Browsing by Subject "VECTORS"

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  • Kopasz, Anna Georgina; Pusztai, David Zsolt; Karkas, Reka; Hudoba, Liza; Abdullah, Khaldoon Sadiq Ahmed; Imre, Gergely; Pankotai-Bodo, Gabriella; Migh, Ede; Nagy, Andrea; Kriston, Andras; German, Peter; Drubi, Andrea Bakne; Molnar, Anna; Fekete, Ildiko; Dani, Virag Eva; Ocsovszki, Imre; Puskas, Laszlo Geza; Horvath, Peter; Sukosd, Farkas; Mates, Lajos (2022)
    Background Understanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice. Results We exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model. Conclusions We developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.
  • Wang, Xin; Ye, Lingling; Lyu, Munan; Ursache, Robertas; Löytynoja, Ari; Mähönen, Ari Pekka (2020)
    Conditional manipulation of gene expression is a key approach to investigating the primary function of a gene in a biological process. While conditional and cell-type-specific overexpression systems exist for plants, there are currently no systems available to disable a gene completely and conditionally. Here, we present a new tool with which target genes can efficiently and conditionally be knocked out by genome editing at any developmental stage. Target genes can also be knocked out in a cell-type-specific manner. Our tool is easy to construct and will be particularly useful for studying genes having null alleles that are non-viable or show pleiotropic developmental defects.
  • Aivelo, Tuomas; Tschirren, Barbara (2020)
    Experimental field studies have demonstrated negative fitness consequences of Hen Flea Ceratophyllus gallinae infestations for bird hosts, yet it is currently unclear whether these negative effects are a direct consequence of flea-induced blood loss or a result of flea-borne pathogen transmission. Here we used a 16S rRNA gene sequencing approach to characterize the bacterial microbiota community of Hen Fleas collected from Great Tit Parus major nests and found that Brevibacterium (Actinobacteria), Staphylococcus (Firmicutes), Stenotrophomonas (Proteobacteria), Massilia (Proteobacteria), as well as the arthropod endosymbionts 'Candidatus Lariskella' and 'Candidatus Midichloria' were most abundant. We found evidence for the occurrence of Staphylococcus spp. in Hen Fleas, which may cause opportunistic infections in bird hosts, but not of other known pathogens commonly transmitted by other flea species, such as Bartonella spp. or Rickettsia spp. However, Hen Fleas might transmit other pathogens (e.g. viruses or bacteria that are not currently recognized as bird pathogens), which may contribute to the negative fitness consequences of Hen Flea infestations in addition to direct blood loss or secondary infections of wounds caused by biting fleas.
  • Ahonen, Marko T.; Diaconu, Iulia; Pesonen, Sari; Kanerva, Anna; Baumann, Marc; Parviainen, Suvi T.; Spiller, Brad; Cerullo, Vincenzo; Hemminki, Akseli (2010)
  • Badazhkova, Veronika D.; Raik, Sergei; Polyakov, Dmitry S.; Poshina, Daria N.; Skorik, Yury A. (2020)
    Recently, much effort has been expended on the development of non-viral gene delivery systems based on polyplexes of nucleic acids with various cationic polymers. Natural polysaccharide derivatives are promising carriers due to their low toxicity. In this work, chitosan was chemically modified by a reaction with 4-formyl-n,n,n-trimethylanilinium iodide and pyridoxal hydrochloride and subsequent reduction of the imine bond with NaBH4. This reaction yielded three novel derivatives, n-[4-(n',n',n'-trimethylammonium)benzyl]chitosan chloride (TMAB-CS), n-[(3-hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridine)methyl]chitosan chloride (Pyr-CS), and n-[4-(n',n',n''-trimethylammonium)benzyl]-n-[(3-hydroxy-5-(hydroxymethyl)-2-methyl-4-pyridine)methyl]chitosan chloride (PyrTMAB-CS). Their structures and degrees of substitution were established by H-1 NMR spectroscopy as DS1 = 0.22 for TMAB-CS, DS2 = 0.28 for Pyr-CS, and DS1 = 0.21, DS2 = 0.22 for PyrTMAB-CS. Dynamic light scattering measurements revealed that the new polymers formed stable polyplexes with plasmid DNA encoding the green fluorescent protein (pEGFP-N3) and that the particles had the smallest size (110-165 nm) when the polymer:DNA mass ratio was higher than 5:1. Transfection experiments carried out in the HEK293 cell line using the polymer:DNA polyplexes demonstrated that Pyr-CS was a rather poor transfection agent at polymer:DNA mass ratios less than 10:1, but it was still more effective than the TMAB-CS and PyrTMAB-CS derivatives that contained a quaternary ammonium group. By contrast, TMAB-CS and PyrTMAB-CS were substantially more effective than Pyr-CS at higher polymer:DNA mass ratios and showed a maximum efficiency at 200:1 (50%-70% transfected cells). Overall, the results show the possibility of combining substituent effects in a single carrier, thereby increasing its efficacy.
