Browsing by Subject "Sf9"

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  • Sjöstedt, Noora; Salminen, Tiina A.; Kidron, Heidi (2019)
    Transport proteins of the ATP-binding cassette (ABC) family are found in all kingdoms of life. In humans, several ABC efflux transporters play a role in drug disposition and excretion. Therefore, in vitro methods have been developed to characterize the substrate and inhibitor properties of drugs with respect to these transporters. In the vesicular transport assay, transport is studied using inverted membrane vesicles produced from transporter overexpressing cell lines of both mammalian and insect origin. Insect cell expression systems benefit from a higher expression compared to background, but are not as well characterized as their mammalian counterparts regarding endogenous transport. Therefore, the contribution of this transport in the assay might be underappreciated. In this study, endogenous transport in membrane vesicles from Spodoptera frugiperda-derived Sf9 cells was characterized using four typical substrates of human ABC transporters: 5(6)-carboxy-2,' 7'-dichlorofluorescein (CDCF), estradiol-17 beta-glucuronide, estrone sulfate and N-methyl-quinidine. Significant ATP-dependent transport was observed for three of the substrates with cholesterol-loading of the vesicles, which is sometimes used to improve the activity of human transporters expressed in Sf9 cells. The highest effect of cholesterol was on CDCF transport, and this transport in the cholesterol-loaded Sf9 vesicles was time and concentration dependent with a Km of 8.06 +/- 1.11 mu M. The observed CDCF transport was inhibited by known inhibitors of human ABCC transporters, but not by ABCB1 and ABCG2 inhibitors verapamil and Ko143, respectively. Two candidate genes for ABCC-type transporters in the S. frugiperda genome (SfABCC2 and SfABCC3) were identified based on sequence analysis as a hypothesis to explain the observed endogenous ABCC-type transport in Sf9 vesicles. Although further studies are needed to verify the role of SfABCC2 and SfABCC3 in Sf9 vesicles, the findings of this study highlight the need to carefully characterize background transport in Sf9 derived membrane vesicles to avoid false positive substrate findings for human ABC transporters studied with this overexpression system.
  • Kaugonen, Olga (Helsingfors universitet, 2017)
    Investigating the role of cell membrane proteins has increased over the last decade, as drugdrug interactions and genetic polymorphisms have been found to cause changes in drug pharmacokinetics and dynamics. In this study the characteristics of the OATP1B1 transporter were reviewed and new in vitro research method to study protein functions was developed. Human Embryonic Kidney cells (HEK) is a human derived mammalian cell-line that is widely used in the study of OATP1B1 transporter. The Sf9 cell line is isolated from Spodoptera frugiperda insect and is one of the standard in vitro tools in a genetic engineering study. In the experimental part of this thesis the goal was to express OATP1B1 transporter in Sf9 and HEK293 cell lines. The wild-type SLCO1B1-gene encoding the OATP1B1 was virulent with baculovirus into the cells by the Bac-to-Bac® Baculovirus Expression System. For expression in the Sf9 cells, the aim of the study was to clone the SLCO1B1-gene into the pFastBac vector. The cloning was not successful in this study although attempts were made for several approaches. The expression of OATP1B1 transporter in HEK293 cells was successful. HEK293 cells expressing OATP1B1 transporter are well suited for the study of the SLCO1B1-gene. The in vitro method developed in this study remains in the research team as a tool to investigate the polymorphisms of the SLCO1B1-gene, the inhibition of the transporter and possible drug interactions.
  • Järvinen, Valtteri (Helsingin yliopisto, 2015)
    Great concern is to be addressed to safety measures in order to guarantee work safety when studying novel, possibly pandemic influenza A viruses. These safety measures slow down the research process and their upkeep is expensive. To overcome these hindrances a virus-like-particle (VLP) can be used as a model to replace the need for a live virus. Because VLPs are non-infectious, they are suitable for being used in research where experiments are done with slighter safety precautions. In addition, VLPs are usually highly immunogenic and thus influenza A VLP may function as a model system for further vaccine development. This research was done in a research group, in which a VLP had previously been made with cloning the genes of hemagglutinin (HA), neuraminidase (NA) and matrix 1 (M1) proteins from the year 2009 pandemic Influenza A virus (H1N1)v to a single baculovirus protein expression vector. During this earlier research project it was found problematic that the expression level of a particular gene could not be controlled. In this research, HA, NA and M1 genes were cloned to different baculovirus protein expression vectors so that the expression level of individual genes could be enhanzed with plaque purification and the multiplicity of infection (MOI) adjusted individually for each vector. It was hypothesized that an optimal configuration of MOI rates between vectors could be found in order to maximise VLP production in Spodoptera frugiperda 9 (Sf9) cells. Baculovirus protein expression vectors were made via traditional cloning of the HA, NA and M1 genes into three pAcYM1 baculovirus transfer vectors under polyhedrin promoter, which has been shown to be a strong promoter. Transfer vectors were used to transfer the genes into linearised baculovirus’ genomes by homologous recombination and the genomes were transfected into Sf9 cells to produce recombinant baculoviruses. These expression vectors were plaque-purified and their titers were amplified. Their expression level was studied using SDS-PAGE and coomassie blue analysis and with metabolic labeling using [S35]-labeled methionine. The formation of VLPs was measured with hemagglutination assay when Sf9 cells were co-infected with all three protein expression vectors. It was found that changing expression vectors MOI between 1, 3 and 5 did not have a great impact on protein expression from individual vectors. The presence of NA protein was found to be necessary for the formation of influenza A virus VLPs with a detectable hemagglutination activity. Differences between VLP formations were obtained when MOI rate compositions were changed, but further study is needed to find the significance of this result. The research to find an optimal configuration of MOI rates between vectors is still to be continued.