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Auswahl der wissenschaftlichen Literatur zum Thema „Genetic vectors“
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Zeitschriftenartikel zum Thema "Genetic vectors"
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Gooding, R. H. „Genetic variation in arthropod vectors of disease-causing organisms: obstacles and opportunities.“ Clinical Microbiology Reviews 9, Nr. 3 (Juli 1996): 301–20. http://dx.doi.org/10.1128/cmr.9.3.301.
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An overview of the genetic variation in arthropods that transmit pathogens to vertebrates is presented, emphasizing the genetics of vector-pathogen relationships and the biochemical genetics of vectors. Vector-pathogen interactions are reviewed briefly as a prelude to a discussion of the genetics of susceptibility and refractoriness in vectors. Susceptibility to pathogens is controlled by maternally inherited factors, sex-linked dominant alleles, and dominant and recessive autosomal genes. There is widespread interpopulation (including intercolony) and temporal variation in susceptibility to pathogens. The amount of biochemical genetic variation in vectors is similar to that found in other invertebrates. However, the amount varies widely among species, among populations within species, and temporally within populations. Biochemical genetic studies show that there is considerable genetic structuring of many vectors at the local, regional, and global levels. It is argued that genetic variation in vectors is critical in understanding vector-pathogen interactions and that genetic variation in vectors creates both obstacles to and opportunities for application of genetic techniques to the control of vectors.
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Yew, Chee-Hong Takahiro, Narmatha Gurumoorthy, Fazlina Nordin, Gee Jun Tye, Wan Safwani Wan Kamarul Zaman, Jun Jie Tan und Min Hwei Ng. „Integrase deficient lentiviral vector: prospects for safe clinical applications“. PeerJ 10 (12.08.2022): e13704. http://dx.doi.org/10.7717/peerj.13704.
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HIV-1 derived lentiviral vector is an efficient transporter for delivering desired genetic materials into the targeted cells among many viral vectors. Genetic material transduced by lentiviral vector is integrated into the cell genome to introduce new functions, repair defective cell metabolism, and stimulate certain cell functions. Various measures have been administered in different generations of lentiviral vector systems to reduce the vector’s replicating capabilities. Despite numerous demonstrations of an excellent safety profile of integrative lentiviral vectors, the precautionary approach has prompted the development of integrase-deficient versions of these vectors. The generation of integrase-deficient lentiviral vectors by abrogating integrase activity in lentiviral vector systems reduces the rate of transgenes integration into host genomes. With this feature, the integrase-deficient lentiviral vector is advantageous for therapeutic implementation and widens its clinical applications. This short review delineates the biology of HIV-1-erived lentiviral vector, generation of integrase-deficient lentiviral vector, recent studies involving integrase-deficient lentiviral vectors, limitations, and prospects for neoteric clinical use.
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Verma, Abhishek, und Ankit Awasthi. „Developing non-viral or viral vectors for efficient and targeted delivery of genetic material, such as DNA or RNA, for gene therapy applications“. Pharmaspire 15, Nr. 04 (2023): 243–56. http://dx.doi.org/10.56933/pharmaspire.2023.15137.
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Gene therapy has emerged as a promising approach for treating a wide range of genetic and acquired diseases by introducing or modifying genetic material within cells. To achieve successful gene therapy, efficient and precise delivery of genetic material, such as DNA or RNA, to target cells is essential. This abstract explores the development of both non-viral and viral vectors for the delivery of genetic material in gene therapy applications. Non-viral vectors, including lipid nanoparticles (LNPs), polymer-based carriers, and cell-penetrating peptides, have gained significant attention due to their safety profile and ease of production. These vectors are designed to protect genetic material from degradation, facilitate cellular uptake, and release the cargo at the desired location. Recent advancements in nanotechnology have enabled the design of customizable non-viral vectors with enhanced delivery efficiency and reduced offtarget effects. Viral vectors, on the other hand, harness the natural infectivity of viruses to transport genetic material into target cells. Retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAVs) are commonly used viral vectors in gene therapy. Viral vectors offer high transduction efficiency but may trigger immune responses or pose risks of insertional mutagenesis. Efforts in vector engineering have led to the development of safer viral vectors with improved targeting capabilities and reduced immunogenicity. AAVs, in particular, have gained prominence due to their ability to achieve long-lasting gene expression with minimal adverse effects. Targeted delivery strategies aim to enhance vector specificity, ensuring that genetic material reaches the intended cell type or tissue. These strategies include modifying vector surface proteins, employing tissue-specific promoters, or utilizing ligand-receptor interactions. In conclusion, the successful application of gene therapy relies on the development of efficient and targeted delivery systems for genetic material. Non-viral and viral vectors offer distinct advantages and continue to evolve to meet the demands of gene therapy applications. Advances in vector design, safety, and targeting strategies hold promise for the continued progress of gene therapy as a transformative medical intervention.
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Perumalsamy, Nandhini, Rohit Sharma, Muthukumaravel Subramanian und Shriram Ananganallur Nagarajan. „Hard Ticks as Vectors: The Emerging Threat of Tick-Borne Diseases in India“. Pathogens 13, Nr. 7 (02.07.2024): 556. http://dx.doi.org/10.3390/pathogens13070556.
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Hard ticks (Ixodidae) play a critical role in transmitting various tick-borne diseases (TBDs), posing significant global threats to human and animal health. Climatic factors influence the abundance, diversity, and vectorial capacity of tick vectors. It is imperative to have a comprehensive understanding of hard ticks, pathogens, eco-epidemiology, and the impact of climatic changes on the transmission dynamics of TBDs. The distribution and life cycle patterns of hard ticks are influenced by diverse ecological factors that, in turn, can be impacted by changes in climate, leading to the expansion of the tick vector’s range and geographical distribution. Vector competence, a pivotal aspect of vectorial capacity, involves the tick’s ability to acquire, maintain, and transmit pathogens. Hard ticks, by efficiently feeding on diverse hosts and manipulating their immunity through their saliva, emerge as competent vectors for various pathogens, such as viruses, parasites and bacteria. This ability significantly influences the success of pathogen transmission. Further exploration of genetic diversity, population structure, and hybrid tick vectors is crucial, as they play a substantial role in influencing vector competence and complicating the dynamics of TBDs. This comprehensive review deals with important TBDs in India and delves into a profound understanding of hard ticks as vectors, their biology, and the factors influencing their vector competence. Given that TBDs continue to pose a substantial threat to global health, the review emphasizes the urgency of investigating tick control strategies and advancing vaccine development. Special attention is given to the pivotal role of population genetics in comprehending the genetic diversity of tick populations and providing essential insights into their adaptability to environmental changes.