  • Heiniö, Camilla; Sorsa, Suvi; Siurala, Mikko; Grönberg-Vähä-Koskela, Susanna; Havunen, Riikka; Haavisto, Elina; Koski, Anniina; Hemminki, Otto; Zafar, Sadia; Cervera-Carrascon, Victor; Munaro, Eleonora; Kanerva, Anna; Hemminki, Akseli (2019)
    After the discovery and characterization of the adenovirus in the 1950s, this prevalent cause of the common cold and other usually mild diseases has been modified and utilized in biomedicine in several ways. To date, adenoviruses are the most frequently used vectors and therapeutic (e.g., oncolytic) agents with a number of beneficial features. They infect both dividing and nondividing cells, enable high-level, transient protein expression, and are easy to amplify to high concentrations. As an important and versatile research tool, it is of essence to understand the limits and advantages that genetic modification of adenovirus vectors may entail. Therefore, a retrospective analysis was performed of adenoviral gene therapy constructs produced in the same laboratory with similar methods. The aim was to assess the impact of various modifications on the physical and functional titer of the virus. It was found that genome size (designed within "the 105% golden rule") did not significantly affect the physical titer of the adenovirus preparations, regardless of the type of transgene (e.g., immunostimulatory vs. other), number of engineered changes, and size of the mutated virus genome. One statistically significant exception was noted, however. Chimeric adenoviruses (5/3) had a slightly lower physical titer compared to Ad5-based viruses, although a trend for the opposite was true for functional titers. Thus, 5/3 chimeric viruses may in fact be appealing from a safety versus efficacy viewpoint. Armed viruses had lower functional and physical titers than unarmed viruses, while five genomic modifications started to decrease functional titer. Importantly, even highly modified armed viruses generally had good titers compatible with clinical testing. In summary, this paper shows the plasticity of adenovirus for various vector, oncolytic, and armed oncolytic uses. These results inform future generations of adenovirus-based drugs for human use. This information is directly transferable to academic laboratories and the biomedical industry involved in vector design and production optimization.
  • Kiljunen, Saija; Pajunen, Maria I.; Dilks, Kieran; Storf, Stefanie; Pohlschroder, Mechthild; Savilahti, Harri (2014)
    Background: Archaea share fundamental properties with bacteria and eukaryotes. Yet, they also possess unique attributes, which largely remain poorly characterized. Haloferax volcanii is an aerobic, moderately halophilic archaeon that can be grown in defined media. It serves as an excellent archaeal model organism to study the molecular mechanisms of biological processes and cellular responses to changes in the environment. Studies on haloarchaea have been impeded by the lack of efficient genetic screens that would facilitate the identification of protein functions and respective metabolic pathways. Results: Here, we devised an insertion mutagenesis strategy that combined Mu in vitro DNA transposition and homologous-recombination-based gene targeting in H. volcanii. We generated an insertion mutant library, in which the clones contained a single genomic insertion. From the library, we isolated pigmentation-defective and auxotrophic mutants, and the respective insertions pinpointed a number of genes previously known to be involved in carotenoid and amino acid biosynthesis pathways, thus validating the performance of the methodologies used. We also identified mutants that had a transposon insertion in a gene encoding a protein of unknown or putative function, demonstrating that novel roles for non-annotated genes could be assigned. Conclusions: We have generated, for the first time, a random genomic insertion mutant library for a halophilic archaeon and used it for efficient gene discovery. The library will facilitate the identification of non-essential genes behind any specific biochemical pathway. It represents a significant step towards achieving a more complete understanding of the unique characteristics of halophilic archaea.
