repeat administration thereby enhancing therapeutic efficacy. In utero gene transfer (IUGT) takes advantage of the immunologic immaturity of the fetus to induce immune tolerance to foreign antigens. In this large animal study, in utero administration of AAV6.2, AAV8 and AAV9 expressing green fluorescent protein (GFP) to ~60 day fetal sheep (term: ~150 days) was performed. Transgene expression and postnatal immune tolerance to GFP and viral antigens were assessed. We demonstrate 1) hepatic expression of GFP 1 month following in utero administration of AAV6.2.GFP and AAV8.GFP, 2) in utero recipients of either AAV6.2.GFP or AAV8.GFP fail to mount an anti-GFP antibody response following postnatal GFP challenge and lack inflammatory cellular infiltrates at the intramuscular site of immunization, 3) a serotype specific anti-AAV neutralizing antibody response is elicited following postnatal challenge of in utero recipients of AAV6.2 or AAV8 with the corresponding AAV serotype, and 4) durable hepatic GFP expression was observed up to 6 months after birth in recipients of AAV8.GFP but expression was lost between 1 and 6 months of age in recipients of AAV6.2.GFP. The current study demonstrates, in a preclinical large animal model, the potential of IUGT to achieve host immune tolerance to the viral vector transgene product but also suggests that a single exposure to the vector capsid proteins at the time of IUGT is inadequate to induce tolerance to viral vector antigens. PMID:28141818

  15. In vitro and in vivo gene therapy with CMV vector-mediated presumed dog beta-nerve growth factor in pyridoxine-induced neuropathy dogs.

    Science.gov (United States)

    Chung, Jin Young; Choi, Jung Hoon; Shin, Il Seob; Choi, Eun Wha; Hwang, Cheol Yong; Lee, Sang Koo; Youn, Hwa Young

    2008-12-01

    Due to the therapeutic potential of gene therapy for neuronal injury, many studies of neurotrophic factors, vectors, and animal models have been performed. The presumed dog beta-nerve growth factor (pdbeta-NGF) was generated and cloned and its expression was confirmed in CHO cells. The recombinant pdbeta-NGF protein reacted with a human beta-NGF antibody and showed bioactivity in PC12 cells. The pdbeta-NGF was shown to have similar bioactivity to the dog beta-NGF. The recombinant pdbeta-NGF plasmid was administrated into the intrathecal space in the gene therapy group. Twenty-four hours after the vector inoculation, the gene therapy group and the positive control group were intoxicated with excess pyridoxine for seven days. Each morning throughout the test period, the dogs' body weight was taken and postural reaction assessments were made. Electrophysiological recordings were performed twice, once before the experiment and once after the test period. After the experimental period, histological analysis was performed. Dogs in the gene therapy group had no weight change and were normal in postural reaction assessments. Electrophysiological recordings were also normal for the gene therapy group. Histological analysis showed that neither the axons nor the myelin of the dorsal funiculus of L4 were severely damaged in the gene therapy group. In addition, the dorsal root ganglia of L4 and the peripheral nerves (sciatic nerve) did not experience severe degenerative changes in the gene therapy group. This study is the first to show the protective effect of NGF gene therapy in a dog model.

  16. Good Laboratory Practice Preclinical Safety Studies for GSK2696273 (MLV Vector-Based Ex Vivo Gene Therapy for Adenosine Deaminase Deficiency Severe Combined Immunodeficiency) in NSG Mice.

    Science.gov (United States)

    Carriglio, Nicola; Klapwijk, Jan; Hernandez, Raisa Jofra; Vezzoli, Michela; Chanut, Franck; Lowe, Rhiannon; Draghici, Elena; Nord, Melanie; Albertini, Paola; Cristofori, Patrizia; Richards, Jane; Staton, Hazel; Appleby, Jonathan; Aiuti, Alessandro; Sauer, Aisha V

    2017-03-01

    GSK2696273 (autologous CD34+ cells transduced with retroviral vector that encodes for the human adenosine deaminase [ADA] enzyme) is a gamma-retroviral ex vivo gene therapy of bone marrow-derived CD34+ cells for the treatment of adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID). ADA-SCID is a severe monogenic disease characterized by immunologic and nonimmunologic symptoms. Bone-marrow transplant from a matched related donor is the treatment of choice, but it is available for only a small proportion of patients. Ex vivo gene therapy of patient bone-marrow CD34+ cells is an alternative treatment. In order to prepare for a marketing authorization application in the European Union, preclinical safety studies in mice were requested by the European Medicines Agency (EMA). A pilot study and a main biodistribution study were performed according to Good Laboratory Practice (GLP) at the San Raffaele Telethon Institute for Gene Therapy test facility. In the main study, human umbilical cord blood (UCB)-derived CD34+ cells were transduced with gamma-retroviral vector used in the production of GSK2696273. Groups of 10 male and 10 female NOD-SCID gamma (NSG) mice were injected intravenously with a single dose of transduced- or mock-transduced UCB CD34+ cells, and they were observed for 4 months. Engraftment and multilineage differentiation of blood cells was observed in the majority of animals in both groups. There was no significant difference in the level of chimerism between the two groups. In the gene therapy group, vector was detectable in lymphohemopoietic and nonlymphohemopoietic tissues, consistent with the presence of gene-modified human hematopoietic donor cells. Given the absence of relevant safety concerns in the data, the nonclinical studies and the clinical experience with GSK2696273 supported a successful application for market authorization in the European Union for the treatment of ADA-SCID patients, for whom no suitable human leukocyte

  17. Adeno-associated virus gene therapy vector scAAVIGF-I for transduction of equine articular chondrocytes and RNA-seq analysis.

    Science.gov (United States)

    Hemphill, D D; McIlwraith, C W; Slayden, R A; Samulski, R J; Goodrich, L R

    2016-05-01

    IGF-I is one of several anabolic factors being investigated for the treatment of osteoarthritis (OA). Due to the short biological half-life, extended administration is required for more robust cartilage healing. Here we create a self-complimentary adeno-associated virus (AAV) gene therapy vector utilizing the transgene for IGF-I. Various biochemical assays were performed to investigate the cellular response to scAAVIGF-I treatment vs an scAAVGFP positive transduction control and a negative for transduction control culture. RNA-sequencing analysis was also performed to establish a differential regulation profile of scAAVIGF-I transduced chondrocytes. Biochemical analyses indicated an average media IGF-I concentration of 608 ng/ml in the scAAVIGF-I transduced chondrocytes. This increase in IGF-I led to increased expression of collagen type II and aggrecan and increased protein concentrations of cellular collagen type II and media glycosaminoglycan vs both controls. RNA-seq revealed a global regulatory pattern consisting of 113 differentially regulated GO categories including those for chondrocyte and cartilage development and regulation of apoptosis. This research substantiates that scAAVIGF-I gene therapy vector increased production of IGF-I to clinically relevant levels with a biological response by chondrocytes conducive to increased cartilage healing. The RNA-seq further established a set of differentially expressed genes and gene ontologies induced by the scAAVIGF-I vector while controlling for AAV infection. This dataset provides a static representation of the cellular transcriptome that, while only consisting of one time point, will allow for further gene expression analyses to compare additional cartilage healing therapeutics or a transient cellular response. Copyright © 2015. Published by Elsevier Ltd.

  18. The db/db mouse: a useful model for the study of diabetic retinal neurodegeneration.

    Directory of Open Access Journals (Sweden)

    Patricia Bogdanov

    Full Text Available BACKGROUND: To characterize the sequential events that are taking place in retinal neurodegeneration in a murine model of spontaneous type 2 diabetes (db/db mouse. METHODS: C57BLKsJ-db/db mice were used as spontaneous type 2 diabetic animal model, and C57BLKsJ-db/+ mice served as the control group. To assess the chronological sequence of the abnormalities the analysis was performed at different ages (8, 16 and 24 weeks. The retinas were evaluated in terms of morphological and functional abnormalities [electroretinography (ERG]. Histological markers of neurodegeneration (glial activation and apoptosis were evaluated by immunohistochemistry. In addition glutamate levels and glutamate/aspartate transporter (GLAST expression were assessed. Furthermore, to define gene expression changes associated with early diabetic retinopathy a transcriptome analyses was performed at 8 week. Furthermore, an additional interventional study to lower blood glucose levels was performed. RESULTS: Glial activation was higher in diabetic than in non diabetic mice in all the stages (p<0.01. In addition, a progressive loss of ganglion cells and a significant reduction of neuroretinal thickness were also observed in diabetic mice. All these histological hallmarks of neurodegeneration were less pronounced at week 8 than at week 16 and 24. Significant ERG abnormalities were present in diabetic mice at weeks 16 and 24 but not at week 8. Moreover, we observed a progressive accumulation of glutamate in diabetic mice associated with an early downregulation of GLAST. Morphological and ERG abnormalities were abrogated by lowering blood glucose levels. Finally, a dysregulation of several genes related to neurotransmission and oxidative stress such as UCP2 were found at week 8. CONCLUSIONS: Our results suggest that db/db mouse reproduce the features of the neurodegenerative process that occurs in the human diabetic eye. Therefore, it seems an appropriate model for investigating the

  19. Ex vivo adenoviral vector-mediated neurotrophin gene transfer to olfactory ensheathing glia : effects on rubrospinal tract regeneration, lesion size, and functional recovery after implantation in the injured rat spinal cord

    NARCIS (Netherlands)

    Ruitenberg, Marc J; Plant, Giles W; Hamers, Frank P T; Wortel, Joke; Blits, Bas; Dijkhuizen, Paul A; Gispen, Willem Hendrik; Boer, Gerard J; Verhaagen, J.

    2003-01-01

    The present study uniquely combines olfactory ensheathing glia (OEG) implantation with ex vivo adenoviral (AdV) vector-based neurotrophin gene therapy in an attempt to enhance regeneration after cervical spinal cord injury. Primary OEG were transduced with AdV vectors encoding rat brain-derived

  20. Differential adenoassociated virus vector-driven expression of a neuropeptide Y gene in primary rat brain astroglial cultures after transfection with Sendai virosomes versus Lipofectin.

    Science.gov (United States)

    de Fiebre, C M; Wu, P; Notabartolo, D; Millard, W J; Meyer, E M

    1994-06-01

    The ability of Sendai virosomes or Lipofectin to introduce an AAV vector into primary rat brain astroglial cultures was characterized. The pJDT95npy vector was constructed by inserting rat NPY cDNA downstream from the indigenous AAV p5, p19 and p40 promoters in pJDT95. Lipofectin-mediated transfection with pJDT95npy (10 micrograms) resulted in pronounced expression of several NPY mRNA species: p5-driven (3.3 kb), p19-driven (2.7 kb) and p40-driven (0.6, 0.8, 1.1, and 1.8 kb). Exposure to virosomally encapsulated pJDT95npy (50 or 100 ng) resulted in transient expression of some p40-driven mRNA species (0.8 and 1.8 kb). Neither method produced astroglia cells which synthesized mature NPY immunoreactivity. This demonstrates that an AAV-derived vector can drive gene expression in astroglia, that Sendai virosomes can infuse vectors into astroglia, but that the amount of DNA infused in this manner may limit long term expression.

  1. CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection

    Science.gov (United States)

    Liu, Jingjing; Zhang, Di; Kimata, Jason T.; Zhou, Paul

    2014-01-01

    CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1. PMID:25541967

  2. Insights into Mechanisms of Chronic Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Abigail B. Diack

    2016-01-01

    Full Text Available Chronic neurodegenerative diseases such as Alzheimer’s disease (AD, Parkinson’s disease (PD, and prion diseases are characterised by the accumulation of abnormal conformers of a host encoded protein in the central nervous system. The process leading to neurodegeneration is still poorly defined and thus development of early intervention strategies is challenging. Unique amongst these diseases are Transmissible Spongiform Encephalopathies (TSEs or prion diseases, which have the ability to transmit between individuals. The infectious nature of these diseases has permitted in vivo and in vitro modelling of the time course of the disease process in a highly reproducible manner, thus early events can be defined. Recent evidence has demonstrated that the cell-to-cell spread of protein aggregates by a “prion-like mechanism” is common among the protein misfolding diseases. Thus, the TSE models may provide insights into disease mechanisms and testable hypotheses for disease intervention, applicable to a number of these chronic neurodegenerative diseases.

  3. Structural neurodegeneration correlates with early diabetic retinopathy

    DEFF Research Database (Denmark)

    Frydkjaer-Olsen, Ulrik; Hansen, Rasmus Søgaard; Peto, Tunde

    2018-01-01

    PURPOSE: To examine differences in structural and functional neurodegenerative measurements between patients with no and early diabetic retinopathy (DR). METHODS: In this cross-sectional study, we examined 103 patients with type 2 diabetes mellitus. In 7-field fundus photographs acquired...... with Topcon TRC-NW6S, a single, certified grader determined the presence of DR according to the Early Treatment Diabetic Retinopathy Study (ETDRS) scale. Retinal neurodegeneration was evaluated by Topcon 3D OCT-2000 spectral domain optical coherence tomography (OCT) and by a RETI-scan multifocal...... electroretinography (mf-ERG) system in rings 1-6. RESULTS: Median age and duration of diabetes were 63.6 and 10 years, respectively, and 46% were men. Median HbA1c was 50 mmol/mol (6.7%), and ETDRS levels were 10 (41.7%, n = 43), 20 (35.0%, n = 36), and 35 (23.3%, n = 24). The duration of diabetes increased...

  4. Near-critical GLUT1 and Neurodegeneration.

    Science.gov (United States)

    Barros, L Felipe; San Martín, Alejandro; Ruminot, Ivan; Sandoval, Pamela Y; Fernández-Moncada, Ignacio; Baeza-Lehnert, Felipe; Arce-Molina, Robinson; Contreras-Baeza, Yasna; Cortés-Molina, Francisca; Galaz, Alex; Alegría, Karin

    2017-11-01

    Recent articles have drawn renewed attention to the housekeeping glucose transporter GLUT1 and its possible involvement in neurodegenerative diseases. Here we provide an updated analysis of brain glucose transport and the cellular mechanisms involved in its acute modulation during synaptic activity. We discuss how the architecture of the blood-brain barrier and the low concentration of glucose within neurons combine to make endothelial/glial GLUT1 the master controller of neuronal glucose utilization, while the regulatory role of the neuronal glucose transporter GLUT3 emerges as secondary. The near-critical condition of glucose dynamics in the brain suggests that subtle deficits in GLUT1 function or its activity-dependent control by neurons may contribute to neurodegeneration. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  5. Loss of circadian clock accelerates aging in neurodegeneration-prone mutants.

    Science.gov (United States)

    Krishnan, Natraj; Rakshit, Kuntol; Chow, Eileen S; Wentzell, Jill S; Kretzschmar, Doris; Giebultowicz, Jadwiga M

    2012-03-01

    Circadian clocks generate rhythms in molecular, cellular, physiological, and behavioral processes. Recent studies suggest that disruption of the clock mechanism accelerates organismal senescence and age-related pathologies in mammals. Impaired circadian rhythms are observed in many neurological diseases; however, it is not clear whether loss of rhythms is the cause or result of neurodegeneration, or both. To address this important question, we examined the effects of circadian disruption in Drosophila melanogaster mutants that display clock-unrelated neurodegenerative phenotypes. We combined a null mutation in the clock gene period (per(01)) that abolishes circadian rhythms, with a hypomorphic mutation in the carbonyl reductase gene sniffer (sni(1)), which displays oxidative stress induced neurodegeneration. We report that disruption of circadian rhythms in sni(1) mutants significantly reduces their lifespan compared to single mutants. Shortened lifespan in double mutants was coupled with accelerated neuronal degeneration evidenced by vacuolization in the adult brain. In addition, per(01)sni(1) flies showed drastically impaired vertical mobility and increased accumulation of carbonylated proteins compared to age-matched single mutant flies. Loss of per function does not affect sni mRNA expression, suggesting that these genes act via independent pathways producing additive effects. Finally, we show that per(01) mutation accelerates the onset of brain pathologies when combined with neurodegeneration-prone mutation in another gene, swiss cheese (sws(1)), which does not operate through the oxidative stress pathway. Taken together, our data suggest that the period gene may be causally involved in neuroprotective pathways in aging Drosophila. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. A vigilant, hypoxia-regulated heme oxygenase-1 gene vector in the heart limits cardiac injury after ischemia-reperfusion in vivo.

    Science.gov (United States)

    Tang, Yao Liang; Qian, Keping; Zhang, Y Clare; Shen, Leping; Phillips, M Ian

    2005-12-01

    The effect of a cardiac specific, hypoxia-regulated, human heme oxygenase-1 (hHO-1) vector to provide cardioprotection from ischemia-reperfusion injury was assessed. When myocardial ischemia and reperfusion is asymptomatic, the damaging effects are cumulative and patients miss timely treatment. A gene therapy approach that expresses therapeutic genes only when ischemia is experienced is a desirable strategy. We have developed a cardiac-specific, hypoxia-regulated gene therapy "vigilant vector'' system that amplifies cardioprotective gene expression. Vigilant hHO-1 plasmids, LacZ plasmids, or saline (n = 40 per group) were injected into mouse heart 2 days in advance of ischemia-reperfusion injury. Animals were exposed to 60 minutes of ischemia followed by 24 hours of reperfusion. For that term (24 hours) effects, the protein levels of HO-1, inflammatory responses, apoptosis, and infarct size were determined. For long-term (3 week) effects, the left ventricular remodeling and recovery of cardiac function were assessed. Ischemia-reperfusion resulted in a timely overexpression of HO-1 protein. Infarct size at 24 hours after ischemia-reperfusion was significantly reduced in the HO-1-treated animals compared with the LacZ-treated group or saline-treated group (P < .001). The reduction of infarct size was accompanied by a decrease in lipid peroxidant activity, inflammatory cell infiltration, and proapoptotic protein level in ischemia-reperfusion-injured myocardium. The long-term study demonstrated that timely, hypoxia-induced HO-1 overexpression is beneficial in conserving cardiac function and attenuating left ventricle remodelling. The vigilant HO-1 vector provides a protective therapy in the heart for reducing cellular damage during ischemia-reperfusion injury and preserving heart function.

  7. Neurodegeneration in drop-dead mutant drosophila melanogaster is associated with the respiratory system but not with Hypoxia.

    Directory of Open Access Journals (Sweden)

    Christine Lynn Sansone

    Full Text Available Mutations in the gene drop-dead (drd cause diverse phenotypes in adult Drosophila melanogaster including early lethality, neurodegeneration, tracheal defects, gut dysfunction, reduced body mass, and female sterility. Despite the identification of the drd gene itself, the causes of early lethality and neurodegeneration in the mutant flies remain unknown. To determine the pattern of drd expression associated with the neurodegenerative phenotype, knockdown of drd with various Gal4 drivers was performed. Early adult lethality and neurodegeneration were observed upon knockdown of drd in the tracheal system with two independent insertions of the breathless-Gal4 driver and upon knockdown in the tracheal system and elsewhere with the DJ717-Gal4 driver. Surprisingly, rescue of drd expression exclusively in the tracheae in otherwise mutant flies rescued the neurodegenerative phenotype but not adult lethality. Gut dysfunction, as measured by defecation rate, was not rescued in these flies, and gut function appeared normal upon tracheal-specific knockdown of drd. Finally, the hypothesis that tracheal dysfunction in drd mutants results in hypoxia was tested. Hypoxia-sensitive reporter transgenes (LDH-Gal4 and LDH-LacZ were placed on a drd mutant background, but enhanced expression of these reporters was not observed. In addition, manipulation of drd expression in the tracheae did not affect expression of the hypoxia-induced genes LDH, tango, and similar. Overall, these results indicate that there are at least two causes of adult lethality in drd mutants, that gut dysfunction and neurodegeneration are independent phenotypes, and that neurodegeneration is associated with tracheal expression of drd but not with hypoxia.

  8. SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis.

    Science.gov (United States)

    Kim, Dohoon; Nguyen, Minh Dang; Dobbin, Matthew M; Fischer, Andre; Sananbenesi, Farahnaz; Rodgers, Joseph T; Delalle, Ivana; Baur, Joseph A; Sui, Guangchao; Armour, Sean M; Puigserver, Pere; Sinclair, David A; Tsai, Li-Huei

    2007-07-11

    A progressive loss of neurons with age underlies a variety of debilitating neurological disorders, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), yet few effective treatments are currently available. The SIR2 gene promotes longevity in a variety of organisms and may underlie the health benefits of caloric restriction, a diet that delays aging and neurodegeneration in mammals. Here, we report that a human homologue of SIR2, SIRT1, is upregulated in mouse models for AD, ALS and in primary neurons challenged with neurotoxic insults. In cell-based models for AD/tauopathies and ALS, SIRT1 and resveratrol, a SIRT1-activating molecule, both promote neuronal survival. In the inducible p25 transgenic mouse, a model of AD and tauopathies, resveratrol reduced neurodegeneration in the hippocampus, prevented learning impairment, and decreased the acetylation of the known SIRT1 substrates PGC-1alpha and p53. Furthermore, injection of SIRT1 lentivirus in the hippocampus of p25 transgenic mice conferred significant protection against neurodegeneration. Thus, SIRT1 constitutes a unique molecular link between aging and human neurodegenerative disorders and provides a promising avenue for therapeutic intervention.

  9. SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis

    Science.gov (United States)

    Kim, Dohoon; Nguyen, Minh Dang; Dobbin, Matthew M; Fischer, Andre; Sananbenesi, Farahnaz; Rodgers, Joseph T; Delalle, Ivana; Baur, Joseph A; Sui, Guangchao; Armour, Sean M; Puigserver, Pere; Sinclair, David A; Tsai, Li-Huei

    2007-01-01

    A progressive loss of neurons with age underlies a variety of debilitating neurological disorders, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), yet few effective treatments are currently available. The SIR2 gene promotes longevity in a variety of organisms and may underlie the health benefits of caloric restriction, a diet that delays aging and neurodegeneration in mammals. Here, we report that a human homologue of SIR2, SIRT1, is upregulated in mouse models for AD, ALS and in primary neurons challenged with neurotoxic insults. In cell-based models for AD/tauopathies and ALS, SIRT1 and resveratrol, a SIRT1-activating molecule, both promote neuronal survival. In the inducible p25 transgenic mouse, a model of AD and tauopathies, resveratrol reduced neurodegeneration in the hippocampus, prevented learning impairment, and decreased the acetylation of the known SIRT1 substrates PGC-1alpha and p53. Furthermore, injection of SIRT1 lentivirus in the hippocampus of p25 transgenic mice conferred significant protection against neurodegeneration. Thus, SIRT1 constitutes a unique molecular link between aging and human neurodegenerative disorders and provides a promising avenue for therapeutic intervention. PMID:17581637

  10. The Regulatory Machinery of Neurodegeneration in In Vitro Models of Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Burcin Ikiz

    2015-07-01

    Full Text Available Neurodegenerative phenotypes reflect complex, time-dependent molecular processes whose elucidation may reveal neuronal class-specific therapeutic targets. The current focus in neurodegeneration has been on individual genes and pathways. In contrast, we assembled a genome-wide regulatory model (henceforth, “interactome”, whose unbiased interrogation revealed 23 candidate causal master regulators of neurodegeneration in an in vitro model of amyotrophic lateral sclerosis (ALS, characterized by a loss of spinal motor neurons (MNs. Of these, eight were confirmed as specific MN death drivers in our model of familial ALS, including NF-κB, which has long been considered a pro-survival factor. Through an extensive array of molecular, pharmacological, and biochemical approaches, we have confirmed that neuronal NF-κB drives the degeneration of MNs in both familial and sporadic models of ALS, thus providing proof of principle that regulatory network analysis is a valuable tool for studying cell-specific mechanisms of neurodegeneration.