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Weklak, Denice, Daniel Pembaur, Georgia Koukou, Franziska Jönsson, Claudia Hagedorn und Florian Kreppel. „Genetic and Chemical Capsid Modifications of Adenovirus Vectors to Modulate Vector–Host Interactions“. Viruses 13, Nr. 7 (02.07.2021): 1300. http://dx.doi.org/10.3390/v13071300.
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Adenovirus-based vectors are playing an important role as efficacious genetic vaccines to fight the current COVID-19 pandemic. Furthermore, they have an enormous potential as oncolytic vectors for virotherapy and as vectors for classic gene therapy. However, numerous vector–host interactions on a cellular and noncellular level, including specific components of the immune system, must be modulated in order to generate safe and efficacious vectors for virotherapy or classic gene therapy. Importantly, the current widespread use of Ad vectors as vaccines against COVID-19 will induce antivector immunity in many humans. This requires the development of strategies and techniques to enable Ad-based vectors to evade pre-existing immunity. In this review article, we discuss the current status of genetic and chemical capsid modifications as means to modulate the vector–host interactions of Ad-based vectors.
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Powell, Jeffrey. „Genetic Variation in Insect Vectors: Death of Typology?“ Insects 9, Nr. 4 (11.10.2018): 139. http://dx.doi.org/10.3390/insects9040139.
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The issue of typological versus population thinking in biology is briefly introduced and defined. It is then emphasized how population thinking is most relevant and useful in vector biology. Three points are made: (1) Vectors, as they exist in nature, are genetically very heterogeneous. (2) Four examples of how this is relevant in vector biology research are presented: Understanding variation in vector competence, GWAS, identifying the origin of new introductions of invasive species, and resistance to inbreeding. (3) The existence of high levels of vector genetic heterogeneity can lead to failure of some approaches to vector control, e.g., use of insecticides and release of sterile males (SIT). On the other hand, vector genetic heterogeneity can be harnessed in a vector control program based on selection for refractoriness.
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Kienesberger, Sabine, Gregor Gorkiewicz, Martina M. Joainig, Sylvia R. Scheicher, Eva Leitner und Ellen L. Zechner. „Development of Experimental Genetic Tools for Campylobacter fetus“. Applied and Environmental Microbiology 73, Nr. 14 (18.05.2007): 4619–30. http://dx.doi.org/10.1128/aem.02407-06.
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ABSTRACT Molecular analysis of the virulence mechanisms of the emerging pathogen Campylobacter fetus has been hampered by the lack of genetic tools. We report the development and functional analysis of Escherichia coli-Campylobacter shuttle vectors that are appropriate for C. fetus. Some vectors were constructed based on the known Campylobacter coli plasmid pIP1455 replicon, which confers a wide host range in Campylobacter spp. Versatility in directing gene expression was achieved by introducing a strong C. fetus promoter. The constructions carry features necessary and sufficient to detect the expression of phenotypic markers, including molecular reporter genes in both subspecies of C. fetus, while retaining function in C. jejuni. The capacity to express several gene products from different vectors in a single host can be advantageous but requires distinct plasmid replicons. To this end, replication features derived from a cryptic plasmid of C. fetus subsp. venerealis strain 4111/108, designated pCFV108, were adapted for a compatible series of constructions. The substitution of the C. coli replication elements reduced vector size while apparently limiting the host range to C. fetus. The complementation of a ciprofloxacin-resistant mutant phenotype via vector-driven gyrA expression was verified. Cocultivation demonstrated that shuttle vectors based on the pCFV108 replicon were compatible with pIP1455 replication functions, and the stable maintenance of two plasmids in a C. fetus subsp. venerealis host over several months was observed. The application of both vector types will facilitate the investigation of the genetics and cellular interactions of the emerging pathogen C. fetus.
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Criscione, Frank, David A. O’Brochta und William Reid. „Genetic technologies for disease vectors“. Current Opinion in Insect Science 10 (August 2015): 90–97. http://dx.doi.org/10.1016/j.cois.2015.04.012.
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Krasnykh, Victor N., Joanne T. Douglas und Victor W. van Beusechem. „Genetic Targeting of Adenoviral Vectors“. Molecular Therapy 1, Nr. 5 (Mai 2000): 391–405. http://dx.doi.org/10.1006/mthe.2000.0062.
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Kojic, Milorad, und William K. Holloman. „Shuttle vectors for genetic manipulations in Ustilago maydis“. Canadian Journal of Microbiology 46, Nr. 4 (01.04.2000): 333–38. http://dx.doi.org/10.1139/w00-002.
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Shuttle vectors with new or improved features were constructed to enable facile genetic manipulations in the plant pathogen Ustilago maydis. Sets of plasmids selectable in media containing geneticin, carboxin, nourseothricin, or hygromycin, able to replicate autonomously, to transform U. maydis by integration, and to express foreign genes under control of the homologous glyceraldehyde-3-phosphate dehydrogenase promoter, were built upon a common pUC19 vector backbone. This permits a large number of choices for a cloning site, blue/white screening for recombinant plasmids, rapid transfer of a cloned DNA fragment between plasmids, and choice of several dominant drug-resistance markers for selection in U. maydis.Key words: G418, carboxin, nourseothricin, hygromycin, expression vectors.
Dissertationen zum Thema "Genetic vectors"
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Theodorides, Kosmas. „Genetic and systematic studies on Cicadellidae vectors“. Thesis, University of East Anglia, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368187.
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Shareck, Julie. „Isolation and characterization of a cryptic plasmid from Lactobacillus plantarum“. Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=84072.
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Lactic acid bacteria (LAB), a group of generally recognized as safe (GRAS) organisms that metabolize sugars into primarily lactic acid, have traditionally been used for the fermentation and preservation of various foods and beverages. There is increasing interest in the genetic manipulation of LAB to improve existing characteristics or introduce novel, industrially pertinent phenotypes. However, because these bacteria have food-related applications, their genetic modification requires the use of food-grade genetic engineering tools. LAB plasmids, self-replicating extrachromosomal DNA molecules, can be used to derive food-grade cloning vectors. The rationale of this research was to develop a food-grade cloning vector using a lactobacilli cryptic plasmid and to investigate its cloning and expression properties. The main objectives were to (i) screen Lactobacillus spp. for plasmids, (ii) isolate and characterize a plasmid, and (iii) use the plasmid replicon to construct a cloning vector and express heterologous genes in various hosts. This is the first step in the development of a new family of food-grade cloning vectors for the genetic modification of lactobacilli.
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Robson, Julia. „The construction of an expression vector for the transformation of the grape chloroplast genome“. Thesis, Stellenbosch : Stellenbosch University, 2003. http://hdl.handle.net/10019.1/53621.