  • Liu, Yixin; Pavic, Ana; Farley, Joshua T.; Lousa, Carine De Marcos; Goldman, Adrian; Postis, Vincent L. G. (Humana, 2021)
    Methods in Molecular Biology
    A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.
  • Kuryk, Lukasz; Moller, Anne-Sophie W.; Vuolanto, Antti; Pesonen, Sari; Garofalo, Mariangela; Cerullo, Vincenzo; Jaderberg, Magnus (2019)
    Oncolytic adenoviruses can trigger lysis of tumor cells, induce an antitumor immune response, bypass classical chemotherapeutic resistance strategies of tumors, and provide opportunities for combination strategies. A major challenge is the development of scalable production methods for viral seed stocks and sufficient quantities of clinical grade viruses. Because of promising clinical signals in a compassionate use program (Advanced Therapy Access Program) which supported further development, we chose the oncolytic adenovirus ONCOS-401 as a testbed for a new approach to scale up. We found that the best viral production conditions in both T-175 flasks and HYPERFlasks included A549 cells grown to 220,000 cells/cm(2) (80% confluency), with ONCOS-401 infection at 30 multiplicity of infection (MOI), and an incubation period of 66 h. The Lysis A harvesting method with benzonase provided the highest viral yield from both T-175 and HYPERFlasks (10,887 +/- 100 and 14,559 +/- 802 infectious viral particles/cell, respectively). T-175 flasks and HYPERFlasks produced up to 2.1 x 10(9) +/- 0.2 and 1.75 x 10(9) +/- 0.08 infectious particles of ONCOS-401 per cm(2) of surface area, respectively. Our findings suggest a suitable stepwise process that can be applied to optimizing the initial production of other oncolytic viruses.
  • Vaha-Koskela, Markus; Tahtinen, Siri; Gronberg-Vaha-Koskela, Susanna; Taipale, Kristian; Saha, Dipongkor; Merisalo-Soikkeli, Maiju; Ahonen, Marko; Rouvinen-Lagerstrom, Noora; Hirvinen, Mari; Veckman, Ville; Matikainen, Sampsa; Zhao, Fang; Pakarinen, Paivi; Salo, Jarmo; Kanerva, Anna; Cerullo, Vincenzo; Hemminki, Akseli (2015)
    Successful cancer control relies on overcoming resistance to cell death and on activation of host antitumor immunity. Oncolytic viruses are particularly attractive in this regard, as they lyse infected tumor cells and trigger robust immune responses during the infection. However, repeated injections of the same virus promote antiviral rather than antitumor immunity and tumors may mount innate antiviral defenses to restrict oncolytic virus replication. In this article, we have explored if alternating the therapy virus could circumvent these problems. We demonstrate in two virus-resistant animal models a substantial delay in antiviral immune- and innate cellular response induction by alternating injections of two immunologically distinct oncolytic viruses, adenovirus, and vaccinia virus. Our results are in support of clinical development of heterologous adeno-/vaccinia virus therapy of cancer.
  • Zhu, Lingping; Pietiainen, Milla; Kontturi, Juha; Turkkelin, Anna; Elomaa, Paula; Teeri, Teemu H. (2022)
    Plant polyketides are well-known for their crucial functions in plants and their importance in the context of human health. They are synthesized by type III polyketide synthases (PKSs) and their final functional diversity is determined by post-PKS tailoring enzymes. Gerbera hybrida is rich in two defense-related polyketides: gerberin and parasorboside. Their synthesis is known to be initiated by GERBERA 2-PYRONE SYNTHASE 1 (G2PS1), but the polyketide reductases (PKRs) that determine their final structure have not yet been identified. We identified two PKR candidates in the pathway, GERBERA REDUCTASE 1 (GRED1) and GRED2. Gene expression and metabolite analysis of different gerbera tissues, cultivars, and transgenic gerbera plants, and in vitro enzyme assays, were performed for functional characterization of the enzymes. GRED1 and GRED2 catalyze the second reduction step in parasorboside biosynthesis. They reduce the proximal keto domain of the linear CoA bound intermediate before lactonization. We identified a crucial tailoring step in an important gerbera PKS pathway and show that plant polyketide biosynthesis shares processing strategies with fungi and bacteria. The two tailoring enzymes are recruited from the ancient sporopollenin biosynthetic pathway to a defense-related PKS pathway in gerbera. Our data provide an example of how plants recruit conserved genes to new functions in secondary metabolism that are important for environmental adaptation.