  11. Virulence test using nematodes to prescreen Nocardia species capable of inducing neurodegeneration and behavioral disorders

    Directory of Open Access Journals (Sweden)

    Claire Bernardin Souibgui

    2017-10-01

    Full Text Available Background Parkinson’s disease (PD is a disorder characterized by dopaminergic neuron programmed cell death. The etiology of PD remains uncertain—some cases are due to selected genes associated with familial heredity, others are due to environmental exposure to toxic components, but over 90% of cases have a sporadic origin. Nocardia are Actinobacteria that can cause human diseases like nocardiosis. This illness can lead to lung infection or central nervous system (CNS invasion in both immunocompromised and immunocompetent individuals. The main species involved in CNS are N. farcinica, N. nova, N. brasiliensis and N. cyriacigeorgica. Some studies have highlighted the ability of N. cyriacigeorgica to induce Parkinson’s disease-like symptoms in animals. Actinobacteria are known to produce a large variety of secondary metabolites, some of which can be neurotoxic. We hypothesized that neurotoxic secondary metabolite production and the onset of PD-like symptoms in animals could be linked. Methods Here we used a method to screen bacteria that could induce dopaminergic neurodegeneration before performing mouse experiments. Results The nematode Caenorhabditis elegans allowed us to demonstrate that Nocardia strains belonging to N. cyriacigeorgica and N. farcinica species can induce dopaminergic neurodegeneration. Strains of interest involved with the nematodes in neurodegenerative disorders were then injected in mice. Infected mice had behavioral disorders that may be related to neuronal damage, thus confirming the ability of Nocardia strains to induce neurodegeneration. These behavioral disorders were induced by N. cyriacigeorgica species (N. cyriacigeorgica GUH-2 and N. cyriacigeorgica 44484 and N. farcinica 10152. Discussion We conclude that C. elegans is a good model for detecting Nocardia strains involved in neurodegeneration. This model allowed us to detect bacteria with high neurodegenerative effects and which should be studied in mice to

  12. Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells).

    Science.gov (United States)

    Howarth, Joanna L; Lee, Youn Bok; Uney, James B

    2010-02-01

    In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described.

  13. Genetic diversity of the mitochondrial cytochrome b gene in Lutzomyia spp., with special reference to Lutzomyia peruensis, a main vector of Leishmania (Viannia) peruviana in the Peruvian Andes.

    Science.gov (United States)

    Yamamoto, Kento; Cáceres, Abraham G; Gomez, Eduardo A; Mimori, Tatsuyuki; Iwata, Hiroyuki; Korenaga, Masataka; Sakurai, Tatsuya; Katakura, Ken; Hashiguchi, Yoshihisa; Kato, Hirotomo

    2013-05-01

    The genetic divergence caused by genetic drift and/or selection is suggested to affect the vectorial capacity and insecticide susceptibility of sand flies, as well as other arthropods. In the present study, cytochrome b (cyt b) gene sequences were determined in 13 species circulating in Peru to establish a basis for analysis of the genetic structure, and the intraspecific genetic diversity was assessed in the Lutzomyia (Lu.) peruensis, a main vector species of Leishmania (Viannia) peruviana in Peruvian Andes. Analysis of intraspecific genetic diversity in the cyt b gene sequences from 36 Lu. peruensis identified 3 highly polymorphic sites in the middle region of the gene. Haplotype and gene network analyses were performed on the cyt b gene sequences of 130 Lu. peruensis in 9 Andean areas from 3 Departments (Ancash, Lima and La Libertad). The results showed that the populations of La Libertad were highly polymorphic and that their haplotypes were distinct from those of Ancash and Lima, where dominant haplotypes were observed, suggesting that a population bottleneck may have occurred in Ancash and Lima, but not in La Libertad. The present study indicated that the middle region of the cyt b gene is useful for the analysis of genetic structure in sand fly populations. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. LINGO-1 and Neurodegeneration: Pathophysiologic Clues for Essential Tremor?

    Directory of Open Access Journals (Sweden)

    Zhou Zhi-dong

    2012-03-01

    Full Text Available Essential tremor (ET, one of the most common adult-onset movement disorders, has been associated with cerebellar Purkinje cell degeneration and formation of brainstem Lewy bodies. Recent findings suggest that genetic variants of the leucine-rich repeat and Ig domain containing 1 (LINGO-1 gene could be risk factors for ET. The LINGO-1 protein contains both leucine-rich repeat (LRR and immunoglobulin (Ig-like domains in its extracellular region, as well as a transmembrane domain and a short cytoplasmic tail. LINGO-1 can form a ternary complex with Nogo-66 receptor (NgR1 and p75. Binding of LINGO-1 with NgR1 can activate the NgR1 signaling pathway, leading to inhibition of oligodendrocyte differentiation and myelination in the central nervous system. LINGO-1 has also been found to bind with epidermal growth factor receptor (EGFR and induce downregulation of the activity of EGFR–PI3K–Akt signaling, which might decrease Purkinje cell survival. Therefore, it is possible that genetic variants of LINGO-1, either alone or in combination with other genetic or environmental factors, act to increase LINGO-1 expression levels in Purkinje cells and confer a risk to Purkinje cell survival in the cerebellum. Here, we provide a concise summary of the link between LINGO-1 and neurodegeneration and discuss various hypotheses as to how this could be potentially relevant to ET pathogenesis.

  15. Cerebellar neurodegeneration in the absence of microRNAs

    Science.gov (United States)

    Schaefer, Anne; O'Carroll, Dónal; Tan, Chan Lek; Hillman, Dean; Sugimori, Mutsuyuki; Llinas, Rodolfo; Greengard, Paul

    2007-01-01

    Genome-encoded microRNAs (miRNAs) are potent regulators of gene expression. The significance of miRNAs in various biological processes has been suggested by studies showing an important role of these small RNAs in regulation of cell differentiation. However, the role of miRNAs in regulation of differentiated cell physiology is not well established. Mature neurons express a large number of distinct miRNAs, but the role of miRNAs in postmitotic neurons has not been examined. Here, we provide evidence for an essential role of miRNAs in survival of differentiated neurons. We show that conditional Purkinje cell–specific ablation of the key miRNA-generating enzyme Dicer leads to Purkinje cell death. Deficiency in Dicer is associated with progressive loss of miRNAs, followed by cerebellar degeneration and development of ataxia. The progressive neurodegeneration in the absence of Dicer raises the possibility of an involvement of miRNAs in neurodegenerative disorders. PMID:17606634

  16. Effective inhibition of foot-and-mouth disease virus (FMDV replication in vitro by vector-delivered microRNAs targeting the 3D gene

    Directory of Open Access Journals (Sweden)

    Cai Xuepeng

    2011-06-01

    Full Text Available Abstract Background Foot-and-mouth disease virus (FMDV causes an economically important and highly contagious disease of cloven-hoofed animals. RNAi triggered by small RNA molecules, including siRNAs and miRNAs, offers a new approach for controlling viral infections. There is no report available for FMDV inhibition by vector-delivered miRNA, although miRNA is believed to have more potential than siRNA. In this study, the inhibitory effects of vector-delivered miRNAs targeting the 3D gene on FMDV replication were examined. Results Four pairs of oligonucleotides encoding 3D-specific miRNA of FMDV were designed and selected for construction of miRNA expression plasmids. In the reporter assays, two of four miRNA expression plasmids were able to significantly silence the expression of 3D-GFP fusion proteins from the reporter plasmid, p3D-GFP, which was cotransfected with each miRNA expression plasmid. After detecting the silencing effects of the reporter genes, the inhibitory effects of FMDV replication were determined in the miRNA expression plasmid-transfected and FMDV-infected cells. Virus titration and real-time RT-PCR assays showed that the p3D715-miR and p3D983-miR plasmids were able to potently inhibit the replication of FMDV when BHK-21 cells were infected with FMDV. Conclusion Our results indicated that vector-delivered miRNAs targeting the 3D gene efficiently inhibits FMDV replication in vitro. This finding provides evidence that miRNAs could be used as a potential tool against FMDV infection.

  17. Genome-wide and expression-profiling analyses suggest the main cytochrome P450 genes related to pyrethroid resistance in the malaria vector, Anopheles sinensis (Diptera Culicidae).

    Science.gov (United States)

    Yan, Zheng-Wen; He, Zheng-Bo; Yan, Zhen-Tian; Si, Feng-Ling; Zhou, Yong; Chen, Bin

    2018-02-02

    Anopheles sinensis is one of the major malaria vectors. However, pyrethroid resistance in An. sinensis is threatening malaria control. Cytochrome P450-mediated detoxification is an important pyrethroid resistance mechanism that has been unexplored in An. sinensis. In this study, we performed a comprehensive analysis of the An. sinensis P450 gene superfamily with special attention to their role in pyrethroid resistance using bioinformatics and molecular approaches. Our data revealed the presence of 112 individual P450 genes in An. sinensis, which were classified into four major clans (mitochondrial, CYP2, CYP3 and CYP4), 18 families and 50 subfamilies. Sixty-seven genes formed nine gene clusters, and genes within the same cluster and the same gene family had a similar gene structure. Phylogenetic analysis showed that most of An. sinensis P450s (82/112) had very close 1: 1 orthology with Anopheles gambiae P450s. Five genes (AsCYP6Z2, AsCYP6P3v1, AsCYP6P3v2, AsCYP9J5 and AsCYP306A1) were significantly upregulated in three pyrethroid-resistant populations in both RNA-seq and RT-qPCR analyses, suggesting that they could be the most important P450 genes involved in pyrethroid resistance in An. sinensis. Our study provides insight on the diversity of An. sinensis P450 superfamily and basis for further elucidating pyrethroid resistance mechanism in this mosquito species. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

  18. Genome-wide identification and characterization of odorant-binding protein (OBP) genes in the malaria vector Anopheles sinensis (Diptera: Culicidae).

    Science.gov (United States)

    He, Xiu; He, Zheng-Bo; Zhang, Yu-Juan; Zhou, Yong; Xian, Peng-Jie; Qiao, Liang; Chen, Bin

    2016-06-01

    Anopheles sinensis is a major malaria vector. Insect odorant-binding proteins (OBPs) may function in the reception of odorants in the olfactory system. The classification and characterization of the An. sinensis OBP genes have not been systematically studied. In this study, 64 putative OBP genes were identified at the whole-genome level of An. sinensis based on the comparison between OBP conserved motifs, PBP_GOBP, and phylogenetic analysis with An. gambiae OBPs. The characterization of An. sinensis OBPs, including the motif's conservation, gene structure, genomic organization and classification, were investigated. A new gene, AsOBP73, belonging to the Plus-C subfamily, was identified with the support of transcript and conservative motifs. These An. sinensis OBP genes were classified into three subfamilies with 37, 15 and 12 genes in the subfamily Classic, Atypical and Plus-C, respectively. The genomic organization of An. sinensis OBPs suggests a clustered distribution across nine different scaffolds. Eight genes (OBP23-28, OBP63-64) might originate from a single gene through a series of historic duplication events at least before divergence of Anopheles, Culex and Aedes. The microsynteny analyses indicate a very high synteny between An. sinensis and An. gambiae OBPs. OBP70 and OBP71 earlier classified under Plus-C in An. gambiae are recognized as belonging to the group Obp59a of the Classic subfamily, and OBP69 earlier classified under Plus-C has been moved to the Atypical subfamily in this study. The study established a basic information frame for further study of the OBP genes in insects as well as in An. sinensis. © 2016 Institute of Zoology, Chinese Academy of Sciences.

  19. Phosphatidylinositol transfer protein alpha and its role in neurodegeneration

    NARCIS (Netherlands)

    Bunte, H.

    2007-01-01

    Selective neuronal loss is a prominent feature in neurodegenerative disorders. Recently, a link between neurodegeneration and a deficiency in the protein phosphatidylinositol transfer protein alpha (PI-TPalpha) has been demonstrated. In this context it is of importance that fibroblasts

  20. Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo

    NARCIS (Netherlands)

    Pfeifer, A.; Brandon, E. P.; Kootstra, N.; Gage, F. H.; Verma, I. M.

    2001-01-01

    The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly, we found a significant reduction in proliferation and an accumulation in the G(2)/M

  1. Inadvertent gene silencing of argininosuccinate synthase (bcass1) in Botrytis cinerea by the pLOB1 vector system

    NARCIS (Netherlands)

    Patel, R.M.; Kan, van J.A.L.; Bailey, A.M.; Foster, G.D.

    2010-01-01

    For several years, researchers working on the plant pathogen Botrytis cinerea and a number of other related fungi have routinely used the pLOB1 vector system, based on hygromycin resistance, under the control of the Aspergillus nidulans oliC promoter and what was reported to be the ß-tubulin (tubA)

  2. A Drosophila melanogaster hobo-white + vector mediates low frequency gene transfer in D. vlrllls with full Interspecific white + complementation

    Science.gov (United States)

    Transformation of a Drosophila virilis white mutant host strain was attempted by using a hobo vector containing the D. melanogaster mini-white+ cassette (H[w+, hawN]) and an unmodified or heat shock regulated hobo transposase helper. Two transformant lines were recovered with the unmodified helper (...

  3. Pyrethroid Resistance in Malaysian Populations of Dengue Vector Aedes aegypti Is Mediated by CYP9 Family of Cytochrome P450 Genes.

    Science.gov (United States)

    Ishak, Intan H; Kamgang, Basile; Ibrahim, Sulaiman S; Riveron, Jacob M; Irving, Helen; Wondji, Charles S

    2017-01-01

    Dengue control and prevention rely heavily on insecticide-based interventions. However, insecticide resistance in the dengue vector Aedes aegypti, threatens the continued effectiveness of these tools. The molecular basis of the resistance remains uncharacterised in many endemic countries including Malaysia, preventing the design of evidence-based resistance management. Here, we investigated the underlying molecular basis of multiple insecticide resistance in Ae. aegypti populations across Malaysia detecting the major genes driving the metabolic resistance. Genome-wide microarray-based transcription analysis was carried out to detect the genes associated with metabolic resistance in these populations. Comparisons of the susceptible New Orleans strain to three non-exposed multiple insecticide resistant field strains; Penang, Kuala Lumpur and Kota Bharu detected 2605, 1480 and 425 differentially expressed transcripts respectively (fold-change>2 and p-value ≤ 0.05). 204 genes were commonly over-expressed with monooxygenase P450 genes (CYP9J27, CYP6CB1, CYP9J26 and CYP9M4) consistently the most up-regulated detoxification genes in all populations, indicating that they possibly play an important role in the resistance. In addition, glutathione S-transferases, carboxylesterases and other gene families commonly associated with insecticide resistance were also over-expressed. Gene Ontology (GO) enrichment analysis indicated an over-representation of GO terms linked to resistance such as monooxygenases, carboxylesterases, glutathione S-transferases and heme-binding. Polymorphism analysis of CYP9J27 sequences revealed a high level of polymorphism (except in Joho Bharu), suggesting a limited directional selection on this gene. In silico analysis of CYP9J27 activity through modelling and docking simulations suggested that this gene is involved in the multiple resistance in Malaysian populations as it is predicted to metabolise pyrethroids, DDT and bendiocarb. The predominant

  4. Epidemiology of neurodegeneration in American-style professional football players

    OpenAIRE

    Lehman, Everett J

    2013-01-01

    The purpose of this article is to review the history of head injuries in relation to American-style football play, summarize recent research that has linked football head injuries to neurodegeneration, and provide a discussion of the next steps for refining the examination of neurodegeneration in football players. For most of the history of football, the focus of media reports and scientific studies on football-related head injuries was on the acute or short-term effects of serious, traumatic...

  5. Comparative study of cellular kinetics of reporter probe [{sup 131}I]FIAU in neonatal cardiac myocytes after transfer of HSV1-tk reporter gene with two vectors

    Energy Technology Data Exchange (ETDEWEB)

    Lan Xiaoli [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: lxl730724@hotmail.com; Yin Xiaohua; Wang Ruihua; Liu Ying [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China); Zhang Yongxue [Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China) and Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022 (China)], E-mail: zhyx1229@163.com

    2009-02-15

    Aim: Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In this study, HSV1-tk (herpes simplex virus type 1 thymidine kinase) and FIAU (2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyl-5-iodouracil) were used as the reporter gene and probe, respectively. Cellular uptakes of radiolabeled FIAU of neonatal rat cardiac myocytes transferred with HSV1-tk were compared between two vectors, adenovirus and liposome. The aims of this study were to choose the better vector and to provide a theoretical basis for good nuclide images. Methods: Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSV1-tk inserted into adenovirus vector (recombinant adenovirus type 5, Ad5-tk) and plasmid (pDC316-tk) coated with Lipofectamine 2000 (pDC316-tk/lipoplex) were developed; thus, HSV1-tk could be transferred into neonatal cardiac myocytes. FAU (2'-fluoro-2'-deoxy-1-{beta}-D-arabinofuranosyluracil) was labeled with {sup 131}I, and the product was assessed after purification with reversed-phase Sep-Pak C-18 column. The uptake rates of [{sup 131}I]FIAU in the transferred cardiac myocytes at different times (0.5, 1, 2, 3, 4 and 5 h) were detected. Furthermore, mRNA expression and protein expression of HSV1-tk were detected by semiquantitative reverse-transcriptase polymerase chain reaction and immunocytochemistry. Results: FAU could be labeled with {sup 131}I, and the labeling efficiency and radiochemical purity rates were 53.82{+-}2.05% and 94.85{+-}1.76%, respectively. Time-dependent increase of the accumulation of [{sup 131}I]FIAU was observed in both the Ad5-tk group and the pDC316/lipoplex group, and the highest uptake rate occurred at 5 h, with peak values of 12.55{+-}0.37% and 2.09{+-}0.34%, respectively. Greater uptakes of [{sup 131}I]FIAU in Ad5-tk-infected cells compared with pDC316/lipoplex-transfected ones occurred at all the time points (t=12.978-38.253, P<.01). The exogenous gene

  6. E1(-)E4(+) adenoviral gene transfer vectors function as a "pro-life" signal to promote survival of primary human endothelial cells.

    Science.gov (United States)

    Ramalingam, R; Rafii, S; Worgall, S; Brough, D E; Crystal, R G

    1999-05-01

    Although endothelial cells are quiescent and long-lived in vivo, when they are removed from blood vessels and cultured in vitro they die within days to weeks. In studies of the interaction of E1(-)E4(+) replication-deficient adenovirus (Ad) vectors and human endothelium, the cells remained quiescent and were viable for prolonged periods. Evaluation of these cultures showed that E1(-)E4(+) Ad vectors provide an "antiapoptotic" signal that, in association with an increase in the ratio of Bcl2 to Bax levels, induces the endothelial cells to enter a state of "suspended animation," remaining viable for at least 30 days, even in the absence of serum and growth factors. Although the mechanisms initiating these events are unclear, the antiapoptoic signal requires the presence of E4 genes in the vector genome, suggesting that one or more E4 open reading frames of subgroup C Ad initiate a "pro-life" program that modifies cultured endothelial cells to survive for prolonged periods.

  7. Neogenesis and proliferation of β-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector

    International Nuclear Information System (INIS)

    Tokui, Yae; Kozawa, Junji; Yamagata, Kazuya; Zhang, Jun; Ohmoto, Hiroshi; Tochino, Yoshihiro; Okita, Kohei; Iwahashi, Hiromi; Namba, Mitsuyoshi; Shimomura, Iichiro; Miyagawa, Jun-ichiro

    2006-01-01

    Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of β-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a β-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of β-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of β-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted β-cell differentiation (mainly from duct cells) and proliferation of pre-existing β-cells, resulting in an increase of the β-cell mass that improved glucose tolerance in diabetic mice

  8. Genome-wide identification, characterization and classification of ionotropic glutamate receptor genes (iGluRs) in the malaria vector Anopheles sinensis (Diptera: Culicidae).

    Science.gov (United States)

    Wang, Ting-Ting; Si, Feng-Ling; He, Zheng-Bo; Chen, Bin

    2018-01-15

    Ionotropic glutamate receptors (iGluRs) are conserved ligand-gated ion channel receptors, and ionotropic receptors (IRs) were revealed as a new family of iGluRs. Their subdivision was unsettled, and their characteristics are little known. Anopheles sinensis is a major malaria vector in eastern Asia, and its genome was recently well sequenced and annotated. We identified iGluR genes in the An. sinensis genome, analyzed their characteristics including gene structure, genome distribution, domains and specific sites by bioinformatic methods, and deduced phylogenetic relationships of all iGluRs in An. sinensis, Anopheles gambiae and Drosophila melanogaster. Based on the characteristics and phylogenetics, we generated the classification of iGluRs, and comparatively analyzed the intron number and selective pressure of three iGluRs subdivisions, iGluR group, Antenna IR and Divergent IR subfamily. A total of 56 iGluR genes were identified and named in the whole-genome of An. sinensis. These genes were located on 18 scaffolds, and 31 of them (29 being IRs) are distributed into 10 clusters that are suggested to form mainly from recent gene duplication. These iGluRs can be divided into four groups: NMDA, non-NMDA, Antenna IR and Divergent IR based on feature comparison and phylogenetic analysis. IR8a and IR25a were suggested to be monophyletic, named as Putative in the study, and moved from the Antenna subfamily in the IR family to the non-NMDA group as a sister of traditional non-NMDA. The generated iGluRs of genes (including NMDA and regenerated non-NMDA) are relatively conserved, and have a more complicated gene structure, smaller ω values and some specific functional sites. The iGluR genes in An. sinensis, An. gambiae and D. melanogaster have amino-terminal domain (ATD), ligand binding domain (LBD) and Lig_Chan domains, except for IR8a that only has the LBD and Lig_Chan domains. However, the new concept IR family of genes (including regenerated Antenna IR, and Divergent

  9. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    International Nuclear Information System (INIS)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku; Okada, Naoki

    2016-01-01

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8"+ CAR-T cells had antigen-specific cytotoxic activity. • CD4"+ CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  10. Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

    Energy Technology Data Exchange (ETDEWEB)

    Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke; Hirobe, Sachiko; Nakagawa, Shinsaku, E-mail: nakagawa@phs.osaka-u.ac.jp; Okada, Naoki, E-mail: okada@phs.osaka-u.ac.jp

    2016-04-22

    Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.