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Thesis (MSc)--University of Stellenbosch, 2003.ENGLISH ABSTRACT: The genetic information of plants is found in the nucleus, the mitochondria, and the plastids. The DNA of plastids is comprised of multiple copies of a double-stranded, circular, prokaryoticallyderived genome of -150 kb. The genome equivalents of plastid organelles in higher plant cells are an attractive target for genetic engineering as high protein expression levels are readily obtained due to the high genome copy number per organelle. The resultant proteins are contained within the plastid organelle and the corresponding transgenes are inherited, in most crop plants, uniparentally, preventing pollen transmission of DNA. Plastid transformation involves the uniform modification of all the plastid genome copies, a process facilitated by homologous recombination and the non-Mendelian segregation of plastids upon cell division. The plastid genomes are in a continuous state of inter- and intra-molecular exchange due to their common genetic complement. This enables the site-specific integration of any piece of DNA flanked by plastid targeting sequences, via homologous recombination. The attainment of homoplasmy, where all genomes are transformed, requires the inclusion of a plastid-specific selectable marker. Selective pressure favouring the propagation of the transformed genome copies, as well as the random segregation of plastids upon cell division, make it feasible to acquire uniformity and hence genetic stability. From this, a complete transplastomie line is obtained where all plastid genome copies present are transgenic, having eliminated all wild-type genome copies. The prokaryotic nature of the chloroplast genetic system enables expression of multiple proteins from polycistronic mRNAs, allowing the introduction of entire operons in a single transformation. Expression cassettes in vectors thus include single regulatory elements of plastid origin, and harbour genes encoding selectable and screenable markers, as well as one or more genes of interest. Each coding region is preceded by an appropriate translation control region to ensure efficient translation from the polycistronic mRNA. The function of a plastid transformation vector is to enable transfer and stable integration of foreign genes into the chloroplast genomes of higher plants. The expression vector constructed in this research is specific for the transformation of the grape chloroplast genome. Vitis vinifera L., from the family, Vitaceae, is the choice species for the production of wine and therefore our target for plastid transformation. All chloroplast derived regulatory elements and sequences included in the vector thus originated from this species.
AFRIKAANSE OPSOMMING: Die genetiese inligting van plante word gevind in die kern, die mitochondria, en die plastiede. Die DNA van plastiede bestaan uit veelvuldige kopieë van 'n ~ 150 kb dubbelstring, sirkulêre genoom van prokariotiese oorsprong. Die genoomekwivalente van plastiede in hoër plante is 'n aantreklike teiken vir genetiese manipulering, aangesien die hoë genoom kopiegetal per organel dit moontlik maak om gereeld hoë vlakke van proteïenuitdrukking te verkry. Hierdie proteïene word tot die plastied beperk, en die ooreenstemmende transgene word in die meeste plante sitoplasmies oorgeërf, sonder die oordrag van DNA deur die stuifmeel. Plastied transformasie behels die uniforme modifikasie van al die plastied genoomkopieë, 'n proses wat deur homoloë rekombinasie en die nie-Mendeliese segregasie van plastiede tydens seldeling gefasiliteer word. As gevolg van die gemeenskaplike genetiese komplement, vind aanhoudende interen intra-molekulêre uitruiling van plastiedgenome plaas. Dit maak die setel-spesifieke integrasie, via homoloë rekombinasie, van enige stuk DNA wat deur plastied teikenvolgordes begrens word, moontlik. Vir die verkrying van homoplasmie, waar alle genome getransformeer is, word die insluiting van 'n plastiedspesifieke selekteerbare merker benodig. Seleksiedruk wat die vermeerdering van die getransformeerde genoomkopieë bevoordeel, en die lukrake segregasie van plastiede tydens seldeling, maak dit moontlik om genetiese stabiliteit en uniformiteit van die genoom te verkry. Dit kan op sy beurt tot die verkryging van 'n volledige transplastomiese lyn lei, waar alle aanwesige plastiedgenome transgenies is, en wilde tipe genoomkopieë geëlimineer is. Die prokariotiese aard van die chloroplas genetiese sisteem maak die uitdrukking van veelvuldige proteïene vanaf polisistroniese mRNAs moontlik, wat die toevoeging van volledige operons in 'n enkele transformasie toelaat. Uitdrukkingskassette in vektore bevat dus enkel regulatoriese elemente van plastied oorsprong, gene wat kodeer vir selekteerbare en sifbare merkers, asook een of meer gene van belang (teikengene). Voor elke koderingsstreek, is daar ook 'n toepaslike translasie beheerstreek om doeltreffende translasie vanaf die polisistroniese mRNA te verseker. Die funksie van 'n plastied transformasie vektor is om die oordrag en stabiele integrasie van transgene in chloroplasgenome van hoër plante moontlik te maak. Die uitdrukkingsvektor wat in hierdie studie gekonstrueer is, is spesifiek vir die transformasie van die druif chloroplasgenoom. Vitis vinifera L., van die familie Vitaceae, is die voorkeur species vir die produksie van wyn, en daarom die teiken vir plastied transformasie. Alle chloroplast-afgeleide regulatoriese elemente en volgordes wat in hierdie vektor ingesluit is, het huloorsprong vanaf VUis vinifera L.
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Ghosh, Arkasubhra. „Rational design of split gene vectors to expand the packaging capacity of adeno-associated viral vectors“. Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/4712.
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Thesis (Ph. D.)--University of Missouri-Columbia, 2007.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2007" Includes bibliographical references.
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Wood, David Rowe Ding Jiahuan. „Design, optimization, and evaluation of conditionally active gene therapy vectors“. Waco, Tex. : Baylor University, 2008. http://hdl.handle.net/2104/5153.
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Mück-Häusl, Martin Andreas. „Genetic engineering of adenoviral vectors for improved therapeutic applications“. Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-138269.
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Wong, Tik-wun Lina. „Construction of an infectious PRRSV cDNA clone and its use as a vector for foreign gene expression“. Click to view the E-thesis via HKUTO, 2010. http://sunzi.lib.hku.hk/hkuto/record/B44251841.
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Van, Eeden C. (Christiaan). „The construction of gene silencing transformation vectors for the introduction of multiple-virus resistance in grapevines“. Thesis, Stellenbosch : Stellenbosch University, 2004. http://hdl.handle.net/10019.1/53764.
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Thesis (MSc)--University of Stellenbosch, 2004.ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies.
AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.
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Warren, Ann. „Transposable genetic elements in the mosquito Aedes aegypti“. Thesis, University of Liverpool, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.237672.