  • Uusitalo, Ruut Jaael; Siljander, Mika; Culverwell, Christine Lorna; Mutai, Noah; Forbes, Kristian Michael; Vapalahti, Olli; Pellikka, Petri Kauko Emil (2019)
    Mosquitoes are vectors for numerous pathogens, which are collectively responsible for millions of human deaths each year. As such, it is vital to be able to accurately predict their distributions, particularly in areas where species composition is unknown. Species distribution modeling was used to determine the relationship between environmental, anthropogenic and distance factors on the occurrence of two mosquito genera, Culex Linnaeus and Stegomyia Theobald (syn. Aedes), in the Taita Hills, southeastern Kenya. This study aims to test whether any of the statistical prediction models produced by the Biomod2 package in R can reliably estimate the distributions of mosquitoes in these genera in the Taita Hills; and to examine which factors best explain their presence. Mosquito collections were acquired from 122 locations between January–March 2016 along transects throughout the Taita Hills. Environmental-, anthropogenic- and distance-based geospatial data were acquired from the Taita Hills geo-database, satellite- and aerial imagery and processed in GIS software. The Biomod2 package in R, intended for ensemble forecasting of species distributions, was used to generate predictive models. Slope, human population density, normalized difference vegetation index, distance to roads and elevation best estimated Culex distributions by a generalized additive model with an area under the curve (AUC) value of 0.791. Mean radiation, human population density, normalized difference vegetation index, distance to roads and mean temperature resulted in the highest AUC (0.708) value in a random forest model for Stegomyia distributions. We conclude that in the process towards more detailed species-level maps, with our study results, general assumptions can be made about the distribution areas of Culex and Stegomyia mosquitoes in the Taita Hills and the factors which influence their distribution.
  • Zhu, Lingping; Zhang, Teng; Teeri, Teemu H. (2021)
    The structurally robust biopolymer sporopollenin is the major constituent of the exine layer of pollen wall and plays a vital role in plant reproductive success. The sporopollenin precursors are synthesized through an ancient polyketide biosynthetic pathway consisting of a series of anther-specific enzymes that are widely present in all land plant lineages. Tetraketide alpha-pyrone reductase 1 (TKPR1) and TKPR2 are two reductases catalyzing the final reduction of the carbonyl group of the polyketide synthase-synthesized tetraketide intermediates to hydroxylated alpha-pyrone compounds, important precursors of sporopollenin. In contrast to the functional conservation of many sporopollenin biosynthesis associated genes confirmed in diverse plant species, TKPR2's role has been addressed only in Arabidopsis, where it plays a minor role in sporopollenin biosynthesis. We identified in gerbera two non-anther-specific orthologues of AtTKPR2, Gerbera reductase 1 (GRED1) and GRED2. Their dramatically expanded expression pattern implies involvement in pathways outside of the sporopollenin pathway. In this study, we show that GRED1 and GRED2 are still involved in sporopollenin biosynthesis with a similar secondary role as AtTKPR2 in Arabidopsis. We further show that this secondary role does not relate to the promoter of the gene, AtTKPR2 cannot rescue pollen development in Arabidopsis even when controlled by the AtTKPR1 promoter. We also identified the gerbera orthologue of AtTKPR1, GTKPR1, and characterized its crucial role in gerbera pollen development. GTKPR1 is the predominant TKPR in gerbera pollen wall formation, in contrast to the minor roles GRED1 and GRED2. GTKPR1 is in fact an excellent target for engineering male-sterile gerbera cultivars in horticultural plant breeding.