  11. Vector analysis

    CERN Document Server

    Newell, Homer E

    2006-01-01

    When employed with skill and understanding, vector analysis can be a practical and powerful tool. This text develops the algebra and calculus of vectors in a manner useful to physicists and engineers. Numerous exercises (with answers) not only provide practice in manipulation but also help establish students' physical and geometric intuition in regard to vectors and vector concepts.Part I, the basic portion of the text, consists of a thorough treatment of vector algebra and the vector calculus. Part II presents the illustrative matter, demonstrating applications to kinematics, mechanics, and e

  12. About vectors

    CERN Document Server

    Hoffmann, Banesh

    1975-01-01

    From his unusual beginning in ""Defining a vector"" to his final comments on ""What then is a vector?"" author Banesh Hoffmann has written a book that is provocative and unconventional. In his emphasis on the unresolved issue of defining a vector, Hoffmann mixes pure and applied mathematics without using calculus. The result is a treatment that can serve as a supplement and corrective to textbooks, as well as collateral reading in all courses that deal with vectors. Major topics include vectors and the parallelogram law; algebraic notation and basic ideas; vector algebra; scalars and scalar p

  13. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Tratschin, J.D.; West, M.H.P.; Sandbank, T.; Carter, B.J.

    1984-10-01

    The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

  14. Adenoviral vectors for highly selective gene expression in central serotonergic neurons reveal quantal characteristics of serotonin release in the rat brain

    Directory of Open Access Journals (Sweden)

    Teschemacher Anja G

    2009-03-01

    Full Text Available Abstract Background 5-hydroxytryptamine (5 HT, serotonin is one of the key neuromodulators in mammalian brain, but many fundamental properties of serotonergic neurones and 5 HT release remain unknown. The objective of this study was to generate an adenoviral vector system for selective targeting of serotonergic neurones and apply it to study quantal characteristics of 5 HT release in the rat brain. Results We have generated adenoviral vectors which incorporate a 3.6 kb fragment of the rat tryptophan hydroxylase-2 (TPH-2 gene which selectively (97% co-localisation with TPH-2 target raphe serotonergic neurones. In order to enhance the level of expression a two-step transcriptional amplification strategy was employed. This allowed direct visualization of serotonergic neurones by EGFP fluorescence. Using these vectors we have performed initial characterization of EGFP-expressing serotonergic neurones in rat organotypic brain slice cultures. Fluorescent serotonergic neurones were identified and studied using patch clamp and confocal Ca2+ imaging and had features consistent with those previously reported using post-hoc identification approaches. Fine processes of serotonergic neurones could also be visualized in un-fixed tissue and morphometric analysis suggested two putative types of axonal varicosities. We used micro-amperometry to analyse the quantal characteristics of 5 HT release and found that central 5 HT exocytosis occurs predominantly in quanta of ~28000 molecules from varicosities and ~34000 molecules from cell bodies. In addition, in somata, we observed a minority of large release events discharging on average ~800000 molecules. Conclusion For the first time quantal release of 5 HT from somato-dendritic compartments and axonal varicosities in mammalian brain has been demonstrated directly and characterised. Release from somato-dendritic and axonal compartments might have different physiological functions. Novel vectors generated in this

  15. Altered expression of the Cdk5 activator-like protein, Cdk5α, causes neurodegeneration, in part by accelerating the rate of aging

    Directory of Open Access Journals (Sweden)

    Joshua Spurrier

    2018-03-01

    Full Text Available Aging is the greatest risk factor for neurodegeneration, but the connection between the two processes remains opaque. This is in part for want of a rigorous way to define physiological age, as opposed to chronological age. Here, we develop a comprehensive metric for physiological age in Drosophila, based on genome-wide expression profiling. We applied this metric to a model of adult-onset neurodegeneration, increased or decreased expression of the activating subunit of the Cdk5 protein kinase, encoded by the gene Cdk5α, the ortholog of mammalian p35. Cdk5α-mediated degeneration was associated with a 27-150% acceleration of the intrinsic rate of aging, depending on the tissue and genetic manipulation. Gene ontology analysis and direct experimental tests revealed that affected age-associated processes included numerous core phenotypes of neurodegeneration, including enhanced oxidative stress and impaired proteostasis. Taken together, our results suggest that Cdk5α-mediated neurodegeneration results from accelerated aging, in combination with cell-autonomous neuronal insults. These data fundamentally recast our picture of the relationship between neurodegeneration and its most prominent risk factor, natural aging.

  16. Cloning of fusion protein gene of Newcastle disease virus into a baculovirus derived bacmid shuttle vector, in order to express it in insect cell line

    Directory of Open Access Journals (Sweden)

    Hashemzadeh MS

    2015-05-01

    Full Text Available Abstract Background: Newcastle disease virus (NDV is one of the major pathogens in poultry and vaccination is intended to control the disease, as an effective solution, yet. Fusion protein (F on surface of NDV, has a fundamental role in virus pathogenicity and can induce protective immunity, alone. With this background, here our aim was to construct a baculovirus derived recombinant bacmid shuttle vector (encoding F-protein in order to express it in insect cell line. Materials and Methods: In this experimental study, at first complete F gene from avirulent strain La Sota of NDV was amplified by RT-PCR to produce F cDNA. The amplicon was cloned into T/A cloning vector and afterwards into pFastBac Dual donor plasmid. After the verification of cloning process by two methods, PCR and enzymatic digestion analysis, the accuracy of F gene sequence was confirmed by sequencing. Finally, F-containing recombinant bacmid was subsequently generated in DH10Bac cell and the construct production was confirmed by a special PCR panel, using F specific primers and M13 universal primers. Results: Analysis of confirmatory tests showed that the recombinant bacmid, expressing of F-protein gene in correct sequence and framework, has been constructed successfully. Conclusion: The product of this F-containing recombinant bacmid, in addition to its independent application in the induction of protective immunity, can be used with the other individual recombinant baculoviruses, expressing HN and NP genes to produce NDV-VLPs in insect cell line.

  17. Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors

    DEFF Research Database (Denmark)

    Cai, Yujia; Laustsen, Anders; Zhou, Yan

    2016-01-01

    -driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34+ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89......% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded 'all-in-one' IDLVs for site-directed gene insertion in stem cell based gene therapies....

  18. Nuevas herramientas para la clasificación taxonómica de los insectos vectores de leishmaniosis: utilidad de los genes mitocondriales

    Directory of Open Access Journals (Sweden)

    Eduar E. Bejarano

    2001-06-01

    Full Text Available La controversia en torno a la posición taxonómica de los flebotomineos, debida en parte a dificultades morfológicas, ha evidenciado la necesidad de buscar nuevas herramientas que permitan no sólo establecer una clasificación más acorde con los procesos evolutivos que han experimentado estos insectos, sino también diferenciar los taxones que conforman los complejos de especies. Con la aparición de las técnicas de biología molecular, el estudio del genoma en insectos vectores de enfermedades se ha convertido en un paso importante en la resolución de muchos conflictos taxonómicos. Las caracteristicas del ADN mitocondrial, entre las que se destacan su rápida evolución y la facilidad con la que se puede aislar y manipular, han favorecido su elección como marcador molecular para estudios de taxonomía. Recientemente, el polimorfisrno de algunos genes mitocondriales comenzó a utilizarse para explorar las relaciones filogenéticas y evolutivas en especies de los géneros Lutzomyia y Phlebotomus. La presente revisión recopila la literatura disponible sobre los principales grupos de flebotomíneos estudiados, los genes mitocondriales más utilizados y su contribución a la clasificación de los vectores de leishmaniosis.

  19. Generation of a high-titer retroviral vector capable of expressing high levels of the human β-globin gene

    NARCIS (Netherlands)

    M. Sadelain (Michel); C.H.J. Wang (Jason); M. Antoniou (Michael); F.G. Grosveld (Frank); R.C. Mulligan

    1995-01-01

    textabstractRetrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and

  20. Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat

    DEFF Research Database (Denmark)

    Pacak, Andrzej; Geisler, Katrin; Jørgensen, Bodil

    2010-01-01

    -expressed genes we wanted to explore the potential of BSMV for silencing genes in root tissues. Furthermore, the newly completed genome sequence of the emerging cereal model species Brachypodium distachyon as well as the increasing amount of EST sequence information available for oat (Avena species) have created...

  1. Ferrous Iron Up-regulation in Fibroblasts of Patients with Beta Propeller Protein-Associated Neurodegeneration (BPAN).

    OpenAIRE

    Ingrassia, Rosaria; Memo, Maurizio; Garavaglia, Barbara

    2017-01-01

    Mutations in WDR45 gene, coding for a beta-propeller protein, have been found in patients affected by Neurodegeneration with Brain Iron Accumulation, NBIA5 (also known as BPAN). BPAN is a movement disorder with Non Transferrin Bound Iron (NTBI) accumulation in the basal ganglia as common hallmark between NBIA classes (Hayflick et al., 2013). WDR45 has been predicted to have a role in autophagy, while the impairment of iron metabolism in the different NBIA subclasses has not currently been cla...

  2. Natural Products Combating Neurodegeneration: Parkinson's Disease.

    Science.gov (United States)

    Solayman, Md; Islam, Md Asiful; Alam, Fahmida; Khalil, Md Ibrahim; Kamal, Mohammad Amjad; Gan, Siew Hua

    2017-01-01

    Parkinson's disease (PD) is characterized by neurodegeneration and a progressive functional impairment of the midbrain nigral dopaminergic neurons. The cause remains unknown; however, several pathological processes and central factors, such as protein aggregation, mitochondrial dysfunction, iron accumulation, neuroinflammation and oxidative stress, have been reported. The current treatment method primarily targets symptoms by using anti-Parkinson drugs such as levodopa, carbidopa, dopamine (DA) agonists, monoamine oxidase type B inhibitors and anticholinergics to replace DA. When drug therapy is not satisfactory, surgical treatments are recommended. Unfortunately, the existing conventional strategies that target PD are associated with numerous side effects and possess an economic burden. Therefore, novel therapeutic approaches that regulate the pathways leading to neuronal death and dysfunction are necessary. For many years, nature has provided the primary resource for the discovery of potential therapeutic agents. Remarkably, many natural products from medicinal plants, fruits and vegetables have been demonstrated to be efficacious anti-Parkinson agents. These products possess neuroprotective properties as a result of not only their wellrecognized anti-oxidative and anti-inflammatory activities but also their inhibitory roles regarding iron accumulation, protein misfolding and the maintenance of proteasomal degradation, as well as mitochondrial homeostasis. The aim of this review is to report the available anti-Parkinson agents based on natural products and delineate their therapeutic actions, which act on various pathways. Overall, this review emphasizes the types of natural products that are potential future resources in the treatment of PD as novel regimens or supplementary agents. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Neurodegeneration and Neuroprotection in Diabetic Retinopathy

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    Abdullah S. Alhomida

    2013-01-01

    Full Text Available Diabetic retinopathy is widely considered to be a neurovascular disease. This is in contrast to its previous identity as solely a vascular disease. Early in the disease progression of diabetes, the major cells in the neuronal component of the retina consist of retinal ganglion cells and glial cells, both of which have been found to be compromised. A number of retinal function tests also indicated a functional deficit in diabetic retina, which further supports dysfunction of neuronal cells. As an endocrinological disorder, diabetes alters metabolism both systemically and locally in several body organs, including the retina. A growing body of evidences indicates increased levels of excitotoxic metabolites, including glutamate, branched chain amino acids and homocysteine in cases of diabetic retinopathy. Also present, early in the disease, are decreased levels of folic acid and vitamin-B12, which are potential metabolites capable of damaging neurons. These altered levels of metabolites are found to activate several metabolic pathways, leading to increases in oxidative stress and decreases in the level of neurotrophic factors. As a consequence, they may damage retinal neurons in diabetic patients. In this review, we have discussed those potential excitotoxic metabolites and their implications in neuronal damage. Possible therapeutic targets to protect neurons are also discussed. However, further research is needed to understand the exact molecular mechanism of neurodegeneration so that effective neuroprotection strategies can be developed. By protecting retinal neurons early in diabetic retinopathy cases, damage of retinal vessels can be protected, thereby helping to ameliorate the progression of diabetic retinopathy, a leading cause of blindness worldwide.

  4. Development of plasmid vector and electroporation condition for gene transfer in sporogenic lactic acid bacterium, Bacillus coagulans.

    Science.gov (United States)

    Rhee, Mun Su; Kim, Jin-Woo; Qian, Yilei; Ingram, L O; Shanmugam, K T

    2007-07-01

    Bacillus coagulans is a sporogenic lactic acid bacterium that ferments glucose and xylose, major components of plant biomass, a potential feedstock for cellulosic ethanol. The temperature and pH for optimum rate of growth of B. coagulans (50 to 55 degrees C, pH 5.0) are very similar to that of commercially developed fungal cellulases (50 degrees C; pH 4.8). Due to this match, simultaneous saccharification and fermentation (SSF) of cellulose to products by B. coagulans is expected to require less cellulase than needed if the SSF is conducted at a sub-optimal temperature, such as 30 degrees C, the optimum for yeast, the main biocatalyst used by the ethanol industry. To fully exploit B. coagulans as a platform organism, we have developed an electroporation method to transfer plasmid DNA into this genetically recalcitrant bacterium. We also constructed a B. coagulans/E. coli shuttle vector, plasmid pMSR10 that contains the rep region from a native plasmid (pMSR0) present in B. coagulans strain P4-102B. The native plasmid, pMSR0 (6823bp), has 9 ORFs, and replicates by rolling-circle mode of replication. Plasmid pNW33N, developed for Geobacillus stearothermophilus, was also transformed into this host and stably maintained while several other Bacillus/Escherichia coli shuttle vector plasmids were not transformed into B. coagulans. The transformation efficiency of B. coagulans strain P4-102B using the plasmids pNW33N or pMSR10 was about 1.5x10(16) per mole of DNA. The availability of shuttle vectors and an electroporation method is expected to aid in genetic and metabolic engineering of B. coagulans.

  5. Elementary vectors

    CERN Document Server

    Wolstenholme, E Œ

    1978-01-01

    Elementary Vectors, Third Edition serves as an introductory course in vector analysis and is intended to present the theoretical and application aspects of vectors. The book covers topics that rigorously explain and provide definitions, principles, equations, and methods in vector analysis. Applications of vector methods to simple kinematical and dynamical problems; central forces and orbits; and solutions to geometrical problems are discussed as well. This edition of the text also provides an appendix, intended for students, which the author hopes to bridge the gap between theory and appl

  6. The Pathway From Genes to Gene Therapy in Glaucoma: A Review of Possibilities for Using Genes as Glaucoma Drugs.

    Science.gov (United States)

    Borrás, Teresa

    2017-01-01

    Treatment of diseases with gene therapy is advancing rapidly. The use of gene therapy has expanded from the original concept of re-placing the mutated gene causing the disease to the use of genes to con-trol nonphysiological levels of expression or to modify pathways known to affect the disease. Genes offer numerous advantages over conventional drugs. They have longer duration of action and are more specific. Genes can be delivered to the target site by naked DNA, cells, nonviral, and viral vectors. The enormous progress of the past decade in molecular bi-ology and delivery systems has provided ways for targeting genes to the intended cell/tissue and safe, long-term vectors. The eye is an ideal organ for gene therapy. It is easily accessible and it is an immune-privileged site. Currently, there are clinical trials for diseases affecting practically every tissue of the eye, including those to restore vision in patients with Leber congenital amaurosis. However, the number of eye trials compared with those for systemic diseases is quite low (1.8%). Nevertheless, judg-ing by the vast amount of ongoing preclinical studies, it is expected that such number will increase considerably in the near future. One area of great need for eye gene therapy is glaucoma, where a long-term gene drug would eliminate daily applications and compliance issues. Here, we review the current state of gene therapy for glaucoma and the possibilities for treating the trabecular meshwork to lower intraocular pressure and the retinal ganglion cells to protect them from neurodegeneration. Copyright© 2017 Asia-Pacific Academy of Ophthalmology.

  7. Vector analysis as a fast and easy method to compare gene expression responses between different experimental backgrounds

    NARCIS (Netherlands)

    Breitling, R.; Armengaud, P.; Amtmann, A.

    2005-01-01

    Background Gene expression studies increasingly compare expression responses between different experimental backgrounds (genetic, physiological, or phylogenetic). By focusing on dynamic responses rather than a direct comparison of static expression levels, this type of study allows a finer

  8. Pantethine treatment is effective in recovering the disease phenotype induced by ketogenic diet in a pantothenate kinase-associated neurodegeneration mouse model

    Science.gov (United States)

    Brunetti, Dario; Dusi, Sabrina; Giordano, Carla; Lamperti, Costanza; Morbin, Michela; Fugnanesi, Valeria; Marchet, Silvia; Fagiolari, Gigliola; Sibon, Ody; Moggio, Maurizio; d’Amati, Giulia

    2014-01-01

    Pantothenate kinase-associated neurodegeneration, caused by mutations in the PANK2 gene, is an autosomal recessive disorder characterized by dystonia, dysarthria, rigidity, pigmentary retinal degeneration and brain iron accumulation. PANK2 encodes the mitochondrial enzyme pantothenate kinase type 2, responsible for the phosphorylation of pantothenate or vitamin B5 in the biosynthesis of co-enzyme A. A Pank2 knockout (Pank2−/−) mouse model did not recapitulate the human disease but showed azoospermia and mitochondrial dysfunctions. We challenged this mouse model with a low glucose and high lipid content diet (ketogenic diet) to stimulate lipid use by mitochondrial beta-oxidation. In the presence of a shortage of co-enzyme A, this diet could evoke a general impairment of bioenergetic metabolism. Only Pank2−/− mice fed with a ketogenic diet developed a pantothenate kinase-associated neurodegeneration-like syndrome characterized by severe motor dysfunction, neurodegeneration and severely altered mitochondria in the central and peripheral nervous systems. These mice also showed structural alteration of muscle morphology, which was comparable with that observed in a patient with pantothenate kinase-associated neurodegeneration. We here demonstrate that pantethine administration can prevent the onset of the neuromuscular phenotype in mice suggesting the possibility of experimental treatment in patients with pantothenate kinase-associated neurodegeneration. PMID:24316510

  9. Novel non-viral vectors for gene delivery: synthesis of a second-generation library of mono-functionalized poly-(guanidinium)amines and their introduction into cationic lipids.

    Science.gov (United States)

    Byk, G; Soto, J; Mattler, C; Frederic, M; Scherman, D

    1998-01-01

    The development of new gene delivery technologies is a prerequisite towards gene therapy clinical trials. Because gene delivery mediated by viral vectors remains of limited scope due to immunological and propagation risks, the development of new non-viral gene delivery systems is of crucial importance. We have synthesized a secondary library of mono-functionalized poly-(guanidinium)amines generated from a library of mono-functionalized polyamines applying the concept of "libraries from libraries." The method allows a quick and easy access to mono-functionalized geometrically varied poly-(guanidinium)amines. The new building blocks were introduced into cationic lipids to obtain novel poly-(guanidinium)amine lipids, which are potential DNA vectors for gene delivery. Copyright 1998 John Wiley & Sons, Inc.

  10. Silencing of Iron and Heme-Related Genes Revealed a Paramount Role of Iron in the Physiology of the Hematophagous Vector Rhodnius prolixus

    Directory of Open Access Journals (Sweden)

    Ana B. Walter-Nuno

    2018-02-01

    Full Text Available Iron is an essential element for most organisms However, free iron and heme, its complex with protoporphyrin IX, can be extremely cytotoxic, due to the production of reactive oxygen species, eventually leading to oxidative stress. Thus, eukaryotic cells control iron availability by regulating its transport, storage and excretion as well as the biosynthesis and degradation of heme. In the genome of Rhodnius prolixus, the vector of Chagas disease, we identified 36 genes related to iron and heme metabolism We performed a comprehensive analysis of these genes, including identification of homologous genes described in other insect genomes. We observed that blood-meal modulates the expression of ferritin, Iron Responsive protein (IRP, Heme Oxygenase (HO and the heme exporter Feline Leukemia Virus C Receptor (FLVCR, components of major pathways involved in the regulation of iron and heme metabolism, particularly in the posterior midgut (PM, where an intense release of free heme occurs during the course of digestion. Knockdown of these genes impacted the survival of nymphs and adults, as well as molting, oogenesis and embryogenesis at different rates and time-courses. The silencing of FLVCR caused the highest levels of mortality in nymphs and adults and reduced nymph molting. The oogenesis was mildly affected by the diminished expression of all of the genes whereas embryogenesis was dramatically impaired by the knockdown of ferritin expression. Furthermore, an intense production of ROS in the midgut of blood-fed insects occurs when the expression of ferritin, but not HO, was inhibited. In this manner, the degradation of dietary heme inside the enterocytes may represent an oxidative challenge that is counteracted by ferritins, conferring to this protein a major antioxidant role. Taken together these results demonstrate that the regulation of iron and heme metabolism is of paramount importance for R. prolixus physiology and imbalances in the levels of

  11. Pretransplant mobilization with granulocyte colony-stimulating factor improves B-cell reconstitution by lentiviral vector gene therapy in SCID-X1 mice.

    Science.gov (United States)

    Huston, Marshall W; Riegman, Adriaan R A; Yadak, Rana; van Helsdingen, Yvette; de Boer, Helen; van Til, Niek P; Wagemaker, Gerard

    2014-10-01

    Hematopoietic stem cell (HSC) gene therapy is a demonstrated effective treatment for X-linked severe combined immunodeficiency (SCID-X1), but B-cell reconstitution and function has been deficient in many of the gene therapy treated patients. Cytoreductive preconditioning is known to improve HSC engraftment, but in general it is not considered for SCID-X1 since the poor health of most of these patients at diagnosis and the risk of toxicity preclude the conditioning used in standard bone marrow stem cell transplantation. We hypothesized that mobilization of HSC by granulocyte colony-stimulating factor (G-CSF) should create temporary space in bone marrow niches to improve engraftment and thereby B-cell reconstitution. In the present pilot study supplementing our earlier preclinical evaluation (Huston et al., 2011), Il2rg(-/-) mice pretreated with G-CSF were transplanted with wild-type lineage negative (Lin(-)) cells or Il2rg(-/-) Lin(-) cells transduced with therapeutic IL2RG lentiviral vectors. Mice were monitored for reconstitution of lymphocyte populations, level of donor cell chimerism, and antibody responses as compared to 2 Gy total body irradiation (TBI), previously found effective in promoting B-cell reconstitution. The results demonstrate that G-CSF promotes B-cell reconstitution similar to low-dose TBI and provides proof of principle for an alternative approach to improve efficacy of gene therapy in SCID patients without adverse effects associated with cytoreductive conditioning.