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Limberis, Maria. „A lentiviral gene transfer vector for the treatment of cystic fibrosis airway disease“. Title page, synopsis and list of contents only, 2002. http://web4.library.adelaide.edu.au/theses/09PH/09phl735.pdf.
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"16th September 2002." Accompanying CD contains 2 MPEG clips with accompanying text, and a copy in PDF format of: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer / M. Limberis ... [et al.], published in Human gene therapy vol. 13 (2002). Bibliography: leaves xxix-li. This thesis focuses on modulating the physical barriers of the airway epithelium with mild detergents, so as to enhance gene transfer by a HIV-1 based lentivirus vector in vivo. The efficiency of the gene transfer was evaluated in the nasal airway of C57B1/6 mice using the Lac Z marker gene. This demonstration of lentivirus-mediated in vivo recovery of CFTR function in CF airway epithelium illustrated the potential of combining a pre-conditioning of the airway surface with a simple and brief HIV-1 based gene transfer vector exposure to produce therapeutic gene expression in the intact airway.
Bücher zum Thema "Genetic vectors"
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L, Hefferon Kathleen, Hrsg. Virus expression vectors. Tribandrum: Transworld Research Network, 2007.
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S, Ruiz Pablo, Hrsg. Genetic vectors research focus. New York: Nova Biomedical Books, 2007.
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David, Curiel, und Douglas Joanne T, Hrsg. Adenoviral vectors for gene therapy. Amsterdam: Academic Press, 2002.
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George, Dickson, Hrsg. Molecular and cell biology of human gene therapeutics. London: Chapman & Hall, 1995.
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Yakov, Gluzman, Hughes Stephen H und Cold Spring Harbor Laboratory, Hrsg. Viral vectors. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory, 1988.
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L, Rodriguez Raymond, und Denhardt David T, Hrsg. Vectors: A survey of molecular cloning vectors and their uses. Boston: Butterworths, 1988.
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D, Richardson Christopher, Hrsg. Baculovirus expression protocols. Totowa, N.J: Humana Press, 1995.
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H, Miller Jeffrey, und Calos Michele P, Hrsg. Gene transfer vectors for mammalian cells. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory, 1987.
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P, Jones, Hrsg. Vectors: Cloning applications. Chichester: Wiley, 1998.
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P, Jones, Hrsg. Vectors: Expression systems. Chichester: John Wiley & Sons, 1998.
Den vollen Inhalt der Quelle findenBuchteile zum Thema "Genetic vectors"
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Benabdellah, Karim, Simone Thomas und Hinrich Abken. „Genetic Engineering of Autologous or Allogeneic Immune Effector Cells“. In The EBMT/EHA CAR-T Cell Handbook, 7–10. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-030-94353-0_2.
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AbstractManufacturing immune effector cells (T or NK cells) with CAR-encoding DNA sequences requires efficient and safe genetic engineering procedures. For this purpose, an appropriate genetic vector is chosen according to numerous factors, including the vector genome packaging capacity, cellular tropism, genomic integration, immune toxicity, and other factors. In clinical trials, genomes integrating viral vectors, in particular vectors based on members of the Retroviridae family, such as retroviruses and lentiviruses, have been successfully used for more than 20 years. These vectors contain an RNA genome that when transcribed into double-stranded DNA by reverse transcriptase integrates into the genome of the transduced cell.
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Vos, Jean-Michel H. „Herpesviruses as Genetic Vectors“. In Viruses in Human Gene Therapy, 109–40. Dordrecht: Springer Netherlands, 1995. http://dx.doi.org/10.1007/978-94-011-0555-2_5.
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Cox, William I., Russell R. Gettig und Enzo Paoletti. „Poxviruses as Genetic Vectors“. In Viruses in Human Gene Therapy, 141–78. Dordrecht: Springer Netherlands, 1995. http://dx.doi.org/10.1007/978-94-011-0555-2_6.
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Bevan, Michael, und Andrew Goldsbrough. „Design and Use of Agrobacterium Transformation Vectors“. In Genetic Engineering, 123–40. Boston, MA: Springer US, 1988. http://dx.doi.org/10.1007/978-1-4615-7081-3_7.
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Shepherd, Robert J. „Caulimoviruses as Potential Gene Vectors for Higher Plants“. In Genetic Engineering, 241–76. Boston, MA: Springer US, 1986. http://dx.doi.org/10.1007/978-1-4615-9456-7_13.
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Dingli, David, und Stephen J. Russell. „Genetic Targeting of Retroviral Vectors“. In Vector Targeting for Therapeutic Gene Delivery, 267–91. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2003. http://dx.doi.org/10.1002/0471234303.ch13.
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Wickham, Thomas J. „Genetic Targeting of Adenoviral Vectors“. In Vector Targeting for Therapeutic Gene Delivery, 143–70. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2003. http://dx.doi.org/10.1002/0471234303.ch7.
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Bouyer, Jérémy, und Eric Marois. „14. Genetic control of vectors“. In Ecology and Control of Vector-borne Diseases, 435–51. The Netherlands: Wageningen Academic Publishers, 2018. http://dx.doi.org/10.3920/978-90-8686-863-6_14.
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Nicklin, Stuart A., und Andrew H. Baker. „Development of Targeted Viral Vectors for Cardiovascular Gene Therapy“. In Genetic Engineering, 15–49. Boston, MA: Springer US, 2003. http://dx.doi.org/10.1007/978-1-4615-0073-5_2.
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Bernstein, Alan, Stuart Berger, Dennis Huszar und John Dick. „Gene Transfer with Retrovirus Vectors“. In Genetic Engineering: Principles and Methods, 235–61. Boston, MA: Springer US, 1985. http://dx.doi.org/10.1007/978-1-4684-4973-0_11.
Der volle Inhalt der QuelleKonferenzberichte zum Thema "Genetic vectors"
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Alshahrani, Mohammed, Spyridon Samothrakis und Maria Fasli. „Identifying idealised vectors for emotion detection using CMA-ES“. In GECCO '19: Genetic and Evolutionary Computation Conference. New York, NY, USA: ACM, 2019. http://dx.doi.org/10.1145/3319619.3322057.
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Funaki, Ryohei, und Hideyuki Takagi. „Application of Gravity Vectors and Moving Vectors for the Acceleration of Both Differential Evolution and Interactive Differential Evolution“. In 2011 Fifth International Conference on Genetic and Evolutionary Computing (ICGEC). IEEE, 2011. http://dx.doi.org/10.1109/icgec.2011.71.
