Ljubljana/subsystems
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Subsystems
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Surface expression of transmembrane receptors CD4, CCR5 and CXCR4
Surface expression of transmembrane receptors was measured by flow cytometry (link na metode, ki jih je opisal Andrej – tam piše tudi o postopku).
We have confirmed expression of the following versions of transmembrane receptors at the surface of cell membrane: CD4(HA)-CUb-GFP, CCR5(c-myc)-NUb and CXCR4(c-myc)-NUb (Fig. 1). Additional constructs with transmembrane receptors were also tested and found to be located at the cell membrane. Expression of transmembrane receptors at the cell surface was crucial for our split systems since it shows appropriate protein folding, thus suggesting that receptors are also functional. Both split-ubiquitin and split-TEV system are activated by heterodimerization of transmembrane receptors after their interaction with viral particles.
The following experiments were done only with CD4 and CCR5 constructs (used by macrophage-tropic viruses). CXCR4 constructs represent receptors for another group of HIV (lymphotropic or dual-tropic viruses) that utilize CXCR4 receptors for entry into cells. Thus we can expand the range of usefulness of our idea to other viral strains.
Results
Fig. 1. Transmembrane receptor constructs CD4, CCR5 and CXCR4 are expressed at the cell surface. Transmembrane receptors were tagged with peptide tags, which allow detection with labeled antibodies. All versions of CD4 were tagged with HA-tag, CXCR4 and CCR5 were tagged with c-myc-tag at the extracellular side of the receptors. Cells were transfected with different receptor constructs and incubated. Surface expressed receptors were detected by using anti-HA (mouse) and anti-c-myc (rabbit) antibodies, followed by fluorescently labelled secondary antibodies (anti-mouse-PE and anti-rabbit-DyeMer). Receptors were detected by flow cytometry. Fluorophores were excited with 488 argon laser and emitted light was detected at 575 and 610 nm, respectively. 10000 events were collected and results are presented as mean fluorescence values at defined emission wavelenght. In comparison with non-transfected cells we can see that our transmembrane receptors are expressed at the cell surface.
T7 promoter (pT7)
T7 promoter is one of the most important aspects of our system since all our reporter and effector proteins are put under its control. Split-ubiquitin, split TEV and HIV protease systems all release T7 RNA polymerase after viral activated heterodimerization of receptors or HIV protease cutting at specific sequence. T7 RNA polymerase with NLS (nuclear localisation sequence) is translocated into the nucleus. T7 RNA polymerase transcribes genes that are controlled by the T7 promoter (reporter, effector and self-amplifying genes).
We wanted to show that:
Results
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