  12. Common defects of mitochondria and iron in neurodegeneration and diabetes (MIND): A paradigm worth exploring

    Science.gov (United States)

    Stroh, Matthew; Swerdlow, Russell H.; Zhu, Hao

    2014-01-01

    A popular, if not centric, approach to the study of an event is to first consider that of the simplest cause. When dissecting the underlying mechanisms governing idiopathic diseases, this generally takes the form of an ab initio genetic approach. To date, this genetic ‘smoking gun’ has remained elusive in diabetes mellitus and for many affected by neurodegenerative diseases. With no single gene, or even subset of genes, conclusively causative in all cases, other approaches to the etiology and treatment of these diseases seem reasonable, including the correlation of a systems’ predisposed sensitivity to particular influence. In the cases of diabetes mellitus and neurodegenerative diseases, overlapping themes of mitochondrial influence or dysfunction and iron dyshomeostasis are apparent and relatively consistent. This mini-review discusses the influence of mitochondrial function and iron homeostasis on diabetes mellitus and neurodegenerative disease, namely Alzheimer’s disease. Also discussed is the incidence of diabetes accompanied by neuropathy and neurodegeneration along with neurodegenerative disorders prone to development of diabetes. Mouse models containing multiple facets of this overlap are also described alongside current molecular trends attributed to both diseases. As a way of approaching the idiopathic and complex nature of these diseases we are proposing the consideration of a MIND (mitochondria, iron, neurodegeneration, and diabetes) paradigm in which systemic metabolic influence, iron homeostasis, and respective genetic backgrounds play a central role in the development of disease. PMID:24361914

  13. Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.

    Science.gov (United States)

    Ali, Yousuf O; Escala, Wilfredo; Ruan, Kai; Zhai, R Grace

    2011-03-11

    Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination. Here we use behavioral assays, including the negative geotaxis assay and the aversive phototaxic suppression assay (APS assay), to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

  14. Forecasting Caspian Sea level changes using satellite altimetry data (June 1992-December 2013) based on evolutionary support vector regression algorithms and gene expression programming

    Science.gov (United States)

    Imani, Moslem; You, Rey-Jer; Kuo, Chung-Yen

    2014-10-01

    Sea level forecasting at various time intervals is of great importance in water supply management. Evolutionary artificial intelligence (AI) approaches have been accepted as an appropriate tool for modeling complex nonlinear phenomena in water bodies. In the study, we investigated the ability of two AI techniques: support vector machine (SVM), which is mathematically well-founded and provides new insights into function approximation, and gene expression programming (GEP), which is used to forecast Caspian Sea level anomalies using satellite altimetry observations from June 1992 to December 2013. SVM demonstrates the best performance in predicting Caspian Sea level anomalies, given the minimum root mean square error (RMSE = 0.035) and maximum coefficient of determination (R2 = 0.96) during the prediction periods. A comparison between the proposed AI approaches and the cascade correlation neural network (CCNN) model also shows the superiority of the GEP and SVM models over the CCNN.

  15. Targeted retrograde transfection of adenovirus vector carrying brain-derived neurotrophic factor gene prevents loss of mouse (twy/twy) anterior horn neurons in vivo sustaining mechanical compression.

    Science.gov (United States)

    Xu, Kan; Uchida, Kenzo; Nakajima, Hideaki; Kobayashi, Shigeru; Baba, Hisatoshi

    2006-08-01

    Immunohistochemical analysis after adenovirus (AdV)-mediated BDNF gene transfer in and around the area of mechanical compression in the cervical spinal cord of the hyperostotic mouse (twy/twy). To investigate the neuroprotective effect of targeted AdV-BDNF gene transfection in the twy mouse with spontaneous chronic compression of the spinal cord motoneurons. Several studies reported the neuroprotective effects of neurotrophins on injured spinal cord. However, no report has described the effect of targeted retrograde neurotrophic gene delivery on motoneuron survival in chronic compression lesions of the cervical spinal cord resembling lesions of myelopathy. LacZ marker gene using adenoviral vector (AdV-LacZ) was used to evaluate retrograde delivery from the sternomastoid muscle in adult twy mice (16-week-old) and (control). Four weeks after the AdV-LacZ or AdV-BDNF injection, the compressed cervical spinal cord was removed en bloc for immunohistologic investigation of b-galactosidase activity and immunoreactivity and immunoblot analyses of BDNF. The number of anterior horn neurons was counted using Nissl, ChAT and AChE staining. Spinal accessory motoneurons between C1 and C3 segments were successfully transfected by AdV-LacZ in both twy and ICR mice after targeted intramuscular injection. Immunoreactivity to BDNF was significantly stronger in AdV-BDNF-gene transfected twy mice than in AdV-LacZ-gene transfected mice. At the cord level showing the maximum compression in AdV-BDNF-transfected twy mice, the number of anterior horn neurons was sinificantly higher in the topographic neuronal cell counting of Nissl-, ChAT-, and AChE-stained samples than in AdV-LacZ-injected twy mice. Targeted AdV-BDNF-gene delivery significantly increased Nissl-stained anterior horn neurons and enhanced cholinergic enzyme activities in the twy. Our results suggest that targeted retrograde AdV-BDNF-gene in vivo delivery may enhance neuronal survival even under chronic mechanical compression.

  16. Neurodegeneration in Autoimmune Optic Neuritis Is Associated with Altered APP Cleavage in Neurons and Up-Regulation of p53.

    Directory of Open Access Journals (Sweden)

    Sabine Herold

    Full Text Available Multiple Sclerosis (MS is a chronic autoimmune inflammatory disease of the central nervous system (CNS. Histopathological and radiological analysis revealed that neurodegeneration occurs early in the disease course. However, the pathological mechanisms involved in neurodegeneration are poorly understood. Myelin oligodendrocyte glycoprotein (MOG-induced experimental autoimmune encephalomyelitis (EAE in Brown Norway rats (BN-rats is a well-established animal model, especially of the neurodegenerative aspects of MS. Previous studies in this animal model indicated that loss of retinal ganglion cells (RGCs, the neurons that form the axons of the optic nerve, occurs in the preclinical phase of the disease and is in part independent of overt histopathological changes of the optic nerve. Therefore, the aim of this study was to identify genes which are involved in neuronal cell loss at different disease stages of EAE. Furthermore, genes that are highly specific for autoimmune-driven neurodegeneration were compared to those regulated in RGCs after optic nerve axotomy at corresponding time points. Using laser capture micro dissection we isolated RNA from unfixed RGCs and performed global transcriptome analysis of retinal neurons. In total, we detected 582 genes sequentially expressed in the preclinical phase and 1150 genes in the clinical manifest EAE (P 1.5. Furthermore, using ingenuity pathway analysis (IPA, we identified amyloid precursor protein (APP as a potential upstream regulator of changes in gene expression in the preclinical EAE but neither in clinical EAE, nor at any time point after optic nerve transection. Therefore, the gene pathway analysis lead to the hypothesis that altered cleavage of APP in neurons in the preclinical phase of EAE leads to the enhanced production of APP intracellular domain (AICD, which in turn acts as a transcriptional regulator and thereby initiates an apoptotic signaling cascade via up-regulation of the target gene p

  17. Synthesis, and Characterization, and Evaluation of Cellular Effects of the FOL-PEG-g-PEI-GAL Nanoparticles as a Potential Non-Viral Vector for Gene Delivery

    International Nuclear Information System (INIS)

    Ghiamkazemi, S.; Amanzadeh, A.; Dinarvand, R.; Rafiee-Tehrani, M.; Amini, M.; Ghiamkazemi, S.; Dinarvand, R.; Rafiee-Tehrani, M.; Ghiamkazemi, S.; Rafiee-Tehrani, M.; Amanzadeh, A.; Rafiee-Tehrani, M.

    2010-01-01

    In this manuscript, we synthesized the potential non viral vector for gene delivery with proper transfection efficiency and low cytotoxicity. Polyethylenimine (PEI) is a well-known cationic polymer which has high positive surface charge for condensing plasmid DNA. However; it is highly cytotoxic in many cell lines because of the high surface charge, non-biodegradability and non-biocompatibility. To enhance PEI biodegradability, the graft copolymer PEG-g-PEI was synthesized. To target cancer liver cells, two targeting ligands folic acid and galactose (lactobionic acid) which are over expressed on human hepatocyte carcinoma were attached to graft copolymer and FOL-PEG-g-PEI-GAL copolymer was synthesized. Composition of this grafted copolymer was characterized using 1H-NMR and FTIR spectra. The molecular weight and zeta potential of this copolymer was compared to PEI. The particle size and zeta potential of FOL-PEG-g-PEI-GAL/DNA complexes at various N/P ratio were measured using dynamic light scattering (DLS). Cytotoxicity of the copolymer was also studied in cultured HepG2 human hepatoblastoma cell line. The FOL-PEG-g-PEI-GAL/DNA complexes at various N/P ratios exhibited no cytotoxicity in HepG2 cell line compared to PEI 25K as a control. The novel copolymer showed enhanced biodegradability in physiological conditions in compared with PEI and targeted cultured HepG2 cells. More importantly, significant transfection efficiency was exhibited in cancer liver cells. Together, our results showed that FOL-PEG-g-PEI-GAL nanoparticles could be considered as a useful non-viral vector for targeted gene delivery.

  18. Unraveling Host-Vector-Arbovirus Interactions by Two-Gene High Resolution Melting Mosquito Bloodmeal Analysis in a Kenyan Wildlife-Livestock Interface.

    Directory of Open Access Journals (Sweden)

    David Omondi

    Full Text Available The blood-feeding patterns of mosquitoes are directly linked to the spread of pathogens that they transmit. Efficient identification of arthropod vector bloodmeal hosts can identify the diversity of vertebrate species potentially involved in disease transmission cycles. While molecular bloodmeal analyses rely on sequencing of cytochrome b (cyt b or cytochrome oxidase 1 gene PCR products, recently developed bloodmeal host identification based on high resolution melting (HRM analyses of cyt b PCR products is more cost-effective. To resolve the diverse vertebrate hosts that mosquitoes may potentially feed on in sub-Saharan Africa, we utilized HRM profiles of both cyt b and 16S ribosomal RNA genes. Among 445 blood-fed Aedeomyia, Aedes, Anopheles, Culex, Mansonia, and Mimomyia mosquitoes from Kenya's Lake Victoria and Lake Baringo regions where many mosquito-transmitted pathogens are endemic, we identified 33 bloodmeal hosts including humans, eight domestic animal species, six peridomestic animal species and 18 wildlife species. This resolution of vertebrate host species was only possible by comparing profiles of both cyt b and 16S markers, as melting profiles of some pairs of species were similar for either marker but not both. We identified mixed bloodmeals in a Culex pipiens from Mbita that had fed on a goat and a human and in two Mansonia africana mosquitoes from Baringo that each had fed on a rodent (Arvicanthis niloticus in addition to a human or baboon. We further detected Sindbis and Bunyamwera viruses in blood-fed mosquito homogenates by Vero cell culture and RT-PCR in Culex, Aedeomyia, Anopheles and Mansonia mosquitoes from Baringo that had fed on humans and livestock. The observed mosquito feeding on both arbovirus amplifying hosts (including sheep and goats and possible arbovirus reservoirs (birds, porcupine, baboons, rodents informs arbovirus disease epidemiology and vector control strategies.

  19. Vector analysis

    CERN Document Server

    Brand, Louis

    2006-01-01

    The use of vectors not only simplifies treatments of differential geometry, mechanics, hydrodynamics, and electrodynamics, but also makes mathematical and physical concepts more tangible and easy to grasp. This text for undergraduates was designed as a short introductory course to give students the tools of vector algebra and calculus, as well as a brief glimpse into these subjects' manifold applications. The applications are developed to the extent that the uses of the potential function, both scalar and vector, are fully illustrated. Moreover, the basic postulates of vector analysis are brou

  20. Developing an Alternanthera mosaic virus vector for efficient clonging of Whitefly cDNA RNAi to screen gene function

    Science.gov (United States)

    Alternanthera mosaic virus (AltMV; genus Potexvirus) is distinguished from the type member of the genus, Potato virus X by features of viral movement and variation within triple gene block protein 1 (TGB1). AltMV TGB1 variants TGB1L88 and TGB1P88 confer strong and weak silencing suppression, respect...

  1. Viral vector-mediated gene expression in olfactory ensheathing glia implants in the lesioned rat spinal cord

    NARCIS (Netherlands)

    Ruitenberg, Marc J; Plant, Giles W; Christensen, C L; Blits, B; Niclou, Simone P; Harvey, Alan R; Boer, G J; Verhaagen, J

    Implantation of olfactory ensheathing glia (OEG) is a promising strategy to augment long-distance regeneration in the injured spinal cord. In this study, implantation of OEG following unilateral hemisection of the dorsal cervical spinal cord was combined with ex vivo gene transfer techniques. We

  2. Minocycline Rescues from Zinc-Induced Nigrostriatal Dopaminergic Neurodegeneration: Biochemical and Molecular Interventions.

    Science.gov (United States)

    Kumar, Vinod; Singh, Brajesh Kumar; Chauhan, Amit Kumar; Singh, Deepali; Patel, Devendra Kumar; Singh, Chetna

    2016-07-01

    Accumulation of zinc (Zn) in dopaminergic neurons is implicated in Parkinson's disease (PD), and microglial activation plays a critical role in toxin-induced Parkinsonism. Oxidative stress is accused in Zn-induced dopaminergic neurodegeneration; however, its connection with microglial activation is still not known. This study was undertaken to elucidate the role and underlying mechanism of microglial activation in Zn-induced nigrostriatal dopaminergic neurodegeneration. Male Wistar rats were treated intraperitoneally with/without zinc sulphate (20 mg/kg) in the presence/absence of minocycline (30 mg/kg), a microglial activation inhibitor, for 2-12 weeks. While neurobehavioral and biochemical indexes of PD and number of dopaminergic neurons were reduced, the number of microglial cells was increased in the substantia nigra of the Zn-exposed animals. Similarly, Zn elevated lipid peroxidation (LPO) and activities of superoxide dismutase (SOD) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; however, catalase activity was reduced. Besides, Zn increased an association of NADPH oxidase subunit p67(phox) with membrane, cytochrome c release from the mitochondria and cleavage of pro-caspase 3. Zn attenuated the expression of tyrosine hydroxylase (TH) and vesicular monoamine transporter-2 (VMAT-2) while augmented the expression of dopamine transporter (DAT) and heme oxygenase-1 (HO-1). Minocycline alleviated Zn-induced behavioural impairments, loss of TH-positive neurons, activated microglial cells and biochemical indexes and modulated the expression of studied genes/proteins towards normalcy. The results demonstrate that minocycline reduces the number of activated microglial cells and oxidative stress, which rescue from Zn-induced changes in the expression of monoamine transporter and nigrostriatal dopaminergic neurodegeneration.

  3. Genetic pathways to Neurodegeneration Models and mechanisms ...

    Indian Academy of Sciences (India)

    Paige Rudich

    Models and mechanisms of repeat expansion disorders: a worm's eye view ..... retardation 1 gene FMR1 gives rise to a spectrum of neurological disorders (Saul and Tarleton ... autism. Shorter repeat expansion lengths from 55-200 cause the.

  4. The nucleotide sequence and a first generation gene transfer vector of species B human adenovirus serotype 3.

    Science.gov (United States)

    Sirena, Dominique; Ruzsics, Zsolt; Schaffner, Walter; Greber, Urs F; Hemmi, Silvio

    2005-12-20

    Human adenovirus (Ad) serotype 3 causes respiratory infections. It is considered highly virulent, accounting for about 13% of all Ad isolates. We report here the complete Ad3 DNA sequence of 35,343 base pairs (GenBank accession DQ086466). Ad3 shares 96.43% nucleotide identity with Ad7, another virulent subspecies B1 serotype, and 82.56 and 62.75% identity with the less virulent species B2 Ad11 and species C Ad5, respectively. The genomic organization of Ad3 is similar to the other human Ads comprising five early transcription units, E1A, E1B, E2, E3, and E4, two delayed early units IX and IVa2, and the major late unit, in total 39 putative and 7 hypothetical open reading frames. A recombinant E1-deleted Ad3 was generated on a bacterial artificial chromosome. This prototypic virus efficiently transduced CD46-positive rodent and human cells. Our results will help in clarifying the biology and pathology of adenoviruses and enhance therapeutic applications of viral vectors in clinical settings.

  5. Vector velocimeter

    DEFF Research Database (Denmark)

    2012-01-01

    The present invention relates to a compact, reliable and low-cost vector velocimeter for example for determining velocities of particles suspended in a gas or fluid flow, or for determining velocity, displacement, rotation, or vibration of a solid surface, the vector velocimeter comprising a laser...

  6. Effects of mild running on substantia nigra during early neurodegeneration.

    Science.gov (United States)

    Almeida, Michael F; Silva, Carolliny M; Chaves, Rodrigo S; Lima, Nathan C R; Almeida, Renato S; Melo, Karla P; Demasi, Marilene; Fernandes, Tiago; Oliveira, Edilamar M; Netto, Luis E S; Cardoso, Sandra M; Ferrari, Merari F R

    2018-06-01

    Moderate physical exercise acts at molecular and behavioural levels, such as interfering in neuroplasticity, cell death, neurogenesis, cognition and motor functions. Therefore, the aim of this study is to analyse the cellular effects of moderate treadmill running upon substantia nigra during early neurodegeneration. Aged male Lewis rats (9-month-old) were exposed to rotenone 1mg/kg/day (8 weeks) and 6 weeks of moderate treadmill running, beginning 4 weeks after rotenone exposure. Substantia nigra was extracted and submitted to proteasome and antioxidant enzymes activities, hydrogen peroxide levels and Western blot to evaluate tyrosine hydroxylase (TH), alpha-synuclein, Tom-20, PINK1, TrkB, SLP1, CRMP-2, Rab-27b, LC3II and Beclin-1 level. It was demonstrated that moderate treadmill running, practiced during early neurodegeneration, prevented the increase of alpha-synuclein and maintained the levels of TH unaltered in substantia nigra of aged rats. Physical exercise also stimulated autophagy and prevented impairment of mitophagy, but decreased proteasome activity in rotenone-exposed aged rats. Physical activity also prevented H 2 O 2 increase during early neurodegeneration, although the involved mechanism remains to be elucidated. TrkB levels and its anterograde trafficking seem not to be influenced by moderate treadmill running. In conclusion, moderate physical training could prevent early neurodegeneration in substantia nigra through the improvement of autophagy and mitophagy.

  7. Genetics Home Reference: fatty acid hydroxylase-associated neurodegeneration

    Science.gov (United States)

    ... Mutat. 2010 Apr;31(4):E1251-60. doi: 10.1002/humu.21205. Citation on PubMed Edvardson S, Hama H, ... Neurol. 2010 Nov;68(5):611-8. doi: 10.1002/ana.22122. Citation on PubMed Schipper HM. Neurodegeneration ...

  8. 4R-cembranoid protects against diisopropylfluorophosphate-mediated neurodegeneration

    OpenAIRE

    Ferchmin, P.A.; Andino, Myrna; Salaman, Rebeca Reyes; Alves, Janaina; Velez-Roman, Joyce; Cuadrado, Brenda; Carrasco, Marimeé; Torres-Rivera, Wilmarie; Segarra, Annabell; Martins, Antonio Henrique; Lee, Jae Eun; Eterovic, Vesna A.

    2014-01-01

    Many organophosphorous esters synthesized for applications in industry, agriculture, or warfare irreversibly inhibit acetylcholinesterase, and acute poisoning with these compounds causes life-threatening cholinergic overstimulation. Following classical emergency treatment with atropine, an oxime, and a benzodiazepine, surviving victims often suffer brain neurodegeneration. Currently, there is no pharmacological treatment to prevent this brain injury. Here we show that a cyclic diterpenoid, (1...

  9. Temporal dynamics of hippocampal neurogenesis in chronic neurodegeneration

    Science.gov (United States)

    Suzzi, Stefano; Vargas-Caballero, Mariana; Fransen, Nina L.; Al-Malki, Hussain; Cebrian-Silla, Arantxa; Garcia-Verdugo, Jose Manuel; Riecken, Kristoffer; Fehse, Boris; Perry, V. Hugh

    2014-01-01

    The study of neurogenesis during chronic neurodegeneration is crucial in order to understand the intrinsic repair mechanisms of the brain, and key to designing therapeutic strategies. In this study, using an experimental model of progressive chronic neurodegeneration, murine prion disease, we define the temporal dynamics of the generation, maturation and integration of new neurons in the hippocampal dentate gyrus, using dual pulse-chase, multicolour γ-retroviral tracing, transmission electron microscopy and patch-clamp. We found increased neurogenesis during the progression of prion disease, which partially counteracts the effects of chronic neurodegeneration, as evidenced by blocking neurogenesis with cytosine arabinoside, and helps to preserve the hippocampal function. Evidence obtained from human post-mortem samples, of both variant Creutzfeldt-Jakob disease and Alzheimer’s disease patients, also suggests increased neurogenic activity. These results open a new avenue into the exploration of the effects and regulation of neurogenesis during chronic neurodegeneration, and offer a new model to reproduce the changes observed in human neurodegenerative diseases. PMID:24941947

  10. The Cytochrome P450 gene CYP6P12 confers pyrethroid resistance in kdr-free Malaysian populations of the dengue vector Aedes albopictus.

    Science.gov (United States)

    Ishak, Intan H; Riveron, Jacob M; Ibrahim, Sulaiman S; Stott, Rob; Longbottom, Joshua; Irving, Helen; Wondji, Charles S

    2016-04-20

    Control of Aedes albopictus, major dengue and chikungunya vector, is threatened by growing cases of insecticide resistance. The mechanisms driving this resistance remain poorly characterised. This study investigated the molecular basis of insecticide resistance in Malaysian populations of Ae. albopictus. Microarray-based transcription profiling revealed that metabolic resistance (cytochrome P450 up-regulation) and possibly a reduced penetration mechanism (consistent over-expression of cuticular protein genes) were associated with pyrethroid resistance. CYP6P12 over-expression was strongly associated with pyrethroid resistance whereas CYP6N3 was rather consistently over-expressed across carbamate and DDT resistant populations. Other detoxification genes also up-regulated in permethrin resistant mosquitoes included a glucuronosyltransferase (AAEL014279-RA) and the glutathione-S transferases GSTS1 and GSTT3. Functional analyses further supported that CYP6P12 contributes to pyrethroid resistance in Ae. albopictus as transgenic expression of CYP6P12 in Drosophila was sufficient to confer pyrethroid resistance in these flies. Furthermore, molecular docking simulations predicted CYP6P12 possessing enzymatic activity towards pyrethroids. Patterns of polymorphism suggested early sign of selection acting on CYP6P12 but not on CYP6N3. The major role played by P450 in the absence of kdr mutations suggests that addition of the synergist PBO to pyrethroids could improve the efficacy of this insecticide class and overcome resistance in field populations of Ae. albopictus.

  11. Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch.