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Madeira, Catarina, Sofia C. Ribeiro, Rui Mendes, Irina S. M. Pinheiro, Claudia L. da Silva und Joaquim M. S. Cabral. „Genetic engineering of stem cells by non-viral vectors“. In 2011 1st Portuguese Meeting in Bioengineering ¿ The Challenge of the XXI Century (ENBENG). IEEE, 2011. http://dx.doi.org/10.1109/enbeng.2011.6026044.
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Pescador-Rojas, Miriam, und Carlos A. Coello Coello. „Studying the effect of techniques to generate reference vectors in many-objective optimization“. In GECCO '18: Genetic and Evolutionary Computation Conference. New York, NY, USA: ACM, 2018. http://dx.doi.org/10.1145/3205651.3205684.
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Sato, Yuji, Mads Midtlyng und Mikiko Sato. „Diffusely Distributed Parallelization of MOEA/D with Edge Weight Vectors Sharing“. In GECCO '23 Companion: Companion Conference on Genetic and Evolutionary Computation. New York, NY, USA: ACM, 2023. http://dx.doi.org/10.1145/3583133.3590738.
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Xianneng Li, S. Mabu, M. K. Mainali und K. Hirasawa. „Probabilistic model building Genetic Network Programming using multiple probability vectors“. In 2010 IEEE Region 10 Conference (TENCON 2010). IEEE, 2010. http://dx.doi.org/10.1109/tencon.2010.5686113.
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Fengli Sun, Quanhua Gao und Jinguo Wang. „Ridgelet Probabilistic Neural Network with Genetic Algorithm selecting center vectors“. In 2011 International Conference on Computer Science and Network Technology (ICCSNT). IEEE, 2011. http://dx.doi.org/10.1109/iccsnt.2011.6182206.
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Tanaka, Mariko, Yuki Yamagishi, Hidetoshi Nagai und Hiroyuki Sato. „Infeasible solution repair and MOEA/D sharing weight vectors for solving multi-objective set packing problems“. In GECCO '18: Genetic and Evolutionary Computation Conference. New York, NY, USA: ACM, 2018. http://dx.doi.org/10.1145/3205651.3208765.
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Sánchez, Alberto Rodríguez, Antonin Ponsich und Antonio López Jaimes. „Generation techniques and a novel on-line adaptation strategy for weight vectors within decomposition-based MOEAs“. In GECCO '19: Genetic and Evolutionary Computation Conference. New York, NY, USA: ACM, 2019. http://dx.doi.org/10.1145/3319619.3322055.
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Wu, Kai, und George Panoutsos. „High Dimensional Many Objective Optimisation through Diverse Creation and Categorisation of Reference Vectors“. In GECCO '23 Companion: Companion Conference on Genetic and Evolutionary Computation. New York, NY, USA: ACM, 2023. http://dx.doi.org/10.1145/3583133.3590612.
Der volle Inhalt der QuelleBerichte der Organisationen zum Thema "Genetic vectors"
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Dawson, William O., und Moshe Bar-Joseph. Creating an Ally from an Adversary: Genetic Manipulation of Citrus Tristeza. United States Department of Agriculture, Januar 2004. http://dx.doi.org/10.32747/2004.7586540.bard.
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Citrus is one of the major agricultural crops common to Israel and the United States, important in terms of nutrition, foreign exchange, and employment. The economy of both citrus industries have been chronically plagued by diseases caused by Citrus tristeza virus (CTV). The short term solution until virus-resistant plants can be used is the use of mild strain cross-protection. We are custom designing "ideal" protecting viruses to immunize trees against severe isolates of CTV by purposely inoculating existing endangered trees and new plantings to be propagated as infected (protected) citrus budwood. We crossed the substantial technological hurdles necessary to accomplish this task which included developing an infectious cDNA clone which allows in vitro manipulation of the virus and methods to then infect citrus plants. We created a series of hybrids between decline-inducing and mild CTV strains, tested them in protoplasts, and are amplifying them to inoculate citrus trees for evaluation and mapping of disease determinants. We also extended this developed technology to begin engineering transient expression vectors based on CTV as tools for genetic improvement of tree crops, in this case citrus. Because of the long periods between genetic transformation and the ultimate assay of mature tree characteristics, there is a great need for an effective system that allows the expression or suppression of target genes in fruiting plants. Virus-based vectors will greatly expedite progress in citrus genetic improvement. We characterized several components of the virus that provides necessary information for designing virus-based vectors. We characterized the requirements of the 3 ’-nontranslated replication promoter and two 3 ’-ORF subgenomic (sg) mRNA controller elements. We discovered a novel type of 5’-terminal sgRNAs and characterized the cis-acting control element that also functions as a strong promoter of a 3 ’-sgRNA. We showed that the p23 gene controls negative-stranded RNA synthesis and expression of 3 ’ genes. We identified which genes are required for infection of plants, which are host range determinants, and which are not needed for plant infection. We continued the characterization of native dRNA populations and showed the presence of five different classes including class III dRNAs that consists of infectious and self-replicating molecules and class V dRNAs that contain all of the 3 ’ ORFs, along with class IV dRNAs that retain non-contiguous internal sequences. We have constructed and tested in protoplasts a series of expression vectors that will be described in this proposal.
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Liu, Zhanjiang John, Rex Dunham und Boaz Moav. Developmental and Evaluation of Advanced Expression Vectors with Both Enhanced Integration and Stable Expression for Transgenic Farmed Fish. United States Department of Agriculture, Dezember 2001. http://dx.doi.org/10.32747/2001.7585196.bard.
