Browsing by Subject "VECTORS"

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  • 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.
  • 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.
  • 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.