    Science.gov (United States)

    Poulain, Adeline; Perret, Sylvie; Malenfant, Félix; Mullick, Alaka; Massie, Bernard; Durocher, Yves

    2017-08-10

    To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHO BRI/rcTA ) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHO BRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900mg/L were reproducibly achieved for a Fc fusion protein and up to 350mg/L for an antibody after 14days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHO BRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  12. C19orf12 mutations in neurodegeneration with brain iron accumulation mimicking juvenile amyotrophic lateral sclerosis.

    Science.gov (United States)

    Deschauer, M; Gaul, C; Behrmann, C; Prokisch, H; Zierz, S; Haack, T B

    2012-11-01

    Mutations in C19orf12 have been recently identified as the molecular genetic cause of a subtype of neurodegeneration with brain iron accumulation (NBIA). Given the mitochondrial localization of the gene product the new NBIA subtype was designated mitochondrial membrane protein-associated neurodegeneration. Frequent features in the patients described so far included extrapyramidal signs and pyramidal tract involvement. Here, we report three C19orf12-mutant patients from two families presenting with predominant upper and lower motor neuron dysfunction mimicking amyotrophic lateral sclerosis with juvenile onset. While extrapyramidal signs were absent, all patients showed neuropsychological abnormalities with disinhibited or impulsive behavior. Optic atrophy was present in the simplex case. T2-weighted cranial MRI showed hypointensities suggestive of iron accumulation in the globi pallidi and the midbrain in all patients. Sequence analysis of C19orf12 revealed a novel mutation, p.Gly66del, compound heterozygous with known mutations in all patients. These patients highlight that C19orf12 defects should be considered as a differential diagnosis in patients with juvenile onset motor neuron diseases. Patients have to be examined carefully for neuropsychological abnormalities, optic neuropathy, and signs of brain iron accumulation in MRI.

  13. Genetic variations of ND5 gene of mtDNA in populations of Anopheles sinensis (Diptera: Culicidae) malaria vector in China

    Science.gov (United States)

    2013-01-01

    Background Anopheles sinensis is a principal vector for Plasmodium vivax malaria in most parts of China. Understanding of genetic structure and genetic differentiation of the mosquito should contribute to the vector control and malaria elimination in China. Methods The present study investigated the genetic structure of An. sinensis populations using a 729 bp fragment of mtDNA ND5 among 10 populations collected from seven provinces in China. Results ND5 was polymorphic by single mutations within three groups of An. sinensis that were collected from 10 different geographic populations in China. Out of 140 specimens collected from 10 representative sites, 84 haplotypes and 71 variable positions were determined. The overall level of genetic differentiation of An. sinensis varied from low to moderate across China and with a FST range of 0.00065 – 0.341. Genealogy analysis clustered the populations of An. sinensis into three main clusters. Each cluster shared one main haplotype. Pairwise variations within populations were higher (68.68%) than among populations (31.32%) and with high fixation index (FST = 0.313). The results of the present study support population growth and expansion in the An. sinensis populations from China. Three clusters of An. sinensis populations were detected in this study with each displaying different proportion patterns over seven Chinese provinces. No correlation between genetic and geographic distance was detected in overall populations of An. sinensis (R2 = 0.058; P = 0.301). Conclusions The results indicate that the ND5 gene of mtDNA is highly polymorphic in An. sinensis and has moderate genetic variability in the populations of this mosquito in China. Demographic and spatial results support evidence of expansion in An. sinensis populations. PMID:24192424

  14. Bifidobacterium breve as a delivery vector of IL-24 gene therapy for head and neck squamous cell carcinoma in vivo.

    Science.gov (United States)

    Wang, L; Vuletic, I; Deng, D; Crielaard, W; Xie, Z; Zhou, K; Zhang, J; Sun, H; Ren, Q; Guo, C

    2017-11-01

    Beneficial bacteria are becoming ever more popular gene delivery method for hypoxia-tumor targeting in vivo. In this study we investigated the therapeutic effect of new recombinant Bifidobacterium breve strain expressing interleukin (IL)-24 gene (B. breve-IL24) on head and neck tumor xenograft in mice. Briefly, B. breve transformants were obtained through electro-transformation. Bacteria-tumor-targeting ability were analyzed in vivo over different time points (1, 3 and 7 days post-bacteria injection). Furthermore, the therapeutic effect of bacteria on tumor cells in vivo were analyzed as follows: 30 Balb/c nude mice bearing subcutaneous tumor were randomly divided in three groups (Drug group, green fluorescent protein (GFP) group and Saline group). The therapy lasted for 2 weeks and included B. breve-IL24 administration via tail vein for Drug group, B. breve-GFP for GFP group and phosphate buffered saline for Saline group. The tumor growth was monitored using standard caliper technique, while the apoptosis induction in vivo was analyzed by Real-time Positron Emission Tomography/Computed Tomography (PET/CT) imaging ([18F]-ML-10 tracer). At the end of the experiment, tumor tissues were collected and analyzed by western blotting. Briefly, our results suggested that our new recombinant bacterium has the capability of targeting tumor tissue in vivo. As for the therapeutic effect, our new strain has revealed to be a promising therapeutic approach against tumor growth in vivo. Briefly, higher tumor growth inhibition and higher tumor cell apoptosis induction were observed in Drug group compared with the GFP and Saline groups. To conclude, a new recombinant strain B. breve-IL24 offers a novel, safe and clinically acceptable therapeutic approach for tumor therapy in vivo.

  15. Equivalent Vectors

    Science.gov (United States)

    Levine, Robert

    2004-01-01

    The cross-product is a mathematical operation that is performed between two 3-dimensional vectors. The result is a vector that is orthogonal or perpendicular to both of them. Learning about this for the first time while taking Calculus-III, the class was taught that if AxB = AxC, it does not necessarily follow that B = C. This seemed baffling. The…

  16. Towards an Integrative Understanding of tRNA Aminoacylation–Diet–Host–Gut Microbiome Interactions in Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Elena L. Paley

    2018-03-01

    Full Text Available Transgenic mice used for Alzheimer’s disease (AD preclinical experiments do not recapitulate the human disease. In our models, the dietary tryptophan metabolite tryptamine produced by human gut microbiome induces tryptophanyl-tRNA synthetase (TrpRS deficiency with consequent neurodegeneration in cells and mice. Dietary supplements, antibiotics and certain drugs increase tryptamine content in vivo. TrpRS catalyzes tryptophan attachment to tRNAtrp at initial step of protein biosynthesis. Tryptamine that easily crosses the blood–brain barrier induces vasculopathies, neurodegeneration and cell death via TrpRS competitive inhibition. TrpRS inhibitor tryptophanol produced by gut microbiome also induces neurodegeneration. TrpRS inhibition by tryptamine and its metabolites preventing tryptophan incorporation into proteins lead to protein biosynthesis impairment. Tryptophan, a least amino acid in food and proteins that cannot be synthesized by humans competes with frequent amino acids for the transport from blood to brain. Tryptophan is a vulnerable amino acid, which can be easily lost to protein biosynthesis. Some proteins marking neurodegenerative pathology, such as tau lack tryptophan. TrpRS exists in cytoplasmic (WARS and mitochondrial (WARS2 forms. Pathogenic gene variants of both forms cause TrpRS deficiency with consequent intellectual and motor disabilities in humans. The diminished tryptophan-dependent protein biosynthesis in AD patients is a proof of our model-based disease concept.

  17. Towards an Integrative Understanding of tRNA Aminoacylation-Diet-Host-Gut Microbiome Interactions in Neurodegeneration.

    Science.gov (United States)

    Paley, Elena L; Perry, George

    2018-03-26

    Transgenic mice used for Alzheimer's disease (AD) preclinical experiments do not recapitulate the human disease. In our models, the dietary tryptophan metabolite tryptamine produced by human gut microbiome induces tryptophanyl-tRNA synthetase (TrpRS) deficiency with consequent neurodegeneration in cells and mice. Dietary supplements, antibiotics and certain drugs increase tryptamine content in vivo. TrpRS catalyzes tryptophan attachment to tRNA trp at initial step of protein biosynthesis. Tryptamine that easily crosses the blood-brain barrier induces vasculopathies, neurodegeneration and cell death via TrpRS competitive inhibition. TrpRS inhibitor tryptophanol produced by gut microbiome also induces neurodegeneration. TrpRS inhibition by tryptamine and its metabolites preventing tryptophan incorporation into proteins lead to protein biosynthesis impairment. Tryptophan, a least amino acid in food and proteins that cannot be synthesized by humans competes with frequent amino acids for the transport from blood to brain. Tryptophan is a vulnerable amino acid, which can be easily lost to protein biosynthesis. Some proteins marking neurodegenerative pathology, such as tau lack tryptophan. TrpRS exists in cytoplasmic (WARS) and mitochondrial (WARS2) forms. Pathogenic gene variants of both forms cause TrpRS deficiency with consequent intellectual and motor disabilities in humans. The diminished tryptophan-dependent protein biosynthesis in AD patients is a proof of our model-based disease concept.

  18. Effective gene expression in the rat dorsal root ganglia with a non-viral vector delivered via spinal nerve injection

    Science.gov (United States)

    Chang, Ming-Fong; Hsieh, Jung-Hsien; Chiang, Hao; Kan, Hung-Wei; Huang, Cho-Min; Chellis, Luke; Lin, Bo-Shiou; Miaw, Shi-Chuen; Pan, Chun-Liang; Chao, Chi-Chao; Hsieh, Sung-Tsang

    2016-01-01

    Delivering gene constructs into the dorsal root ganglia (DRG) is a powerful but challenging therapeutic strategy for sensory disorders affecting the DRG and their peripheral processes. The current delivery methods of direct intra-DRG injection and intrathecal injection have several disadvantages, including potential injury to DRG neurons and low transfection efficiency, respectively. This study aimed to develop a spinal nerve injection strategy to deliver polyethylenimine mixed with plasmid (PEI/DNA polyplexes) containing green fluorescent protein (GFP). Using this spinal nerve injection approach, PEI/DNA polyplexes were delivered to DRG neurons without nerve injury. Within one week of the delivery, GFP expression was detected in 82.8% ± 1.70% of DRG neurons, comparable to the levels obtained by intra-DRG injection (81.3% ± 5.1%, p = 0.82) but much higher than those obtained by intrathecal injection. The degree of GFP expression by neurofilament(+) and peripherin(+) DRG neurons was similar. The safety of this approach was documented by the absence of injury marker expression, including activation transcription factor 3 and ionized calcium binding adaptor molecule 1 for neurons and glia, respectively, as well as the absence of behavioral changes. These results demonstrated the efficacy and safety of delivering PEI/DNA polyplexes to DRG neurons via spinal nerve injection. PMID:27748450

  19. Radiation-Induced Upregulation of Gene Expression From Adenoviral Vectors Mediated by DNA Damage Repair and Regulation

    International Nuclear Information System (INIS)

    Nokisalmi, Petri; Rajecki, Maria; Pesonen, Sari; Escutenaire, Sophie; Soliymani, Rabah; Tenhunen, Mikko; Ahtiainen, Laura; Hemminki, Akseli

    2012-01-01

    Purpose: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. Methods and Materials: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. Results: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. Conclusions: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.

  20. Usefulness of intra-arterial embolization method using gelfoam particles in effective gene transduction of adenoviral vector for liver-directed gene therapy: an preliminary animal study in dogs

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jin Hwa; Park, Byeong Ho; Kim, Chan Sung [Dong-A University College of Medicine, Pusan (Korea, Republic of)

    2003-02-01

    Liver-directed gene therapy is being actively pursued and developed as a method of treating various liver diseases. A number of aspects, including gene intervention, an efficient gene delivery system, and stable transgene expression are key to the success of the chosen strategy, and to overcome problems in these areas, several tactics can be used. In this study, we assess the utility of transarterial embolization using gelfoam particles soaked in an adenovirus vector as a gene-delivery method. Using the angiographic approach, three dogs each weighing 9.5-11kg were superselectively catheterized at the left hepatic artery using a 3-F microcatheter and the coaxial method. Two of the dogs were embolized at the left hepatic artery using 3x2x2-mm and 2x1x1-mm gelfoam particles soaked in 2x10{sup 11} particles/kg of recombinant adv. CMV.LacZ(LacZ-adv). The left hepatic artery of the remaining animal, used as a control, was infused with the same dose of lacZ-adv in the same way as before but without embolization of the left hepatic artery. Three days after embolization or the infusion of LacZ-adv, the dogs were sacrificed prior to harvest of the entire liver for the evaluation of gene transduction. X-gal staining of the liver tissue obtained was positive for hepatocytes, but the pattern and degree of gene transduction differed according to gelfoam particle size. Where this was 3x2x2 mm, gene transduction along the liver hilum varied, but where 2x1x1-mm particles were used, transduction was more even. No pathologic hepatic tissue injury or inflammation was apparent, and control liver tissue was not stained by X-gal. Serum SGOT and SGPT levels were slightly higher one day after the procedure, but had normalized by day 3. Intrahepatic transarterial embolization using gelfoam particles soaked in LacZ-adv appears to be a good method for effective liver-targed gene therapy.

  1. Usefulness of intra-arterial embolization method using gelfoam particles in effective gene transduction of adenoviral vector for liver-directed gene therapy: an preliminary animal study in dogs

    International Nuclear Information System (INIS)

    Lee, Jin Hwa; Park, Byeong Ho; Kim, Chan Sung

    2003-01-01

    Liver-directed gene therapy is being actively pursued and developed as a method of treating various liver diseases. A number of aspects, including gene intervention, an efficient gene delivery system, and stable transgene expression are key to the success of the chosen strategy, and to overcome problems in these areas, several tactics can be used. In this study, we assess the utility of transarterial embolization using gelfoam particles soaked in an adenovirus vector as a gene-delivery method. Using the angiographic approach, three dogs each weighing 9.5-11kg were superselectively catheterized at the left hepatic artery using a 3-F microcatheter and the coaxial method. Two of the dogs were embolized at the left hepatic artery using 3x2x2-mm and 2x1x1-mm gelfoam particles soaked in 2x10 11 particles/kg of recombinant adv. CMV.LacZ(LacZ-adv). The left hepatic artery of the remaining animal, used as a control, was infused with the same dose of lacZ-adv in the same way as before but without embolization of the left hepatic artery. Three days after embolization or the infusion of LacZ-adv, the dogs were sacrificed prior to harvest of the entire liver for the evaluation of gene transduction. X-gal staining of the liver tissue obtained was positive for hepatocytes, but the pattern and degree of gene transduction differed according to gelfoam particle size. Where this was 3x2x2 mm, gene transduction along the liver hilum varied, but where 2x1x1-mm particles were used, transduction was more even. No pathologic hepatic tissue injury or inflammation was apparent, and control liver tissue was not stained by X-gal. Serum SGOT and SGPT levels were slightly higher one day after the procedure, but had normalized by day 3. Intrahepatic transarterial embolization using gelfoam particles soaked in LacZ-adv appears to be a good method for effective liver-targed gene therapy

  2. Comparison of Human Sodium/Iodide Symporter (hNIS) Gene Expressions between Lentiviral and Adenoviral Vectors in Rat Mesenchymal Stem Cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, So Yeon; Lee, Won Woo; Kim, Hyun Joo; Chung, June Key; Kim, Sang Eun [Seoul National University College of Medicine, Seoul (Korea, Republic of); Kim, Sung Jin; Lee, Heui Ran [Medical Research Center, Seoul National University, Seoul (Korea, Republic of)

    2008-10-15

    Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirusmediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was 19.1{+-}4.7%, 54.0{+-}6.4%, 85.7{+-}8.7%, and 98.4{+-}1.3% at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and I-125 uptake. Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro I-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC (29,704{+-}6,659 picomole/10{sup 6} cells) was greater than that in adeno-hNIS-rMSC at MOI 100 (6,168{+-}2,134 picomole/10{sup 6} cells). Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression.

  3. Comparison of Human Sodium/Iodide Symporter (hNIS) Gene Expressions between Lentiviral and Adenoviral Vectors in Rat Mesenchymal Stem Cells

    International Nuclear Information System (INIS)

    Park, So Yeon; Lee, Won Woo; Kim, Hyun Joo; Chung, June Key; Kim, Sang Eun; Kim, Sung Jin; Lee, Heui Ran

    2008-01-01

    Quantitative comparison of transgene expression within stem cells between lentivirus and adenovirusmediated delivery systems has not been reported. Here, we evaluated the human sodium iodide symporter (hNIS) gene expression in rat mesenchymal stem cell (rMSC) transduced by lentivirus or adenovirus, and compared the hNIS expression quantitatively between the two delivery systems. Lentiviral-mediated hNIS expressing rMSC (lenti-hNIS-rMSC) was constructed by cloning hNIS gene into pLenti6/UbC/V5-DEST (Invitrogen) to obtain pLenti-hNIS, transducing rMSC with the pLenti-hNIS, and selecting with blasticidin for 3 weeks. Recombinant adenovirus expressing hNIS gene (Rad-hNIS) was produced by homologous recombination and transduction efficiency of Rad-hNIS into rMSC evaluated by Rad-GFP was 19.1±4.7%, 54.0±6.4%, 85.7±8.7%, and 98.4±1.3% at MOI 1, 5, 20, and 100, respectively. The hNIS expressions in lenti-hNIS-rMSC or adeno-hNIS-rMSC were assessed by immunocytochemistry, western blot, and I-125 uptake. Immunocytochemistry and western blot analyses revealed that hNIS expressions in lenti-hNIS-rMSC were greater than those in adeno-hNIS-rMSC at MOI 20 but lower than at MOI 50. However in vitro I-125 uptake test demonstrated that iodide uptake in lenti-hNIS-rMSC (29,704±6,659 picomole/10 6 cells) was greater than that in adeno-hNIS-rMSC at MOI 100 (6,168±2,134 picomole/10 6 cells). Despite lower amount of expressed protein, hNIS function in rMSC was greater by lentivirus than by adenovirus mediated expression. Stem cell tracking using hNIS as a reporter gene should be conducted in consideration of relative vector efficiency for transgene expression

  4. Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway

    Directory of Open Access Journals (Sweden)

    Gaigalat Lars

    2006-08-01

    Full Text Available Abstract Background Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. Results A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4-monophosphatases (EC 3.1.3.25. Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown

  5. Construction of expression vectors carrying mouse peroxisomal ...

    African Journals Online (AJOL)

    The aim of this study was to construct expression vectors carrying mouse peroxisomal protein gene (PEP-cDNA) in prokaryotic and mammalian expression vectors in ... pGEX6p2-PEP and pUcD3-FLAG-PEP constructed vectors were transformed into the one shot TOP10 and JM105 bacterial competent cells, respectively.

  6. Inhibition of HBV replication by delivering the dual-gene expression vector pHsa-miR16-siRNA in HepG2.2.15 cells.

    Science.gov (United States)

    Wei, Wei; Wang, Su-Fei; Yu, Bing; Ni, Ming

    2017-12-01

    This study aimed to construct the dual-gene expression vector pHsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The expression vectors siR-1583 and pHsa-miR16-siRNA were designed and constructed. HepG2.2.15 cells were transfected with the empty vector, siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively. ELISA was performed to measure the expression of HBsAg and HBeAg in the culture supernatant 48 and72 h post transfection. Fluorescence quantitative PCR was used to measure the HBV mRNA degradation efficiency and HBV DNA copy number. The results showed that the expression of HBV genes was significantly inhibited in HepG2.2.15 cells transfected with siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively, when compared with that in cells transfected with the empty vectors, with the inhibitory effect of pHsa-miR16-siRNA being the most significant. ELISA showed that the inhibitory rates of HBsAg and HBeAg in pHsa-miR16-siRNA transfected cells were correspondingly 87.3% and 85.0% at 48 h, and 88.6% and 86.5% at 72 h post transfection (PHBV mRNA decreased by 80.2% (t=-99.22, PHBV DNA by 92.8% (t=-73.06, PHBV DNA copy number by 89.8% (t=-47.13, PHBV more efficiently than a single-gene expression vector.

  7. A vector carrying the GFP gene (Green fluorescent protein as a yeast marker for fermentation processes Um vetor com o gene da GFP (Green fluorescent protein para a marcação de leveduras em processos fermentativos

    Directory of Open Access Journals (Sweden)

    Luiz Humberto Gomes

    2000-12-01

    Full Text Available Contaminant yeasts spoil pure culture fermentations and cause great losses in quality and product yields. They can be detected by a variety of methods although none being so efficient for early detection of contaminant yeast cells that appear at low frequency. Pure cultures bearing genetic markers can ease the direct identification of cells and colonies among contaminants. Fast and easy detection are desired and morphological markers would even help the direct visualization of marked pure cultures among contaminants. The GFP gene for green fluorescent protein of Aquorea victoria, proved to be a very efficient marker to visualize transformed cells in mixed populations and tissues. To test this marker in the study of contaminated yeast fermentations, the GFP gene was used to construct a vector under the control of the ADH2 promoter (pYGFP3. Since ADH2 is repressed by glucose the expression of the protein would not interfere in the course of fermentation. The transformed yeasts with the vector pYGFP3 showed high stability and high bioluminescence to permit identification of marked cells among a mixed population of cells. The vector opens the possibility to conduct further studies aiming to develop an efficient method for early detection of spoilage yeasts in industrial fermentative processes.Leveduras contaminantes podem causar grandes perdas em processos fermentativos quando infectam culturas puras e degradam a qualidade do produto final. Estas leveduras podem ser detectadas por diversos métodos mas nenhum deles oferece resultados com a exatidão e precisão necessárias, quando os contaminantes estão em baixa freqüência. Culturas puras contendo um gene marcador podem ser utilizadas para a direta identificação de células e colônias contaminantes. Detecção rápida e fácil é desejada e marcadores morfológicos podem auxiliar na visualização da cultura marcada. O gene da GFP (green fluorescent protein extraído da Aequorea victoria

  8. Microglial cell dysregulation in brain aging and neurodegeneration

    OpenAIRE

    von Bernhardi, Rommy; Eugen?n-von Bernhardi, Laura; Eugen?n, Jaime

    2015-01-01

    Aging is the main risk factor for neurodegenerative diseases. In aging, microglia undergoes phenotypic changes compatible with their activation. Glial activation can lead to neuroinflammation, which is increasingly accepted as part of the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease (AD). We hypothesize that in aging, aberrant microglia activation leads to a deleterious environment and neurodegeneration. In aged mice, microglia exhibit an increased expression of c...

  9. Multiproteinopathy, neurodegeneration and old age: a case study.

    Science.gov (United States)

    Rojas, Julio C; Stephens, Melanie L; Rabinovici, Gil D; Kramer, Joel H; Miller, Bruce L; Seeley, William W

    2018-02-01

    A complex spectrum of mixed brain pathologies is common in older people. This clinical pathologic conference case study illustrates the challenges of formulating clinicopathologic correlations in late-onset neurodegenerative diseases featuring cognitive-behavioral syndromes with underlying multiple proteinopathy. Studies on the co-existence and interactions of Alzheimer's disease (AD) with neurodegenerative non-AD pathologies in the aging brain are needed to understand the pathogenesis of neurodegeneration and to support the development of diagnostic biomarkers and therapies.