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The objectives of the project were to develop expression vectors using the Sleeping Beauty transposon technology and the genetic border elements to provide both enhanced integration rate and stable transgene expression, and to evaluate the application of such vectors in farmed fish such as catfish and carp. The panel recommended adding the objective of evaluating the endogenous transposable elements, particularly in catfish, in order to evaluate the applicability of the expression vectors while reduc1ng efforts in real production of transgenic fish considering the focus of the project was to develop the vector and evaluation of its applicability, not producing transgenic fish. Efficient production of transgenic farmed fish is hindered by two major problems: mosaicism due to delayed integration after single-cell stage, and silencing of transgene expression. In this project, we proposed to combat these problems by coupling the Sleeping Beauty transposon technology that can enhance integration rate and the border elements that can insulate transgene from position effect. Our major objective was to develop a new generation of expression vector that contains both of these elements. We have developed expression vectors containing both the Sleeping Beauty transposon signals, inverted repeats and direct repeats (IR and DR, respectively), and the border elements, scs and scs'. Growth hormone minigene has been cloned into this vector for applications of such vectors in growth enhancement. Luc reporter gene has been also cloned into this vector cascades for relative easy evaluation of transgene expression. Transgenic fish have been produced using these expression vectors in both catfish (US) and carp (Israel). Much effort was also devoted to evaluation of the endogenous transposable elements in catfish as recommended by the BARD grant panel. Multiple families of Tcl-like transposons were identified from catfish. Surprisingly, many Tc I-related transcripts were identified. Among these transcripts, both the sense and antisense transcripts were present. Some of the transcripts may be useful for development of novel transposase-based technology for aquaculture applications in the future. This project has both scientific and aquaculture implications. First, to develop expression vectors containing both IR/DR and scs/scs' repeated elements have been reported being extremely technically difficult due to excision of the repeated sequences by the E. coli host during cloning processes. We have successfully constructed this advanced vector that contained very complex cascades for both gene integration and gene regulation. We have produced transgenic fish using such vectors. This advanced expression vector should be useful for production of transgenic fish. By simply replacing the growth hormone gene, any gene of interest can be readily inserted in this vector. Thus this vector should provide technological possibility for early integration and stable expression of any economically important genes in aquaculture. We have also evaluated the applications of the Sleeping Beauty-based vectors in terms of the impact of gene size and found that the size of trans gene drastically affects transposition. The system will be only useful for transferring genes smaller than 5.6 kb. We have also identified novel transposase-related transcripts that may be useful for the development of novel transposase-based technologies for general scientific research and for aquaculture applications.
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Jung, Carina, Karl Indest, Matthew Carr, Richard Lance, Lyndsay Carrigee und Kayla Clark. Properties and detectability of rogue synthetic biology (SynBio) products in complex matrices. Engineer Research and Development Center (U.S.), September 2022. http://dx.doi.org/10.21079/11681/45345.
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Synthetic biology (SynBio) aims to rationally engineer or modify traits of an organism or integrate the behaviors of multiple organisms into a singular functional organism through advanced genetic engineering techniques. One objective of this research was to determine the environmental persistence of engineered DNA in the environment. To accomplish this goal, the environmental persistence of legacy engineered DNA building blocks were targeted that laid the foundation for SynBio product development and application giving rise to “post-use products.” These building blocks include genetic constructs such as cloning and expression vectors, promoter/terminator elements, selectable markers, reporter genes, and multi-cloning sites. Shotgun sequencing of total DNA from water samples of pristine sites was performed and resultant sequence data mined for frequency of legacy recombinant DNA signatures. Another objective was to understand the fate of a standardized contemporary synthetic genetic construct (SC) in the context of various chassis systems/genetic configurations representing different degrees of “genetic bioavailability” to the environmental landscape. These studies were carried out using microcosms representing different environmental matrices (soils, waters, wastewater treatment plant (WWTP) liquor) and employed a novel genetic reporter system based on volatile organic compounds (VOC) detection to assess proliferation and persistence of the SC in the matrix over time.
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Perl, Avichai, Bruce I. Reisch und Ofra Lotan. Transgenic Endochitinase Producing Grapevine for the Improvement of Resistance to Powdery Mildew (Uncinula necator). United States Department of Agriculture, Januar 1994. http://dx.doi.org/10.32747/1994.7568766.bard.
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The original objectives are listed below: 1. Design vectors for constitutive expression of endochitinase from Trichoderma harzianum strain P1. Design vectors with signal peptides to target gene expression. 2. Extend transformation/regeneration technology to other cultivars of importance in the U.S. and Israel. 3. Transform cultivars with the endochitinase constructs developed as part of objective 1. A. Characterize foliar powdery mildew resistance in transgenic plants. Background of the topic Conventional breeding of grapevines is a slow and imprecise process. The long generation cycle, large space requirements and poor understanding of grapevine genetics prevent rapid progress. There remains great need to improve existing important cultivars without the loss of identity that follows from hybridization. Powdery mildew (Uncinula necator) is the most important fungal pathogen of grapevines, causing economic losses around the world. Genetic control of powdery mildew would reduce the requirement for chemical or cultural control of the disease. Yet, since the trait is under polygenic control, it is difficult to manipulate through hybridization and breeding. Also, because grapevines are heterozygous and vegetatively propagated cultivar identity is lost in the breeding process. Therefore, there is great need for techniques to produce transgenic versions of established cultivars with heterologous genes conferring disease resistance. Such a gene is now available for control of powdery mildew of grapevines. The protein coded by the Endochitinase gene, derived from Trichoderma harzianum, is very effective in suppressing U. necator growth. The goal of this proposal is to develop transgenic grapevines with this antifungal gene, and to test the effect of this gene on resistance to powdery mildew. Conclusions, achievements and implications Gene transfer technology for grape was developed using commercial cultivars for both wine and table grapes. It paved the way for a new tool in grapevine genetic studies enabling the alteration of specific important traits while maintaining the essential features of existing elite cultivars. Regeneration and transformation technologies were developed and are currently at an advanced stage for USA wine and Israeli seedless cultivars, representing the cutting edge of grape genetic engineering studies worldwide. Transgenic plants produced are tested for powdery mildew resistance in greenhouse and field experiments at both locations. It is our ultimate goal to develop transgenic grapes which will be more efficient and economical for growers to produce, while also providing consumers with familiar products grown with reduced chemical inputs.
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Ullman, Diane, James Moyer, Benjamin Raccah, Abed Gera, Meir Klein und Jacob Cohen. Tospoviruses Infecting Bulb Crops: Evolution, Diversity, Vector Specificity and Control. United States Department of Agriculture, September 2002. http://dx.doi.org/10.32747/2002.7695847.bard.