  10. Astrocytic Pathological Calcium Homeostasis and Impaired Vesicle Trafficking in Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Nina Vardjan

    2017-02-01

    Full Text Available Although the central nervous system (CNS consists of highly heterogeneous populations of neurones and glial cells, clustered into diverse anatomical regions with specific functions, there are some conditions, including alertness, awareness and attention that require simultaneous, coordinated and spatially homogeneous activity within a large area of the brain. During such events, the brain, representing only about two percent of body mass, but consuming one fifth of body glucose at rest, needs additional energy to be produced. How simultaneous energy procurement in a relatively extended area of the brain takes place is poorly understood. This mechanism is likely to be impaired in neurodegeneration, for example in Alzheimer’s disease, the hallmark of which is brain hypometabolism. Astrocytes, the main neural cell type producing and storing glycogen, a form of energy in the brain, also hold the key to metabolic and homeostatic support in the central nervous system and are impaired in neurodegeneration, contributing to the slow decline of excitation-energy coupling in the brain. Many mechanisms are affected, including cell-to-cell signalling. An important question is how changes in cellular signalling, a process taking place in a rather short time domain, contribute to the neurodegeneration that develops over decades. In this review we focus initially on the slow dynamics of Alzheimer’s disease, and on the activity of locus coeruleus, a brainstem nucleus involved in arousal. Subsequently, we overview much faster processes of vesicle traffic and cytosolic calcium dynamics, both of which shape the signalling landscape of astrocyte-neurone communication in health and neurodegeneration.

  11. Switch-Like Roles for Polycomb Proteins from Neurodevelopment to Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Anke Hoffmann

    2017-12-01

    Full Text Available Polycomb Group (PcG proteins are best-known for maintaining repressive or active chromatin states that are passed on across multiple cell divisions, and thus sustain long-term memory of gene expression. PcG proteins engage different, partly gene- and/or stage-specific, mechanisms to mediate spatiotemporal gene expression during central nervous system development. In the course of this, PcG proteins bind to various cis-regulatory sequences (e.g., promoters, enhancers or silencers and coordinate, as well the interactions between distantly separated genomic regions to control chromatin function at different scales ranging from compaction of the linear chromatin to the formation of topological hubs. Recent findings show that PcG proteins are involved in switch-like changes in gene expression states of selected neural genes during the transition from multipotent to differentiating cells, and then to mature neurons. Beyond neurodevelopment, PcG proteins sustain mature neuronal function and viability, and prevent progressive neurodegeneration in mice. In support of this view, neuropathological findings from human neurodegenerative diseases point to altered PcG functions. Overall, improved insight into the multiplicity of PcG functions may advance our understanding of human neurodegenerative diseases and ultimately pave the way to new therapies.

  12. Optic neuropathies: the tip of the neurodegeneration iceberg

    Science.gov (United States)

    Carelli, Valerio; La Morgia, Chiara; Ross-Cisneros, Fred N.; Sadun, Alfredo A.

    2017-01-01

    Abstract The optic nerve and the cells that give origin to its 1.2 million axons, the retinal ganglion cells (RGCs), are particularly vulnerable to neurodegeneration related to mitochondrial dysfunction. Optic neuropathies may range from non-syndromic genetic entities, to rare syndromic multisystem diseases with optic atrophy such as mitochondrial encephalomyopathies, to age-related neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease where optic nerve involvement has, until recently, been a relatively overlooked feature. New tools are available to thoroughly investigate optic nerve function, allowing unparalleled access to this part of the central nervous system. Understanding the molecular pathophysiology of RGC neurodegeneration and optic atrophy, is key to broadly understanding the pathogenesis of neurodegenerative disorders, for monitoring their progression in describing the natural history, and ultimately as outcome measures to evaluate therapies. In this review, the different layers, from molecular to anatomical, that may contribute to RGC neurodegeneration and optic atrophy are tackled in an integrated way, considering all relevant players. These include RGC dendrites, cell bodies and axons, the unmyelinated retinal nerve fiber layer and the myelinated post-laminar axons, as well as olygodendrocytes and astrocytes, looked for unconventional functions. Dysfunctional mitochondrial dynamics, transport, homeostatic control of mitobiogenesis and mitophagic removal, as well as specific propensity to apoptosis may target differently cell types and anatomical settings. Ultimately, we can envisage new investigative approaches and therapeutic options that will speed the early diagnosis of neurodegenerative diseases and their cure. PMID:28977448

  13. Xeroderma Pigmentosum: defective DNA repair causes skin cancer and neurodegeneration

    International Nuclear Information System (INIS)

    Robbins, J.H.

    1988-01-01

    Xeroderma pigmentosum is a rare autosomal recessive disease with numerous malignancies on sun-exposed areas of the skin and eye because of an inability to repair DNA damage inflicted by harmful ultraviolet (UV) radiation of the sun. Because it is the only disease in which cancer is known to result from defective DNA repair, XP has received intense clinical and biochemical study during the last two decades. Furthermore, some patients with XP develop a primary neuronal degeneration, probably due to the inability of nerve cells to repair damage to their DNA caused by intraneuronal metabolites and physicochemical events that mimic the effects of UV radiation. Studies of XP neurodegeneration and DNA-repair defects have led to the conclusion that efficient DNA repair is required to prevent premature death of human nerve cells. Since XP neurodegeneration has similarities to premature death of nerve cells that occurs in such neurodegenerative disorders, XP may be the prototype for these more common neurodegenerations. Recent studies indicate that these degenerations also may have DNA-repair defects

  14. In Vitro Transduction and Target-Mutagenesis Efficiency of HIV-1 pol Gene Targeting ZFN and CRISPR/Cas9 Delivered by Various Plasmids and/or Vectors: Toward an HIV Cure.

    Science.gov (United States)

    Okee, Moses; Bayiyana, Alice; Musubika, Carol; Joloba, Moses L; Ashaba-Katabazi, Fred; Bagaya, Bernard; Wayengera, Misaki

    2018-01-01

    Efficiency of artificial restriction enzymes toward curing HIV has only been separately examined, using differing delivery vehicles. We compared the in vitro transduction and target-mutagenesis efficiency of consortium plasmid and adenoviral vector delivered HIV-1 pol gene targeting zinc finger nuclease (ZFN) with CRISPR/Cas, Custom-ZFN, CRISPR-Cas-9, and plasmids and vectors (murCTSD_pZFN, pGS-U-gRNA, pCMV-Cas-D01A, Ad5-RGD); cell lines (TZM-bl and ACH-2/J-Lat cells); and the latency reversing agents prostratin, suberoylanilide hydroxamic acid, and phorbol myristate acetate. Cell lines were grown in either Dulbecco's modified Eagle's medium or Roswell Park Memorial Institute with the antibiotics kanamycin, zeocin, and efavirenz. Efficiency was assayed by GFP/luciferase activity and/or validated by yeast MEL1 reporter assay, CEL1 restriction fragment assay, and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Ad5-RGD vectors had better transduction efficiency than murCTSD and pGS-U-gRNA/pCMV-Cas-D01A plasmids. CRISPR/Cas9 exhibited better target-mutagenesis efficiency relative to ZFN (delivered by either plasmid or Ad5 vector) based on gel electrophoresis of pol gene amplicons within ACH-2 and J-Lat cells. Ad-5-RGD vectors enhanced target mutagenesis of ZFN, relative to murCTSD_pZFN plasmids, to levels of CRISPR/Cas9 plasmids. Similar reduction of luciferase activity among TZM-bl treated with Ad5-ZFN vectors relative to CRISPR/Cas-9 and murCTSD_pZFN plasmids was observed on challenge with HIV-1. qRT-PCR of HIV-1 pol gene transcripts affirmed that Ad5 (RGD) vectors enhanced target mutagenesis of ZFN. Whereas CRISPR/Cas-9 may possess inherent superior target-mutagenesis efficiency; the efficiency of ZFN (off-target toxicity withstanding) can be enhanced by altering delivery vehicle from plasmid to Ad5 (RGD) vectors.

  15. Vector geometry

    CERN Document Server

    Robinson, Gilbert de B

    2011-01-01

    This brief undergraduate-level text by a prominent Cambridge-educated mathematician explores the relationship between algebra and geometry. An elementary course in plane geometry is the sole requirement for Gilbert de B. Robinson's text, which is the result of several years of teaching and learning the most effective methods from discussions with students. Topics include lines and planes, determinants and linear equations, matrices, groups and linear transformations, and vectors and vector spaces. Additional subjects range from conics and quadrics to homogeneous coordinates and projective geom

  16. Construction and characterization in vitro of a bicistronic retroviral vector coding endostatin and interleukin-2 for use in gene therapy; Construcao e caracterizacao in vitro de um vetor retroviral bicistronico codificando endostatina e interleucina-2 para utilizacao em terapia genica

    Energy Technology Data Exchange (ETDEWEB)

    Calvo, Fernanda Bernardes

    2009-07-01

    Gene therapy has been used in preclinical studies and clinical trials in order to alleviate or cure a disease. Retroviral vectors are a tool for gene transfer is widely used. Bicistronic vectors are an attractive alternative for treatment of complex diseases. A variety of options exists to simultaneously express two genes in genetically modified cells. The most common approach relies on bicistronic vectors in which the genes are linked to each other by an internal ribosome entry site allowing co-translational expression of both cistrons. Endostatin, the C-terminal fragment of collagen XVIII, is a potent angiogenesis inhibitor. At present, ES has been widely used in anti-angiogenic in a variety of experimental tumor models, and clinical trials to test it as an anti-tumor agent are already under way. Immunotherapy has been used as adjuvant treatment for tumors and has been used in several preclinical studies and clinical trials. The objective of this project was to construct and characterize 'in vitro' an IRES-based bicistronic retroviral vector encoding endostatin and interleukin-2. The construction of the vector was performed in three stages, the final construction was analyzed by restriction analysis and sequencing. Packaging cells were prepared. The endostatin and interleukin-2 levels were determined by Dot blot. Monocistronic and bicistronic mRNA expression were analyzed by real time RT-PCR. Bicistronic vector showed high levels of virus trites, ranging from 4.20x10{sup 5} to 1.53x10{sup 6}UFC/ml. Secreted levels of endostatin and interleukin-2 ranged from 1.08 to 2.08{mu}g/10{sup 6}cells.24h and 0.66 - 0.89{mu}g/10{sup 6}cells.24h, respectively. The mRNA expression of ES in the NIH3T3 clone pLend-IRES-IL2SN was 2 times higher than the level presented by the NIH3T3 clone pLendSN. The endostatin promoted inhibition (40%) of endothelial cell proliferation. Interleukin-2 promoted a proliferation of 10.6% lymphocytes CD4 and 8.9% of CD8. We conclude that

  17. Epidemiology of neurodegeneration in American-style professional football players.

    Science.gov (United States)

    Lehman, Everett J

    2013-01-01

    The purpose of this article is to review the history of head injuries in relation to American-style football play, summarize recent research that has linked football head injuries to neurodegeneration, and provide a discussion of the next steps for refining the examination of neurodegeneration in football players. For most of the history of football, the focus of media reports and scientific studies on football-related head injuries was on the acute or short-term effects of serious, traumatic head injuries. Beginning about 10 years ago, a growing concern developed among neurologists and researchers about the long-term effects that playing professional football has on the neurologic health of the players. Autopsy-based studies identified a pathologically distinct neurodegenerative disorder, chronic traumatic encephalopathy, among athletes who were known to have experienced concussive and subconcussive blows to the head during their playing careers. Football players have been well represented in these autopsy findings. A mortality study of a large cohort of retired professional football players found a significantly increased risk of death from neurodegeneration. Further analysis found that non-line players were at higher risk than line players, possibly because of an increased risk of concussion. Although the results of the studies reviewed do not establish a cause effect relationship between football-related head injury and neurodegenerative disorders, a growing body of research supports the hypothesis that professional football players are at an increased risk of neurodegeneration. Significant progress has been made in the last few years on detecting and defining the pathology of neurodegenerative diseases. However, less progress has been made on other factors related to the progression of those diseases in football players. This review identifies three areas for further research: (a) quantification of exposure - a consensus is needed on the use of clinically

  18. VECTOR INTEGRATION

    NARCIS (Netherlands)

    Thomas, E. G. F.

    2012-01-01

    This paper deals with the theory of integration of scalar functions with respect to a measure with values in a, not necessarily locally convex, topological vector space. It focuses on the extension of such integrals from bounded measurable functions to the class of integrable functions, proving

  19. Diabetes and Neurodegeneration in Wolfram Syndrome

    Science.gov (United States)

    Rohayem, Julia; Ehlers, Christian; Wiedemann, Bärbel; Holl, Reinhard; Oexle, Konrad; Kordonouri, Olga; Salzano, Giuseppina; Meissner, Thomas; Burger, Walter; Schober, Edith; Huebner, Angela; Lee-Kirsch, Min Ae

    2011-01-01

    OBJECTIVE To describe the diabetes phenotype in Wolfram syndrome compared with type 1 diabetes, to investigate the effect of glycemic control on the neurodegenerative process, and to assess the genotype-phenotype correlation. RESEARCH DESIGN AND METHODS The clinical data of 50 patients with Wolfram syndrome-related diabetes (WSD) were reviewed and compared with the data of 24,164 patients with type 1 diabetes. Patients with a mean HbA1c during childhood and adolescence of ≤7.5 and >7.5% were compared with respect to the occurrence of additional Wolfram syndrome symptoms. The wolframin (WFS1) gene was screened for mutations in 39 patients. WFS1 genotypes were examined for correlation with age at onset of diabetes. RESULTS WSD was diagnosed earlier than type 1 diabetes (5.4 ± 3.8 vs. 7.9 ± 4.2 years; P diabetes (NS). Severe hypoglycemia occurred in 37 vs. 7.9% (P 7.5% (P = 0.031). Thirteen novel WSF1 mutations were identified. Predicted functional consequence of WFS1 mutations correlated with age at WSD onset (P = 0.028). CONCLUSIONS Endoplasmic reticulum stress–mediated decline of β-cells in WSD occurs earlier in life than autoimmune-mediated β-cell destruction in type 1 diabetes. This study establishes a role for WFS1 in determining the age at onset of diabetes in Wolfram syndrome and identifies glucose toxicity as an accelerating feature in the progression of disease. PMID:21602428

  20. Adeno-associated viral vector-mediated neurotrophin gene transfer in the injured adult rat spinal cord improves hind-limb function

    NARCIS (Netherlands)

    Blits, B; Oudega, M.; Boer, G J; Bartlett Bunge, M; Verhaagen, J

    2003-01-01

    To foster axonal growth from a Schwann cell bridge into the caudal spinal cord, spinal cells caudal to the implant were transduced with adeno-associated viral (AAV) vectors encoding for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (AAV-NT-3). Control rats received AAV vectors encoding

  1. Defining SNAP by cross-sectional and longitudinal definitions of neurodegeneration

    OpenAIRE

    Wisse, L.E.M.; Das, S.R.; Davatzikos, C.; Dickerson, B.C.; Xie, S.X.; Yushkevich, P.A.; Wolk, D.A.

    2018-01-01

    Introduction: Suspected non-Alzheimer's pathophysiology (SNAP) is a biomarker driven designation that represents a heterogeneous group in terms of etiology and prognosis. SNAP has only been identified by cross-sectional neurodegeneration measures, whereas longitudinal measures might better reflect “active” neurodegeneration and might be more tightly linked to prognosis. We compare neurodegeneration defined by cross-sectional ‘hippocampal volume’ only (SNAP/L−) versus both cross-sectional and ...

  2. Mucosal immunization with recombinant adenoviral vectors expressing murine gammaherpesvirus-68 genes M2 and M3 can reduce latent viral load.

    Science.gov (United States)

    Hoegh-Petersen, Mette; Thomsen, Allan R; Christensen, Jan P; Holst, Peter J

    2009-11-12

    Gammaherpesviruses establish life-long latent infections in their hosts. If the host becomes immunosuppressed, these viruses may reactivate and cause severe disease, and even in immunocompetent individuals the gammaherpesviruses are presumed to have an oncogenic potential. Murine gammaherpesvirus-68 (MHV-68) is a member of the Gammaherpesvirinae subfamily and represents a useful murine model for this category of infections, in which new vaccination strategies may initially be evaluated. Two attenuated variants of MHV-68 have successfully been used as vaccines, but the oncogenic potential of the gammaherpesvirinae speaks against using a similar approach in humans. DNA immunization with plasmids encoding the MHV-68 genes M2 or M3 caused a reduction in either acute or early latent viral load, respectively, but neither immunization had an effect at times later than 14 days post-infection. Adenovirus-based vaccines are substantially more immunogenic than DNA vaccines and can be applied to induce mucosal immunity. Here we show that a significant reduction of the late viral load in the spleens, at 60 days post-infection, was achieved when immunizing mice both intranasally and subcutaneously with adenoviral vectors encoding both M2 and M3. Additionally we show that M3 immunization prevented the usual development of virus-induced splenomegaly at 2-3 weeks post-infection. This is the first time that immunization with a non-replicating vaccine has lead to a significantly reduced viral load at time points beyond 14 days post-infection, and thus demonstrates that a non-replicating vaccine may successfully be employed to reduce the viral burden during chronic gammaherpesvirus infection.

  3. [In vitro differentiation of synovial-derived mesenchymal stem cells infected by adenovirus vector mediated by bone morphogenetic protein 2/7 genes into fibrocartilage cells in rabbits].

    Science.gov (United States)

    Fu, Peiliang; Zhang, Lei; Wu, Haishan; Cong, Ruijun; Chen, Song; Ding, Zheru; Hu, Kaimen

    2013-03-01

    To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro. SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 +/- 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining. SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells. It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.

  4. An introduction to vectors, vector operators and vector analysis

    CERN Document Server

    Joag, Pramod S

    2016-01-01

    Ideal for undergraduate and graduate students of science and engineering, this book covers fundamental concepts of vectors and their applications in a single volume. The first unit deals with basic formulation, both conceptual and theoretical. It discusses applications of algebraic operations, Levi-Civita notation, and curvilinear coordinate systems like spherical polar and parabolic systems and structures, and analytical geometry of curves and surfaces. The second unit delves into the algebra of operators and their types and also explains the equivalence between the algebra of vector operators and the algebra of matrices. Formulation of eigen vectors and eigen values of a linear vector operator are elaborated using vector algebra. The third unit deals with vector analysis, discussing vector valued functions of a scalar variable and functions of vector argument (both scalar valued and vector valued), thus covering both the scalar vector fields and vector integration.

  5. Nitrosamine exposure exacerbates high fat diet-mediated type 2 diabetes mellitus, non-alcoholic steatohepatitis, and neurodegeneration with cognitive impairment

    Directory of Open Access Journals (Sweden)

    de la Monte Suzanne M

    2009-12-01

    Full Text Available Abstract Background The current epidemics of type 2 diabetes mellitus (T2DM, non-alcoholic steatohepatitis (NASH, and Alzheimer's disease (AD all represent insulin-resistance diseases. Previous studies linked insulin resistance diseases to high fat diets or exposure to streptozotocin, a nitrosamine-related compound that causes T2DM, NASH, and AD-type neurodegeneration. We hypothesize that low-level exposure to nitrosamines that are widely present in processed foods, amplifies the deleterious effects of high fat intake in promoting T2DM, NASH, and neurodegeneration. Methods Long Evans rat pups were treated with N-nitrosodiethylamine (NDEA by i.p. Injection, and upon weaning, they were fed with high fat (60%; HFD or low fat (5%; LFD chow for 6 weeks. Rats were evaluated for cognitive impairment, insulin resistance, and neurodegeneration using behavioral, biochemical, molecular, and histological methods. Results NDEA and HFD ± NDEA caused T2DM, NASH, deficits in spatial learning, and neurodegeneration with hepatic and brain insulin and/or IGF resistance, and reductions in tau and choline acetyltransferase levels in the temporal lobe. In addition, pro-ceramide genes, which promote insulin resistance, were increased in livers and brains of rats exposed to NDEA, HFD, or both. In nearly all assays, the adverse effects of HFD+NDEA were worse than either treatment alone. Conclusions Environmental and food contaminant exposures to low, sub-mutagenic levels of nitrosamines, together with chronic HFD feeding, function synergistically to promote major insulin resistance diseases including T2DM, NASH, and AD-type neurodegeneration. Steps to minimize human exposure to nitrosamines and consumption of high-fat content foods are needed to quell these costly and devastating epidemics.

  6. Tobacco Smoke Exposure Impairs Brain Insulin/IGF Signaling: Potential Co-Factor Role in Neurodegeneration.

    Science.gov (United States)

    Deochand, Chetram; Tong, Ming; Agarwal, Amit R; Cadenas, Enrique; de la Monte, Suzanne M

    2016-01-01

    Human studies suggest tobacco smoking is a risk factor for cognitive impairment and neurodegeneration, including Alzheimer's disease (AD). However, experimental data linking tobacco smoke exposures to underlying mediators of neurodegeneration, including impairments in brain insulin and insulin-like growth factor (IGF) signaling in AD are lacking. This study tests the hypothesis that cigarette smoke (CS) exposures can impair brain insulin/IGF signaling and alter expression of AD-associated proteins. Adult male A/J mice were exposed to air for 8 weeks (A8), CS for 4 or 8 weeks (CS4, CS8), or CS8 followed by 2 weeks recovery (CS8+R). Gene expression was measured by qRT-PCR analysis and proteins were measured by multiplex bead-based or direct binding duplex ELISAs. CS exposure effects on insulin/IGF and insulin receptor substrate (IRS) proteins and phosphorylated proteins were striking compared with the mRNA. The main consequences of CS4 or CS8 exposures were to significantly reduce insulin R, IGF-1R, IRS-1, and tyrosine phosphorylated insulin R and IGF-1R proteins. Paradoxically, these effects were even greater in the CS8+R group. In addition, relative levels of S312-IRS-1, which inhibits downstream signaling, were increased in the CS4, CS8, and CS8+R groups. Correspondingly, CS and CS8+R exposures inhibited expression of proteins and phosphoproteins required for signaling through Akt, PRAS40, and/or p70S6K, increased AβPP-Aβ, and reduced ASPH protein, which is a target of insulin/IGF-1 signaling. Secondhand CS exposures caused molecular and biochemical abnormalities in brain that overlap with the findings in AD, and many of these effects were sustained or worsened despite short-term CS withdrawal.

  7. Production of lentiviral vectors

    Directory of Open Access Journals (Sweden)

    Otto-Wilhelm Merten

    2016-01-01

    Full Text Available Lentiviral vectors (LV have seen considerably increase in use as gene therapy vectors for the treatment of acquired and inherited diseases. This review presents the state of the art of the production of these vectors with particular emphasis on their large-scale production for clinical purposes. In contrast to oncoretroviral vectors, which are produced using stable producer cell lines, clinical-grade LV are in most of the cases produced by transient transfection of 293 or 293T cells grown in cell factories. However, more recent developments, also, tend to use hollow fiber reactor, suspension culture processes, and the implementation of stable producer cell lines. As is customary for the biotech industry, rather sophisticated downstream processing protocols have been established to remove any undesirable process-derived contaminant, such as plasmid or host cell DNA or host cell proteins. This review compares published large-scale production and purification processes of LV and presents their process performances. Furthermore, developments in the domain of stable cell lines and their way to the use of production vehicles of clinical material will be presented.

  8. Safety profile, efficacy, and biodistribution of a bicistronic high-capacity adenovirus vector encoding a combined immunostimulation and cytotoxic gene therapy as a prelude to a phase I clinical trial for glioblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Puntel, Mariana [Department of Neurosurgery, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Department of Cell and Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Ghulam, Muhammad A.K.M. [Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Farrokhi, Catherine [Department of Psychiatry and Behavioral Neurosciences, Cedars Sinai Medical Center, Los Angeles, CA 90048 (United States); VanderVeen, Nathan; Paran, Christopher; Appelhans, Ashley [Department of Neurosurgery, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Department of Cell and Developmental Biology, The University of Michigan School of Medicine, MSRB II, RM 4570C, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5689 (United States); Kroeger, Kurt M.; Salem, Alireza [Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Lacayo, Liliana [Department of Psychiatry and Behavioral Neurosciences, Cedars Sinai Medical Center, Los Angeles, CA 90048 (United States); Pechnick, Robert N. [Department of Psychiatry and Behavioral Neurosciences, Cedars Sinai Medical Center, Los Angeles, CA 90048 (United States); Department of Psychiatry and Behavioral Neurosciences, David Geffen School of Medicine, University of California, Los Angeles, CA (United States); Kelson, Kyle R.; Kaur, Sukhpreet; Kennedy, Sean [Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 (United States); Palmer, Donna; Ng, Philip [Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030 (United States); and others

    2013-05-01

    Adenoviral vectors (Ads) are promising gene delivery vehicles due to their high transduction efficiency; however, their clinical usefulness has been hampered by their immunogenicity and the presence of anti-Ad immunity in humans. We reported the efficacy of a gene therapy approach for glioma consisting of intratumoral injection of Ads encoding conditionally cytotoxic herpes simplex type 1 thymidine kinase (Ad-TK) and the immunostimulatory cytokine fms-like tyrosine kinase ligand 3 (Ad-Flt3L). Herein, we report the biodistribution, efficacy, and neurological and systemic effects of a bicistronic high-capacity Ad, i.e., HC-Ad-TK/TetOn-Flt3L. HC-Ads elicit sustained transgene expression, even in the presence of anti-Ad immunity, and can encode large therapeutic cassettes, including regulatory elements to enable turning gene expression “on” or “off” according to clinical need. The inclusion of two therapeutic transgenes within a single vector enables a reduction of the total vector load without adversely impacting efficacy. Because clinically the vectors will be delivered into the surgical cavity, normal regions of the brain parenchyma are likely to be transduced. Thus, we assessed any potential toxicities elicited by escalating doses of HC-Ad-TK/TetOn-Flt3L (1 × 10{sup 8}, 1 × 10{sup 9}, or 1 × 10{sup 10} viral particles [vp]) delivered into the rat brain parenchyma. We assessed neuropathology, biodistribution, transgene expression, systemic toxicity, and behavioral impact at acute and chronic time points. The results indicate that doses up to 1 × 10{sup 9} vp of HC-Ad-TK/TetOn-Flt3L can be safely delivered into the normal rat brain and underpin further developments for its implementation in a phase I clinical trial for glioma. - Highlights: ► High capacity Ad vectors elicit sustained therapeutic gene expression in the brain. ► HC-Ad-TK/TetOn-Flt3L encodes two therapeutic genes and a transcriptional switch. ► We performed a dose escalation study at

  9. Deletion of A44L, A46R and C12L Vaccinia Virus Genes from the MVA Genome Improved the Vector Immunogenicity by Modifying the Innate Immune Response Generating Enhanced and Optimized Specific T-Cell Responses

    Directory of Open Access Journals (Sweden)

    María Pía Holgado

    2016-05-01

    Full Text Available MVA is an attenuated vector that still retains immunomodulatory genes. We have previously reported its optimization after deleting the C12L gene, coding for the IL-18 binding-protein. Here, we analyzed the immunogenicity of MVA vectors harboring the simultaneous deletion of A44L, related to steroid synthesis and A46R, a TLR-signaling inhibitor (MVAΔA44L-A46R; or also including a deletion of C12L (MVAΔC12L/ΔA44L-A46R. The absence of biological activities of the deleted genes in the MVA vectors was demonstrated. Adaptive T-cell responses against VACV epitopes, evaluated in spleen and draining lymph-nodes of C57Bl/6 mice at acute/memory phases, were of higher magnitude in those animals that received deleted MVAs compared to MVAwt. MVAΔC12L/ΔA44L-A46R generated cellular specific memory responses of higher quality characterized by bifunctionality (CD107a/b+/IFN-γ+ and proliferation capacity. Deletion of selected genes from MVA generated innate immune responses with higher levels of determining cytokines related to T-cell response generation, such as IL-12, IFN-γ, as well as IL-1β and IFN-β. This study describes for the first time that simultaneous deletion of the A44L, A46R and C12L genes from MVA improved its immunogenicity by enhancing the host adaptive and innate immune responses, suggesting that this approach comprises an appropriate strategy to increase the MVA vaccine potential.

  10. Brain and Peripheral Atypical Inflammatory Mediators Potentiate Neuroinflammation and Neurodegeneration.

    Science.gov (United States)

    Kempuraj, Duraisamy; Thangavel, Ramasamy; Selvakumar, Govindhasamy P; Zaheer, Smita; Ahmed, Mohammad E; Raikwar, Sudhanshu P; Zahoor, Haris; Saeed, Daniyal; Natteru, Prashant A; Iyer, Shankar; Zaheer, Asgar

    2017-01-01

    Neuroinflammatory response is primarily a protective mechanism in the brain. However, excessive and chronic inflammatory responses can lead to deleterious effects involving immune cells, brain cells and signaling molecules. Neuroinflammation induces and accelerates pathogenesis of Parkinson's disease (PD), Alzheimer's disease (AD) and Multiple sclerosis (MS). Neuroinflammatory pathways are indicated as novel therapeutic targets for these diseases. Mast cells are immune cells of hematopoietic origin that regulate inflammation and upon activation release many proinflammatory mediators in systemic and central nervous system (CNS) inflammatory conditions. In addition, inflammatory mediators released from activated glial cells induce neurodegeneration in the brain. Systemic inflammation-derived proinflammatory cytokines/chemokines and other factors cause a breach in the blood brain-barrier (BBB) thereby allowing for the entry of immune/inflammatory cells including mast cell progenitors, mast cells and proinflammatory cytokines and chemokines into the brain. These peripheral-derived factors and intrinsically generated cytokines/chemokines, α-synuclein, corticotropin-releasing hormone (CRH), substance P (SP), beta amyloid 1-42 (Aβ1-42) peptide and amyloid precursor proteins can activate glial cells, T-cells and mast cells in the brain can induce additional release of inflammatory and neurotoxic molecules contributing to chronic neuroinflammation and neuronal death. The glia maturation factor (GMF), a proinflammatory protein discovered in our laboratory released from glia, activates mast cells to release inflammatory cytokines and chemokines. Chronic increase in the proinflammatory mediators induces neurotoxic Aβ and plaque formation in AD brains and neurodegeneration in PD brains. Glial cells, mast cells and T-cells can reactivate each other in neuroinflammatory conditions in the brain and augment neuroinflammation. Further, inflammatory mediators from the brain can

  11. Gene therapy with recombinant adeno-associated vectors for neovascular age-related macular degeneration: 1 year follow-up of a phase 1 randomised clinical trial.

    Science.gov (United States)

    Rakoczy, Elizabeth P; Lai, Chooi-May; Magno, Aaron L; Wikstrom, Matthew E; French, Martyn A; Pierce, Cora M; Schwartz, Steven D; Blumenkranz, Mark S; Chalberg, Thomas W; Degli-Esposti, Mariapia A; Constable, Ian J

    2015-12-12

    Neovascular, or wet, age-related macular degeneration causes central vision loss and represents a major health problem in elderly people, and is currently treated with frequent intraocular injections of anti-VEGF protein. Gene therapy might enable long-term anti-VEGF therapy from a single treatment. We tested the safety of rAAV.sFLT-1 in treatment of wet age-related macular degeneration with a single subretinal injection. In this single-centre, phase 1, randomised controlled trial, we enrolled patients with wet age-related macular degeneration at the Lions Eye Institute and the Sir Charles Gairdner Hospital (Nedlands, WA, Australia). Eligible patients had to be aged 65 years or older, have age-related macular degeneration secondary to active subfoveal choroidal neovascularisation, with best corrected visual acuity (BCVA) of 3/60-6/24 and 6/60 or better in the other eye. Patients were randomly assigned (3:1) to receive either 1 × 10(10) vector genomes (vg; low-dose rAAV.sFLT-1 group) or 1 × 10(11) vg (high-dose rAAV.sFLT-1 group), or no gene-therapy treatment (control group). Randomisation was done by sequential group assignment. All patients and investigators were unmasked. Staff doing the assessments were masked to the study group at study visits. All patients received ranibizumab at baseline and week 4, and rescue treatment during follow-up based on prespecified criteria including BCVA measured on the Early Treatment Diabetic Retinopathy Study (EDTRS) scale, optical coherence tomography, and fluorescein angiography. The primary endpoint was ocular and systemic safety. This trial is registered with ClinicalTrials.gov, number NCT01494805. From Dec 16, 2011, to April 5, 2012, we enrolled nine patients of whom eight were randomly assigned to receive either intervention (three patients in the low-dose rAAV.sFLT-1 group and three patients in the high-dose rAAV.sFLT-1 group) or no treatment (two patients in the control group). Subretinal injection of r

  12. The experimental study of reporter probe 131I-FIAU in neonatal cardiac myocytes after transfer of herpes simplex virus type 1 thymidine kinase reporter gene by different vectors

    International Nuclear Information System (INIS)

    Yin Xiaohua; Lan Xiaoli; Wang Ruihua; Liu Ying; Zhang Yongxue

    2009-01-01

    Objective: Reporter gene imaging is a promising approach for noninvasive monitoring of cardiac gene therapy. In the present study, the recombinant plasmid and adenoviral vector carrying reporter gene. herpes simplex virus type 1 thymidine kinase (HSV1-tk), were constructed and transferred into nee-natal cardiac myocytes, and a series of in vitro studies were carried out on the cells transferred to evaluate the uptake of radiolabeled reporter probe and to compare both vectors for cardiac reporter gene imaging. Methods: Neonatal cardiac myocytes were obtained from rat heart by single collagenase digestion. HSVI-tk. chosen as the reporter gene.was inserted into adenovirus vector (Ad5-tk) and plasmid (pDC316-tk), thus it could be transferred into neonatal cardiac myocytes. Recombinant adenovirus containing enhanced green fluorescent protein (Ad5-EGFP) was used as control. Recombinant plasmid was coated with lipofectamine TM 2000 (pDC316-tk/lipoplex). The specific reporter probe of HSV1-tk, 2'-fluoro-2'-deoxy-l-β-D-arabinofuranosyl-uracil (FAU), was labeled with 131 I by solid phase oxidation with lodogen. Product wag purified on a reverse. phase Sep-Pak C18 column and the radiochemical purity wag then assessed. The accumulation of it in the transferred cardiac myocytes wag detected as uptake rate. Furthermore, mRNA expression of HSV1-tk was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), while its protein expression wag located by immunocytochemistry. Results: FAU could be labeled with 131 I and the labeling efficiency was (53.82 ±2.05)%. The radiochemical purity was (94.85 ± 1.76)% after purification, and it kept stable in vitro for at least 24h. Time-dependent increase of the ac- cumulation of 131 I-FIAU was observed in both Ad5-tk group and pDC316-tk/lipoplex group. and the highest uptake rate occurred at 5h, with peak values of (12.55 ± 0.37)% and (2.09 ± 0.34)% respectively. However, it also indicated that greater

  13. Gene Transfer of Glutamic Acid Decarboxylase 67 by Herpes Simplex Virus Vectors Suppresses Neuropathic Pain Induced by Human Immunodeficiency Virus gp120 Combined with ddC in Rats.

    Science.gov (United States)

    Kanao, Megumi; Kanda, Hirotsugu; Huang, Wan; Liu, Shue; Yi, Hyun; Candiotti, Keith A; Lubarsky, David A; Levitt, Roy C; Hao, Shuanglin

    2015-06-01

    Human immunodeficiency virus (HIV)-related painful sensory neuropathies primarily consist of the HIV infection-related distal sensory polyneuropathy and antiretroviral toxic neuropathies. Pharmacotherapy provides only partial relief of pain in patients with HIV/acquired immune deficiency syndrome because little is known about the exact neuropathological mechanisms for HIV-associated neuropathic pain (NP). Hypofunction of γ-aminobutyric acid (GABA) GABAergic inhibitory mechanisms has been reported after peripheral nerve injury. In this study, we tested the hypothesis that HIV gp120 combined with antiretroviral therapy reduces spinal GABAergic inhibitory tone and that restoration of GABAergic inhibitory tone will reduce HIV-related NP in a rat model. The application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve plus systemic ddC (one antiretroviral drug) induced mechanical allodynia. The hind paws of rats were inoculated with replication-defective herpes simplex virus (HSV) vectors genetically encoding gad1 gene to express glutamic acid decarboxylase 67 (GAD67), an enzyme that catalyzes the decarboxylation of glutamate to GABA. Mechanical threshold was tested using von Frey filaments before and after treatments with the vectors. The expression of GAD67 in both the lumbar spinal cord and the L4-5 dorsal root ganglia was examined using western blots. The expression of mitochondrial superoxide in the spinal dorsal horn was examined using MitoSox imaging. The immunoreactivity of spinal GABA, pCREB, and pC/EBPβ was tested using immunohistochemistry. In the gp120 with ddC-induced neuropathic pain model, GAD67 expression mediated by the HSV vector caused an elevation of mechanical threshold that was apparent on day 3 after vector inoculation. The antiallodynic effect of the single HSV vector inoculation expressing GAD67 lasted >28 days. The area under the time-effect curves in the HSV vector expressing GAD67 was increased compared with that in the

  14. Correction of acid beta-galactosidase deficiency in GM1 gangliosidosis human fibroblasts by retrovirus vector-mediated gene transfer: higher efficiency of release and cross-correction by the murine enzyme.

    Science.gov (United States)

    Sena-Esteves, M; Camp, S M; Alroy, J; Breakefield, X O; Kaye, E M

    2000-03-20

    Mutations in the lysosomal acid beta-galactosidase (EC 3.2.1.23) underlie two different disorders: GM1 gangliosidosis, which involves the nervous system and visceral organs to varying extents, and Morquio's syndrome type B (Morquio B disease), which is a skeletal-connective tissue disease without any CNS symptoms. This article shows that transduction of human GM1 gangliosidosis fibroblasts with retrovirus vectors encoding the human acid beta-galactosidase cDNA leads to complete correction of the enzymatic deficiency. The newly synthesized enzyme is correctly processed and targeted to the lysosomes in transduced cells. Cross-correction experiments using retrovirus-modified cells as enzyme donors showed, however, that the human enzyme is transferred at low efficiencies. Experiments using a different retrovirus vector carrying the human cDNA confirmed this observation. Transduction of human GM1 fibroblasts and mouse NIH 3T3 cells with a retrovirus vector encoding the mouse beta-galactosidase cDNA resulted in high levels of enzymatic activity. Furthermore, the mouse enzyme was found to be transferred to human cells at high efficiency. Enzyme activity measurements in medium conditioned by genetically modified cells suggest that the human beta-galactosidase enzyme is less efficiently released to the extracellular space than its mouse counterpart. This study suggests that lysosomal enzymes, contrary to the generalized perception in the field of gene therapy, may differ significantly in their properties and provides insights for design of future gene therapy interventions in acid beta-galactosidase deficiency.

  15. Cerebrospinal fluid biomarkers of neurodegeneration are decreased or normal in narcolepsy

    DEFF Research Database (Denmark)

    Jennum, Poul Jørgen; Pedersen, Lars Østergaard; Bahl, Justyna Maria Czarna

    2017-01-01

    OBJECTIVES: To investigate whether cerebrospinal fluid (CSF) biomarkers of neurodegeneration are altered in narcolepsy in order to evaluate whether the hypocretin deficiency and abnormal sleep-wake pattern in narcolepsy leads to neurodegeneration. METHODS: Twenty-one patients with central...... that hypocretin deficiency and an abnormal sleep-wake pattern alter the turnover of these proteins in CNS....

  16. Autophagy and Microglia: Novel Partners in Neurodegeneration and Aging.

    Science.gov (United States)

    Plaza-Zabala, Ainhoa; Sierra-Torre, Virginia; Sierra, Amanda

    2017-03-09

    Autophagy is emerging as a core regulator of Central Nervous System (CNS) aging and neurodegeneration. In the brain, it has mostly been studied in neurons, where the delivery of toxic molecules and organelles to the lysosome by autophagy is crucial for neuronal health and survival. However, we propose that the (dys)regulation of autophagy in microglia also affects innate immune functions such as phagocytosis and inflammation, which in turn contribute to the pathophysiology of aging and neurodegenerative diseases. Herein, we first describe the basic concepts of autophagy and its regulation, discuss key aspects for its accurate monitoring at the experimental level, and summarize the evidence linking autophagy impairment to CNS senescence and disease. We focus on acute, chronic, and autoimmunity-mediated neurodegeneration, including ischemia/stroke, Alzheimer's, Parkinson's, and Huntington's diseases, and multiple sclerosis. Next, we describe the actual and potential impact of autophagy on microglial phagocytic and inflammatory function. Thus, we provide evidence of how autophagy may affect microglial phagocytosis of apoptotic cells, amyloid-β, synaptic material, and myelin debris, and regulate the progression of age-associated neurodegenerative diseases. We also discuss data linking autophagy to the regulation of the microglial inflammatory phenotype, which is known to contribute to age-related brain dysfunction. Overall, we update the current knowledge of autophagy and microglia, and highlight as yet unexplored mechanisms whereby autophagy in microglia may contribute to CNS disease and senescence.

  17. Impact of aging immune system on neurodegeneration and potential immunotherapies.

    Science.gov (United States)

    Liang, Zhanfeng; Zhao, Yang; Ruan, Linhui; Zhu, Linnan; Jin, Kunlin; Zhuge, Qichuan; Su, Dong-Ming; Zhao, Yong

    2017-10-01

    The interaction between the nervous and immune systems during aging is an area of avid interest, but many aspects remain unclear. This is due, not only to the complexity of the aging process, but also to a mutual dependency and reciprocal causation of alterations and diseases between both the nervous and immune systems. Aging of the brain drives whole body systemic aging, including aging-related changes of the immune system. In turn, the immune system aging, particularly immunosenescence and T cell aging initiated by thymic involution that are sources of chronic inflammation in the elderly (termed inflammaging), potentially induces brain aging and memory loss in a reciprocal manner. Therefore, immunotherapeutics including modulation of inflammation, vaccination, cellular immune therapies and "protective autoimmunity" provide promising approaches to rejuvenate neuroinflammatory disorders and repair brain injury. In this review, we summarize recent discoveries linking the aging immune system with the development of neurodegeneration. Additionally, we discuss potential rejuvenation strategies, focusing aimed at targeting the aging immune system in an effort to prevent acute brain injury and chronic neurodegeneration during aging. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Adenoviral vectors expressing fusogenic membrane glycoproteins activated via matrix metalloproteinase cleavable linkers have significant antitumor potential in the gene therapy of gliomas.

    Science.gov (United States)

    Allen, Cory; McDonald, Cari; Giannini, Caterina; Peng, Kah Whye; Rosales, Gabriela; Russell, Stephen J; Galanis, Evanthia

    2004-11-01

    Fusogenic membrane glycoproteins (FMG) such as the gibbon ape leukemia virus envelope (GALV) glycoprotein are potent therapeutic transgenes with potential utility in the gene therapy of gliomas. Transfection of glioma cell lines with FMG expression constructs results in fusion with massive syncytia formation followed by cytotoxic cell death. Nevertheless, ubiquitous expression of the GALV receptor, Pit-1, makes targeting desirable in order to increase the specificity of the observed cytopathic effect. Here we report on use of matrix metalloproteinase (MMP)-cleavable linkers to target the cytotoxicity of FMG-expressing adenoviral vectors against gliomas. Replication-defective adenoviruses (Ad) were constructed expressing the hyperfusogenic version of the GALV glycoprotein linked to a blocking ligand (C-terminal extracellular domain of CD40 ligand) through either an MMP-cleavable linker (AdM40) or a non-cleavable linker (AdN40). Both viruses also co-expressed the green fluorescent protein (GFP) via an internal ribosomal entry site. The glioma cell lines U87, U118, and U251 characterized by zymography and MMP-2 activity assay as high, medium and low MMP expressors, respectively, the MMP-poor cell lines TE671 and normal human astrocytes were infected with AdM40 and AdN40 at different multiplicities of infection (MOIs) from 1-30. Fusion was quantitated by counting both number and size of syncytia. Infection of these cell lines with AdN40 did not result in fusion or cytotoxic cell death, despite the presence of infection, as demonstrated by GFP positivity, therefore indicating that the displayed CD40 ligand blocked GALV-induced fusion. Fusion was restored after infection of glioma cells with AdM40 at an MOI as low as 1 to an extent dependent on MMP expression and coxsackie adenovirus receptor (CAR) expression in the specific cell line. Western immunoblotting demonstrated the presence of the cleaved CD40 ligand in the supernatant of fused glioma cells. Use of the MMP

  19. Timing of neurodegeneration and beta-amyloid (Abeta) peptide deposition in the brain of aging kokanee salmon.

    Science.gov (United States)

    Maldonado, Tammy A; Jones, Richard E; Norris, David O

    2002-10-01

    Brains of kokanee salmon (Oncorhynchus nerka kennerlyi) in one of four reproductive stages (sexually immature, maturing, sexually mature, and spawning) were stained with cresyl violet and silver stain to visualize neurodegeneration. These reproductive stages correlate with increasing somatic aging of kokanee salmon, which die after spawning. Twenty-four regions of each brain were examined. Brains of sexually immature fish exhibited low levels of neurodegeneration, whereas neurodegeneration was more marked in maturing fish and greatest in spawning fish. Neurodegeneration was present in specific regions of the telencephalon, diencephalon, mesencephalon, and rhombencephalon. Pyknotic neurons were observed in all regions previously reported to be immunopositive for A beta. Regions that did not exhibit neurodegeneration during aging included the magnocellular vestibular nucleus, the nucleus lateralis tuberis of the hypothalamus, and Purkinje cells of the cerebellum, all of which also lack A beta; perhaps these regions are neuroprotected. In 14 of 16 brain areas for which data were available on both the increase in A beta deposition and pyknosis, neurodegeneration preceded or appeared more or less simultaneously with A beta production, whereas in only two regions did A beta deposition precede neurodegeneration. This information supports the hypothesis that A beta deposition is a downstream product of neurodegeneration in most brain regions. Other conclusions are that the degree of neurodegeneration varies among brain regions, neurodegeneration begins in maturing fish and peaks in spawning fish, the timing of neurodegeneration varies among brain regions, and some regions do not exhibit accelerated neurodegeneration during aging. Copyright 2002 Wiley Periodicals, Inc.