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Objectives. The overall goal of the proposed research was to develop a mechanistic understanding of tospovirus evolution, diversity and vector specificity that could be applied to development of novel methods for limiting virus establishment and spread. Our specific objectives were: 1) To characterize newly intercepted tospoviruses in onion, Hippeastrum and other bulb crops and compare them with the known tomato spotted wilt virus (TSWV) and its isolates; 2) To characterize intra- and interspecific variation in the virus transmission by thrips of the new and distinct tospoviruses. and, 3) To determine the basis of vector specificity using biological, cellular and molecular approaches. Background. New tospoviruses infecting bulb crops were detected in Israel and the US in the mid-90s. Their plant host ranges and relationships with thrips vectors showed they differed from the type member of the Tospovirus genus, tomato spotted wilt virus (TSWV). Outbreaks of these new viruses caused serious crop losses in both countries, and in agricultural and ornamental crops elsewhere. In the realm of plant infecting viruses, the tospoviruses (genus: Tospovirus , family: Bunyaviridae ) are among the most aggressive emerging viruses. Tospoviruses are transmitted by several species of thrips in a persistent, propagative fashion and the relationships between the viruses and their thrips vectors are often specific. With the emergence of new tospoviruses, new thrips vector/tospovirus relationships have also arisen and vector specificities have changed. There is known specificity between thrips vector species and particular tospoviruses, although the cellular and molecular bases for this specificity have been elusive. Major conclusions, solutions and achievements. We demonstrated that a new tospovirus, iris yellow spot virus (IYSV) caused "straw bleaching" in onion (Allium cepa) and lisianthus necrosis in lisianthus (Eustoma russellianum). Characterization of virus isolates revealed genetic diversity among US, Brazilian, Dutch and Israeli isolates. IYSV was not seed transmitted, and in Israel, was not located in bulbs of infected plants. In the US, infected plants were generated from infected bulbs. The relationship between IYSV and Thrips tabaci was shown to be specific. Frankliniella occidentalis, the primary vector of many other tospoviruses, did not transmit IYSV isolates in Israel or the US. Furthermore, 1': tabaci populations varied in their transmission ability. Transmission was correlated to IYSV presence in thrips salivary glands. In Israel, surveys in onion fields revealed that the onion thrips, Thrips tabaci Lindeman was the predominant species and that its incidence was strongly related to that of IYSV infection. In contrast, in the U.S., T. tabaci and F. occidentalis were present in high numbers during the times sampled. In Israel, insecticides reduced onion thrips population and caused a significant yield increase. In the US, a genetic marker system that differentiates non-thrips transmissible isolates from thrips transmissible isolate demonstrated the importance of the M RNA to thrips transmission of tospoviruses. In addition, a symbiotic Erwinia was discovered in thrips and was shown to cause significant artifacts in certain types of virus binding experiments. Implications, scientific and agricultural. Rapid emergence of distinct tospoviruses and new vector relationships is profoundly important to global agriculture. We advanced the understanding of IYSV in bulb crops and its relationships with thrips vector species. The knowledge gained provided growers with new strategies for control and new tools for studying the importance of particular viral proteins in thrips specificity and transmission efficiency.
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Singh, Anjali. What Is Optogenetics and How Does It Work? ConductScience, Juli 2022. http://dx.doi.org/10.55157/cs20220704.
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Optogenetics is a biotechnological method that combines optical systems and genetic engineering to control and monitor the functions of cells, tissues, and organisms. It involves using light-sensitive proteins called opsins to manipulate specific cells or regions with precision. This technique has revolutionized neuroscience, allowing researchers to study neural circuits and behavior by turning cells on and off. Opsins are categorized into microbial and animal types, each with specific functions. Optogenetic experiments require opsins, suitable plasmids or viral vectors, and a light source. This method has broad applications in neurology, animal behavior, and physiology, providing insights into various biological processes. It is used to map neural circuits, study diseases, and understand behaviors.
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Mawassi, Munir, Baozhong Meng und Lorne Stobbs. Development of Virus Induced Gene Silencing Tools for Functional Genomics in Grapevine. United States Department of Agriculture, Juli 2013. http://dx.doi.org/10.32747/2013.7613887.bard.
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Grapevine is perhaps the most widely grown fruit crop. To understand the genetic make-up so as to improve the yield and quality of grapes and grape products, researchers in Europe have recently sequenced the genomes of Pinot noir and its inbred. As expected, function of many grape genes is unknown. Functional genomics studies have become the major focus of grape researchers and breeders. Current genetic approaches for gene function studies include mutagenesis, crossing and genetic transformation. However, these approaches are difficult to apply to grapes and takes long periods of time to accomplish. It is thus imperative to seek new ways for grape functional genomics studies. Virus-induced gene silencing (VIGS) offers an attractive alternative for this purpose and has proven highly effective in several herbaceous plant species including tomato, tobacco and barley. VIGS offers several advantages over existing functional genomics approaches. First, it does not require transformation to silence a plant gene target. Instead, it induces silencing of a plant gene through infection with a virus that contains the target gene sequence, which can be accomplished within a few weeks. Second, different plant genes can be readily inserted into the viral genome via molecular cloning and functions of a large number of genes can be identified within a short period of time. Our long-term goal of this research is to develop VIGS-based tools for grapevine functional genomics, made of the genomes of Grapevine virus A (GVA) from Israel and Grapevine rupestris stem pitting-associated virus (GRSPaV) from Canada. GVA and GRSPaV are members of the Flexiviridae. Both viruses have single-stranded, positive sense RNA genomes, which makes them easy to manipulate genetically and excellent candidates as VIGS vectors. In our three years research, several major breakthroughs have been made by the research groups involved in this project. We have engineered a cDNA clone of GVA into a binary vector that is infectious upon delivery into plantlets of micropropagated Vitis viniferacv. Prime. We further developed the GVA into an expression vector that successfully capable to silence endogenous genes. We also were able to assemble an infectious full-length cDNA clones of GRSPaV. In the following sections Achievements and Detailed description of the research activities, we are presenting the outcome and results of this research in details.
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Mawassi, Munir, und Valerian V. Dolja. Role of the viral AlkB homologs in RNA repair. United States Department of Agriculture, Juni 2014. http://dx.doi.org/10.32747/2014.7594396.bard.
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AlkB proteins that repair DNA via reversing methylation damage are conserved in a broad range of prokaryotes and eukaryotes including plants. Surprisingly, AlkB-domains were discovered in the genomes of numerous plant positive-strand RNA viruses, majority of which belong to the family Flexiviridae. The major goal of this research was to reveal the AlkB functions in the viral infection cycle using a range of complementary genetic and biochemical approaches. Our hypotheses was that AlkB is required for efficient replication and genetic stability of viral RNA genomes The major objectives of the research were to identify the functions of GVA AlkB domain throughout the virus infection cycle in N. benthamiana and grapevine, to investigate possible RNA silencing suppression activity of the viral AlkBs, and to characterize the RNA demethylation activity of the mutated GVA AlkBs in vitro and in vivo to determine methylation status of the viral RNA. Over the duration of project, we have made a very substantial progress with the first two objectives. Because of the extreme low titer of the virus particles in plants infected with the AlkB mutant viruses, we were unable to analyze RNA demethylation activity and therefore had to abandon third objective. The major achievements with our objectives were demonstration of the AlkB function in virus spread and accumulation in both experimental and natural hosts of GVA, discovery of the functional cooperation and physical interaction between AlkB and p10 AlkB in suppression of plant RNA silencing response, developing a powerful virus vector technology for grapevine using GLRaV-2-derived vectors for functional genomics and pathogen control in grapevine, and in addition we used massive parallel sequencing of siRNAs to conduct comparative analysis of the siRNA populations in grape plants infected with AlkB-containing GLRaV-3 versus GLRaV-2 that does not encode AlkB. This analysis revealed dramatically reduced levels of virus-specific siRNAs in plants infected with GLRaV-3 compared to that in GLRaV-2 infection implicating AlkB in suppression of siRNA formation. We are pleased to report that BARD funding resulted in 5 publications directly supported by BARD, one US patent, and 9 more publications also relevant to project. Moreover, two joint manuscripts that summarize work on GVA AlkB (led by Israeli PI) and on viral siRNAs in grapevine (led by US PI in collaboration with University of Basel) are in preparation.