  20. Endothelial Cell-Targeted Adenoviral Vector for Suppressing Breast Malignancies

    National Research Council Canada - National Science Library

    Huang, Shuang

    2004-01-01

    .... Our proposal is designed to develop an endothelial cell-targeted adenoviral vector and to use the targeted vector to express high levels of anticancer therapeutic genes in the sites of angiogenenic...

  1. Vaccine protection of chickens against antigenically diverse H5 highly pathogenic avian influenza isolates with a live HVT vector vaccine expressing the influenza hemagglutinin gene derived from a clade 2.2 avian influenza virus.

    Science.gov (United States)

    Kapczynski, Darrell R; Esaki, Motoyuki; Dorsey, Kristi M; Jiang, Haijun; Jackwood, Mark; Moraes, Mauro; Gardin, Yannick

    2015-02-25

    Vaccination is an important tool in the protection of poultry against avian influenza (AI). For field use, the overwhelming majority of AI vaccines produced are inactivated whole virus formulated into an oil emulsion. However, recombinant vectored vaccines are gaining use for their ability to induce protection against heterologous isolates and ability to overcome maternal antibody interference. In these studies, we compared protection of chickens provided by a turkey herpesvirus (HVT) vector vaccine expressing the hemagglutinin (HA) gene from a clade 2.2 H5N1 strain (A/swan/Hungary/4999/2006) against homologous H5N1 as well as heterologous H5N1 and H5N2 highly pathogenic (HP) AI challenge. The results demonstrated all vaccinated birds were protected from clinical signs of disease and mortality following homologous challenge. In addition, oral and cloacal swabs taken from challenged birds demonstrated that vaccinated birds had lower incidence and titers of viral shedding compared to sham-vaccinated birds. Following heterologous H5N1 or H5N2 HPAI challenge, 80-95% of birds receiving the HVT vector AI vaccine at day of age survived challenge with fewer birds shedding virus after challenge than sham vaccinated birds. In vitro cytotoxicity analysis demonstrated that splenic T lymphocytes from HVT-vector-AI vaccinated chickens recognized MHC-matched target cells infected with H5, as well as H6, H7, or H9 AI virus. Taken together, these studies provide support for the use of HVT vector vaccines expressing HA to protect poultry against multiple lineages of HPAI, and that both humoral and cellular immunity induced by live vaccines likely contributes to protection. Published by Elsevier Ltd.

  2. Neuronal dark matter: The emerging role of microRNAs in neurodegeneration

    Directory of Open Access Journals (Sweden)

    Emily Frances Goodall

    2013-10-01

    Full Text Available MicroRNAs (miRNAs are small, abundant RNA molecules that constitute part of the cell’s non-coding RNA dark matter. In recent years, the discovery of miRNAs has revolutionised the traditional view of gene expression and our understanding of miRNA biogenesis and function has expanded. Altered expression of miRNAs is increasingly recognised as a feature of many disease states, including neurodegeneration. Here, we review the emerging role for miRNA dysfunction in Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and Huntington’s disease pathogenesis. We emphasise the complex nature of gene regulatory networks and the need for systematic studies, with larger sample cohorts than have so far been reported, to reveal the most important miRNA regulators in disease. Finally, miRNA diversity and their potential to target multiple pathways, offers novel clinical applications for miRNAs as biomarkers and therapeutic agents in neurodegenerative diseases.

  3. A saposin deficiency model in Drosophila: Lysosomal storage, progressive neurodegeneration and sensory physiological decline.

    Science.gov (United States)

    Hindle, Samantha J; Hebbar, Sarita; Schwudke, Dominik; Elliott, Christopher J H; Sweeney, Sean T

    2017-02-01

    Saposin deficiency is a childhood neurodegenerative lysosomal storage disorder (LSD) that can cause premature death within three months of life. Saposins are activator proteins that promote the function of lysosomal hydrolases that mediate the degradation of sphingolipids. There are four saposin proteins in humans, which are encoded by the prosaposin gene. Mutations causing an absence or impaired function of individual saposins or the whole prosaposin gene lead to distinct LSDs due to the storage of different classes of sphingolipids. The pathological events leading to neuronal dysfunction induced by lysosomal storage of sphingolipids are as yet poorly defined. We have generated and characterised a Drosophila model of saposin deficiency that shows striking similarities to the human diseases. Drosophila saposin-related (dSap-r) mutants show a reduced longevity, progressive neurodegeneration, lysosomal storage, dramatic swelling of neuronal soma, perturbations in sphingolipid catabolism, and sensory physiological deterioration. Our data suggests a genetic interaction with a calcium exchanger (Calx) pointing to a possible calcium homeostasis deficit in dSap-r mutants. Together these findings support the use of dSap-r mutants in advancing our understanding of the cellular pathology implicated in saposin deficiency and related LSDs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Whey protein concentrate supplementation protects rat brain against aging-induced oxidative stress and neurodegeneration.

    Science.gov (United States)

    Garg, Geetika; Singh, Sandeep; Singh, Abhishek Kumar; Rizvi, Syed Ibrahim

    2018-05-01

    Whey protein concentrate (WPC) is a rich source of sulfur-containing amino acids and is consumed as a functional food, incorporating a wide range of nutritional attributes. The purpose of this study is to evaluate the neuroprotective effect of WPC on rat brain during aging. Young (4 months) and old (24 months) male Wistar rats were supplemented with WPC (300 mg/kg body weight) for 28 days. Biomarkers of oxidative stress and antioxidant capacity in terms of ferric reducing antioxidant potential (FRAP), lipid hydroperoxide (LHP), total thiol (T-SH), protein carbonyl (PC), reactive oxygen species (ROS), nitric oxide (NO), and acetylcholinesterase (AChE) activity were measured in brain of control and experimental (WPC supplemented) groups. In addition, gene expression and histopathological studies were also performed. The results indicate that WPC augmented the level of FRAP, T-SH, and AChE in old rats as compared with the old control. Furthermore, WPC-treated groups exhibited significant reduction in LHP, PC, ROS, and NO levels in aged rats. WPC supplementation also downregulated the expression of inflammatory markers (tumor necrosis factor alpha, interleukin (IL)-1β, IL-6), and upregulated the expression of marker genes associated with autophagy (Atg3, Beclin-1, LC3B) and neurodegeneration (neuron specific enolase, Synapsin-I, MBP-2). The findings suggested WPC to be a potential functional nutritional food supplement that prevents the progression of age-related oxidative damage in Wistar rats.

  5. Enhancing poxvirus vectors vaccine immunogenicity.

    Science.gov (United States)

    García-Arriaza, Juan; Esteban, Mariano

    2014-01-01

    Attenuated recombinant poxvirus vectors expressing heterologous antigens from pathogens are currently at various stages in clinical trials with the aim to establish their efficacy. This is because these vectors have shown excellent safety profiles, significant immunogenicity against foreign expressed antigens and are able to induce protective immune responses. In view of the limited efficacy triggered by some poxvirus strains used in clinical trials (i.e, ALVAC in the RV144 phase III clinical trial for HIV), and of the restrictive replication capacity of the highly attenuated vectors like MVA and NYVAC, there is a consensus that further improvements of these vectors should be pursuit. In this review we considered several strategies that are currently being implemented, as well as new approaches, to improve the immunogenicity of the poxvirus vectors. This includes heterologous prime/boost protocols, use of co-stimulatory molecules, deletion of viral immunomodulatory genes still present in the poxvirus genome, enhancing virus promoter strength, enhancing vector replication capacity, optimizing expression of foreign heterologous sequences, and the combined use of adjuvants. An optimized poxvirus vector triggering long-lasting immunity with a high protective efficacy against a selective disease should be sought.

  6. Transcriptional Silencing of Retroviral Vectors

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M.; Pedersen, F.S.

    1996-01-01

    . Extinction of long-term vector expression has been observed after implantation of transduced hematopoietic cells as well as fibroblasts, myoblasts and hepatocytes. Here we review the influence of vector structure, integration site and cell type on transcriptional silencing. While down-regulation of proviral...... transcription is known from a number of cellular and animal models, major insight has been gained from studies in the germ line and embryonal cells of the mouse. Key elements for the transfer and expression of retroviral vectors, such as the viral transcriptional enhancer and the binding site for the t......RNA primer for reverse transcription may have a major influence on transcriptional silencing. Alterations of these elements of the vector backbone as well as the use of internal promoter elements from housekeeping genes may contribute to reduce transcriptional silencing. The use of cell culture and animal...

  7. A common multiple cloning site in a set of vectors for expression of eukaryotic genes in mammalian, insect and bacterial cells

    DEFF Research Database (Denmark)

    Pallisgaard, N; Pedersen, FS; Birkelund, Svend

    1994-01-01

    a start Met codon was included in the same reading frame as in lambda gt11Sfi-Not to support expression of partial cDNA clones. Thus a cDNA insert of lambda gt11Sfi-Not could be shuttled among the new vectors for expression. The other set of vectors without a start codon were suitable for expression of c......DNA carrying their own start Met codon. By Western blot analysis and by transactivation of a reporter plasmid in co-transfections we show that cDNA is very efficiently expressed in NIH 3T3 cells under control of the elongation factor 1 alpha promoter....

  8. Some remarks on a generalized vector product

    OpenAIRE

    ACOSTA-HUMÁNEZ, PRIMITIVO; ARANDA, MOISÉS; NÚÑEZ, REINALDO

    2011-01-01

    Abstract. In this paper we use a generalized vector product to construct an exterior form ⊥ : , where Finally, for n = k - 1 we introduce the reversing operation to study this generalized vector product over palindromic and antipalindromic vectors. Resumen. En este art&íacute;culo usamos un producto vectorial generalizado para construir una forma exterior ⊥ : , en donde como es natural, Finalmente, para n = k - 1 introducimos la operación reversar para estudiar este producto vectorial gene...

  9. Dementia, preclinical studies in neurodegeneration and its potential for translational medicine in SouthAmerica

    Directory of Open Access Journals (Sweden)

    Gloria Patricia Cardona Gomez

    2016-12-01

    Full Text Available Latin-American people with dementia will increase in a 368% in 2050, higher than USA and Europe. In addition, to sporadic dementia type Alzheimer and vascular dementia progression after Cerebrovascular disease, the statistics are increased in Colombia by specific populations affected with pure neurodegenerative and vascular dementias like autosomical dominant familial Alzheimer´s disease and CADASIL. In spite of the enormous human and economical effort and investment, neither sporadic nor genetic kinds of dementia progression have been prevented or blocked yet. Currently, exist several animal models that partially solve the understanding of the neurodegenerative etiopathogenesis and its treatment. However, when the potential therapies are translated to humans, those do not work or present a limited action. Main difficulties are the diverse comorbility associated to the cause and/or several affected brain regions, reducing the efficacy of some therapies which are limited to a tissue-specific action or modulating a kind of neurotransmission. Global investigation suggests that a general prevention could be achieved with the improvement in the quality of lifestyle, including healthy diet, physical and mental activity, and avoiding mechanical or chemical pro-inflammatory events in an early stage in the most of non-communicable diseases. In this review, we present some molecular targets and preclinical studies in animal models to propose strategies that could be useful in a future translation to prevent or block neurodegeneration: One is gene therapy silencing pathogenic genes in critical brain areas where excitotoxicity arise and spread. Another is to take advantage of the natural source and its wide biodiversity of natural products some of them identified by the blocking and prevention of neurodegeneration. On the other side, the casuistic of pure dementias in the Latin-American region give an exceptional opportunity to understand the pathogenesis

  10. Efficient delivery and stable gene expression in a hematopoietic cell line using a chimeric serotype 35 fiber pseudotyped helper-dependent adenoviral vector

    International Nuclear Information System (INIS)

    Balamotis, Michael Andrew; Huang, Katie; Mitani, Kohnosuke

    2004-01-01

    Certain human cell populations have remained difficult to infect with human adenovirus (Ad) serotype 5 because of their lack of coxsackievirus B-adenovirus receptor (CAR). Native adenovirus fiber compositions, although diverse, cannot infect all tissue types. Recently, a chimeric Ad5/35 fiber was created, which displays an altered tropism from Ad5. We incorporated this chimeric fiber into a helper-dependent (HD) adenovirus vector system and compared HD to E1-deleted (E1Δ) vectors by transgene expression, cell transduction efficiency, and cytotoxicity. K562 cells were infected ∼50 times more efficiently with the chimeric Ad5/35 fiber compared with the Ad5 fiber. Short-term transgene expression was sustained longer from HD Ad5/35 than E1Δ Ad5/35 vector after in vitro infection of actively dividing K562 cells. Rapid loss of transgene expression from E1Δ Ad5/35 infection was not due to the loss of vector genomes, as determined by quantitative real-time PCR (QRT-PCR), or cytotoxicity, but rather through a putative silencing mechanism

  11. Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

    Science.gov (United States)

    Nallapareddy, Sreedhar R; Singh, Kavindra V; Murray, Barbara E

    2006-01-01

    Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the

  12. Age-Related Neurodegeneration and Memory Loss in Down Syndrome

    Directory of Open Access Journals (Sweden)

    Jason P. Lockrow

    2012-01-01

    Full Text Available Down syndrome (DS is a condition where a complete or segmental chromosome 21 trisomy causes variable intellectual disability, and progressive memory loss and neurodegeneration with age. Many research groups have examined development of the brain in DS individuals, but studies on age-related changes should also be considered, with the increased lifespan observed in DS. DS leads to pathological hallmarks of Alzheimer's disease (AD by 40 or 50 years of age. Progressive age-related memory deficits occurring in both AD and in DS have been connected to degeneration of several neuronal populations, but mechanisms are not fully elucidated. Inflammation and oxidative stress are early events in DS pathology, and focusing on these pathways may lead to development of successful intervention strategies for AD associated with DS. Here we discuss recent findings and potential treatment avenues regarding development of AD neuropathology and memory loss in DS.

  13. Deep Brain Stimulation for Pantothenate Kinase-Associated Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Pedro J. Garcia-Ruiz

    2015-01-01

    Full Text Available Pantothenate kinase-associated neurodegeneration (PKAN is usually associated with dystonia, which is typically severe and progressive over time. Pallidal stimulation (GPi DBS has been carried out in selected cases of PKAN with drug-resistant dystonia with variable results. We report a 30-month follow-up study of a 30-year-old woman with PKAN-related dystonia treated with GPi DBS. Postoperatively, the benefit quickly became evident, as the patient exhibited a marked improvement in her dystonia, including her writing difficulty. This result has been maintained up to the present. GPi DBS should be considered in dystonic PKAN patients provided fixed contractures and/or pyramidal symptoms are not present.

  14. Chromosome 13 dementia syndromes as models of neurodegeneration

    DEFF Research Database (Denmark)

    Ghiso, J.; Revesz, T.; Holton, J.

    2001-01-01

    Two hereditary conditions, familial British dementia (FBD) and familial Danish dementia (FDD), are associated with amyloid deposition in the central nervous system and neurodegeneration. The two amyloid proteins, ABri and ADan, are degradation products of the same precursor molecule BriPP bearing......-terminus. Neurofibrillary tangles containing the classical paired helical filaments as well as neuritic components in many instances co-localize with the amyloid deposits. In both disorders, the pattern of hyperphosphorylatedtau immunoreactivity is almost indistinguishable from that seen in Alzheimer's disease....... These issues argue for the primary importance of the amyloid deposits in the mechanism(s) of neuronal cell loss. We propose FBD and FDD, the chromosome 13 dementia syndromes, as models to study the molecular basis of neurofibrillary degeneration, cell death and amyloid formation in the brain....

  15. Autophagy and Neurodegeneration: Pathogenic Mechanisms and Therapeutic Opportunities.

    Science.gov (United States)

    Menzies, Fiona M; Fleming, Angeleen; Caricasole, Andrea; Bento, Carla F; Andrews, Stephen P; Ashkenazi, Avraham; Füllgrabe, Jens; Jackson, Anne; Jimenez Sanchez, Maria; Karabiyik, Cansu; Licitra, Floriana; Lopez Ramirez, Ana; Pavel, Mariana; Puri, Claudia; Renna, Maurizio; Ricketts, Thomas; Schlotawa, Lars; Vicinanza, Mariella; Won, Hyeran; Zhu, Ye; Skidmore, John; Rubinsztein, David C

    2017-03-08

    Autophagy is a conserved pathway that delivers cytoplasmic contents to the lysosome for degradation. Here we consider its roles in neuronal health and disease. We review evidence from mouse knockout studies demonstrating the normal functions of autophagy as a protective factor against neurodegeneration associated with intracytoplasmic aggregate-prone protein accumulation as well as other roles, including in neuronal stem cell differentiation. We then describe how autophagy may be affected in a range of neurodegenerative diseases. Finally, we describe how autophagy upregulation may be a therapeutic strategy in a wide range of neurodegenerative conditions and consider possible pathways and druggable targets that may be suitable for this objective. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Microglial cell dysregulation in Brain Aging and Neurodegeneration.

    Directory of Open Access Journals (Sweden)

    Rommy eVon Bernhardi

    2015-07-01

    Full Text Available Aging is the main risk factor for neurodegenerative diseases. In aging, microglia undergo phenotypic changes compatible with their activation. Glial activation can lead to neuroinflammation, which is increasingly accepted as part of the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease (AD. We hypothesize that in aging, aberrant microglia activation leads to a deleterious environment and neurodegeneration. In aged mice, microglia exhibit an increased expression of cytokines and an exacerbated inflammatory response to pathological changes. Whereas LPS increases nitric oxide secretion in microglia from young mice, induction of reactive oxygen species (ROS predominates in older mice. Furthermore, there is accumulation of DNA oxidative damage in mitochondria of microglia during aging, and also an increased intracellular ROS production. Increased ROS activates the redox-sensitive nuclear factor kappa B, which promotes more neuroinflammation, and can be translated in functional deficits, such as cognitive impairment. Mitochondria-derived ROS and cathepsin B, are also necessary for the microglial cell production of interleukin-1β, a key inflammatory cytokine. Interestingly, whereas the regulatory cytokine TGFβ1 is also increased in the aged brain, neuroinflammation persists. Assessing this apparent contradiction, we have reported that TGFβ1 induction and activation of Smad3 signaling after inflammatory stimulation are reduced in adult mice. Other protective functions, such as phagocytosis, although observed in aged animals, become not inducible by inflammatory stimuli and TGFβ1. Here, we discuss data suggesting that mitochondrial and endolysosomal dysfunction could at least partially mediate age-associated microglial cell changes, and, together with the impairment of the TGFβ1-Smad3 pathway, could result in a reduction of protective activation and a facilitation of cytotoxic activation of microglia, resulting in the

  17. The multiple sclerosis visual pathway cohort: understanding neurodegeneration in MS.

    Science.gov (United States)

    Martínez-Lapiscina, Elena H; Fraga-Pumar, Elena; Gabilondo, Iñigo; Martínez-Heras, Eloy; Torres-Torres, Ruben; Ortiz-Pérez, Santiago; Llufriu, Sara; Tercero, Ana; Andorra, Magi; Roca, Marc Figueras; Lampert, Erika; Zubizarreta, Irati; Saiz, Albert; Sanchez-Dalmau, Bernardo; Villoslada, Pablo

    2014-12-15

    Multiple Sclerosis (MS) is an immune-mediated disease of the Central Nervous System with two major underlying etiopathogenic processes: inflammation and neurodegeneration. The latter determines the prognosis of this disease. MS is the main cause of non-traumatic disability in middle-aged populations. The MS-VisualPath Cohort was set up to study the neurodegenerative component of MS using advanced imaging techniques by focusing on analysis of the visual pathway in a middle-aged MS population in Barcelona, Spain. We started the recruitment of patients in the early phase of MS in 2010 and it remains permanently open. All patients undergo a complete neurological and ophthalmological examination including measurements of physical and disability (Expanded Disability Status Scale; Multiple Sclerosis Functional Composite and neuropsychological tests), disease activity (relapses) and visual function testing (visual acuity, color vision and visual field). The MS-VisualPath protocol also assesses the presence of anxiety and depressive symptoms (Hospital Anxiety and Depression Scale), general quality of life (SF-36) and visual quality of life (25-Item National Eye Institute Visual Function Questionnaire with the 10-Item Neuro-Ophthalmic Supplement). In addition, the imaging protocol includes both retinal (Optical Coherence Tomography and Wide-Field Fundus Imaging) and brain imaging (Magnetic Resonance Imaging). Finally, multifocal Visual Evoked Potentials are used to perform neurophysiological assessment of the visual pathway. The analysis of the visual pathway with advance imaging and electrophysilogical tools in parallel with clinical information will provide significant and new knowledge regarding neurodegeneration in MS and provide new clinical and imaging biomarkers to help monitor disease progression in these patients.

  18. Patient Affected by Beta-Propeller Protein-Associated Neurodegeneration: A Therapeutic Attempt with Iron Chelation Therapy

    Directory of Open Access Journals (Sweden)

    Mattia Fonderico

    2017-08-01

    Full Text Available Here, we report the case of a 36-year-old patient with a diagnosis of de novo mutation of the WDR45 gene, responsible for beta-propeller protein-associated neurodegeneration, a phenotypically distinct, X-linked dominant form of Neurodegeneration with Brain Iron Accumulation. The clinical history is characterized by a relatively stable intellectual disability and a hypo-bradykinetic and hypertonic syndrome with juvenile onset. Genetic investigations and T1 and T2-weighted MR images align with what is described in literature. The patient was also subjected to PET with 18-FDG investigation and DaT-Scan study. In reporting relevant clinical data, we want to emphasize the fact that the patient received a chelation therapy with deferiprone (treatment already used in other forms of NBIA with encouraging results, which, however, had to be interrupted because the parkinsonian symptoms worsened. Conversely, the patient has benefited from non-drug therapies and, in particular, from an adapted motor activity with assisted pedaling (method in the process of validation in treatments of parkinsonian syndromes, which started before the treatment with deferiprone and still continues.

  19. Pantothenate kinase-associated neurodegeneration: altered mitochondria membrane potential and defective respiration in Pank2 knock-out mouse model.

    Science.gov (United States)

    Brunetti, Dario; Dusi, Sabrina; Morbin, Michela; Uggetti, Andrea; Moda, Fabio; D'Amato, Ilaria; Giordano, Carla; d'Amati, Giulia; Cozzi, Anna; Levi, Sonia; Hayflick, Susan; Tiranti, Valeria

    2012-12-15