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Bar-Joseph, Moshe, William O. Dawson und Munir Mawassi. Role of Defective RNAs in Citrus Tristeza Virus Diseases. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575279.bard.
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This program focused on citrus tristeza virus (CTV), the largest and one of the most complex RNA-plant-viruses. The economic importance of this virus to the US and Israeli citrus industries, its uniqueness among RNA viruses and the possibility to tame the virus and eventually turn it into a useful tool for the protection and genetic improvement of citrus trees justify these continued efforts. Although the overall goal of this project was to study the role(s) of CTV associated defective (d)-RNAs in CTV-induced diseases, considerable research efforts had to be devoted to the engineering of the helper virus which provides the machinery to allow dRNA replication. Considerable progress was made through three main lines of complementary studies. For the first time, the generation of an engineered CTV genetic system that is capable of infecting citrus plants with in vitro modified virus was achieved. Considering that this RNA virus consists of a 20 kb genome, much larger than any other previously developed similar genetic system, completing this goal was an extremely difficult task that was accomplished by the effective collaboration and complementarity of both partners. Other full-length genomic CTV isolates were sequenced and populations examined, resulting in a new level of understanding of population complexities and dynamics in the US and Israel. In addition, this project has now considerably advanced our understanding and ability to manipulate dRNAs, a new class of genetic elements of closteroviruses, which were first found in the Israeli VT isolate and later shown to be omnipresent in CTV populations. We have characterized additional natural dRNAs and have shown that production of subgenomic mRNAs can be involved in the generation of dRNAs. We have molecularly cloned natural dRNAs and directly inoculated citrus plants with 35S-cDNA constructs and have shown that specific dRNAs are correlated with specific disease symptoms. Systems to examine dRNA replication in protoplasts were developed and the requirements for dRNA replication were defined. Several artificial dRNAs that replicate efficiently with a helper virus were created from infectious full-genomic cDNAs. Elements that allow the specific replication of dRNAs by heterologous helper viruses also were defined. The T36-derived dRNAs were replicated efficiently by a range of different wild CTV isolates and hybrid dRNAs with heterologous termini are efficiently replicated with T36 as helper. In addition we found: 1) All CTV genes except of the p6 gene product from the conserved signature block of the Closteroviridae are obligate for assembly, infectivity, and serial protoplast passage; 2) The p20 protein is a major component of the amorphous inclusion bodies of infected cells; and 3) Novel 5'-Co-terminal RNAs in CTV infected cells were characterized. These results have considerably advanced our basic understanding of the molecular biology of CTV and CTV-dRNAs and form the platform for the future manipulation of this complicated virus. As a result of these developments, the way is now open to turn constructs of this viral plant pathogen into new tools for protecting citrus against severe CTV terms and development of virus-based expression vectors for other citrus improvement needs. In conclusion, this research program has accomplished two main interconnected missions, the collection of basic information on the molecular and biological characteristics of the virus and its associated dRNAs toward development of management strategies against severe diseases caused by the virus and building of novel research tools to improve citrus varieties. Reaching these goals will allow us to advance this project to a new phase of turning the virus from a pathogen to an ally.
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Knowles, Donald, und Monica Leszkowicz Mazuz. Transfected Babesia bovis expressing the anti-tick Bm86 antigen as a vaccine to limit tick infestation and protect against virulent challenge. United States Department of Agriculture, Januar 2014. http://dx.doi.org/10.32747/2014.7598160.bard.
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Bovine babesiosis, caused by the apicomplexan parasites Babesiabovisand B. bigemina, is a major tick borne disease of cattle with significant economic importance globally. The vectors of Babesia parasites are R. (Boophilus) annulatusand R. microplus. In Israel these parasites are transmitted manly by R. annulatus. The main goal of the proposal was developing and testing a novel B. bovisvaccine based on stably transfected attenuated B. bovisexpressing the anti-tick Bm86 antigen. This required generating a transfected- attenuated B. bovisparasite containing a bidirectional promoter expressing both, the gfp- bsd selectable marker and the tick vaccine antigen Bm86. The vaccine was tested for its ability to elicit protective immune responses against T. annulatusticks. Efficient control of babesiosis is based on a complex scheme of integrated management, including preventive immunization, anti-babesial chemotherapy and control of tick populations. Live vaccines based on attenuated parasites are the most effective measure to control babesiosis, and are currently used in several countries, including Israel. Live attenuated parasites lead to a chronic infection and development of strong and long term immunity in vaccinated cattle. Still, live vaccines have several limitations, including the difficulty to distinguish among vaccinated and naturally infected cattle and potential for sporadic outbreaks in vaccinated animals. Tick limitation is essential to control babesiosis but the main measure to reduce tick infestation is traditionally approached using acaricides, which is limited by environmental concerns and the development of resistance by the ticks. Alternative tick-control measures including the use of anti-tick vaccines are emerging, and at least partial protective immunity has been achieved against tick vectors by vaccination with recombinant protective tick antigens (ie: Bm86). In addition, the Babesia vaccine development toolbox has been recently expanded with the development of transfection technology in Babesia parasites. In this approved proposal we successfully developed a Babesia live attenuated transfected vaccine, which is able to express a B. bovisMSA-1 signal-Bm86 chimera and eGFP genes under the control of the B. bovisef- 1 and actin promoters respectively. Genetic analysis demonstrated specific stable integration of the transfected genes in the expected ef-1 locus, and immunofluorescence analysis confirmed expression of Bm86 in the surface of transfected parasites. When applied to splenectomized calves, the transfected parasites were able to cause persistent B. bovisinfection with production of antibodies reactive with Bm86 for at least six months. In addition, partial protection against ticks was also observed upon challenging the vaccinated animals with R. annulatuslarvae. However, when used on intact calves, the vaccine failed to elicit detectable immune responses against Bm86, and we are still in the process of interpreting the data and make necessary changes in our experimental approaches. Overall, the results obtained here represent a step forward towards the development of integrated vaccines against both ticks and tick –borne pathogens, using the Babesia attenuated parasites as a platform to the delivery of exogenous protective antigens