Ljubljana/methods

In addition to basic parts, we also designed dozens of composite parts, mainly constructs expressing chimeric proteins. Composite parts were assembled by three-point ligations <font color="red">(REFERENCA?)</font>, leaving a 6 nucleotides long <i>Spe</i>I-<i>Xba</i>I mixed restriction site in-between the two domains. Some of our composite parts contain as many as 6 basic parts. Such composite parts were constructed by repeating three point ligations.

       <p class="p1">In our experiments two different cell lines were used: human embryonic kidney cell line HEK293T and human cervical cancer cell line HeLa. Cells were grown in DMEM medium containing 10% FBS and antibiotics streptomycin and penicillin, except during transfection when antibiotics were absent from the medium. For luciferase assays, cells were typically seeded in 96-well plates, for microscopy in slide chambers, whereas for Western blotting and cytometry, where larger numbers of cells were needed, they were grown in 6-well plates, 35 mm dishes or even 10 cm² dishes. Depending on the type of experiment Lipofectamine 2000 or GeneJuice were used for transfection according to manufacturer´s protocol. Cells were transfected with the desired plasmids, stimulated if necessary and left to grow before detection.</p><br>

       <p class="p1">Bioluminescence is emission of light produced by an enzymatic reaction. In molecular biology, genes coding for enzymes which catalyze reactions where light is emitted are used as reporter genes. The most widely used reporter enzymes are luciferases.<br>

In our experiments we used firefly luciferase placed under a specific inducible promoter of interest. The amount of synthesized luciferase was therefore dependent on the activation rate of cells and was semiquantified by measuring emitted light after substrate cleavage. As the number of cells and transfection efficiency vary, the results were normalized by a second reporter luciferase (the <i>Renilla</i> luciferase) placed under a constitutive promoter (expression of which is therefore cell activation-independent) and with specificity for a different substrate. The measurements were conducted directly on 96-well plates in a luminometer.<br>

The luciferase assay proved to be our most valuable tool due to the extremely low detection limits. We used this assay to test both our split protein and HIV-protease systems. Cells were transfected with CD4 and CCR5 receptors fused with components of split ubiquitin or split TEV protease systems and reporter constructs. T7 promoter-luciferase reporter construct was used to measure the cellular response to viral attack. HIV surface protein g120 induces cleavage of T7 RNA polymerase fused with a transmembrane segment and its translocation to the nucleus, where it starts to transcribe the reporter gene. </p><br>

       <p class="p1">Western blotting is a method of protein transfer from SDS-PAGE (SDS-polyacrylamide gel electrophoresis) gels to a nitrocellulose membrane, which enables detection of proteins of interest in cell lysates using specific primary antibodies (pAb) and enzyme-conjugated secondary antibodies (sAb), which bind to pAb. In the first step, transfected cells grown in cell cultures were lysed and the insoluble membrane fraction was removed by centrifugation. Loading buffer containing SDS and a reducing agent was then added to the cleared lysate and the samples were denatured by boiling. Next, samples were applied to PAGE, where proteins were separated according to their molecular weight. Separated proteins were then transferred to a nitrocellulose membrane by electrical current. The efficiency of the transfer was determined either by staining the gel with Ponceau dye or by using the pre-stained molecular weight markers, which are visible on the membrane after the transfer. After blotting, the free binding sites on the membrane were blocked by 5% non-fat dry milk in order to prevent unspecific binding of antibodies to the membrane. Then, the nitrocellulose membrane was incubated with pAb for 1 h at room temperature or overnight at 4^oC with gentle shaking. The membrane was then washed to remove the unbound pAb and incubated with horseradish peroxidase-labelled sAb for 1 h at room temperature with gentle shaking. The signal was finally detected on the film by using a chemiluminescent substrate.<br>

Western blotting proved to be a very useful technique for determining whether proteolytic cleavage has occured. In the split ubiquitin assay, CMV-CD4-CUb-GFP and CMV-CCR5-NUb constructs were used. By comparison to molecular weight standards, we could detect whether the GFP was cut off  from the transmembrane segment or remained attached to it when using anti-GFP antibodies. This method was also used in the HIV-protease project; the construct CMV-CD4-cleavage site-GFP was used and again GFPs of different sizes were detected when cotransfection with plasmid expressing HIV protease was performed.</p><br>

       <p class="p1">The basis of fluorescence as a technique is excitation of a fluorophore, which then emits light of a specific wavelength. The main feature of this technique is its very high sensitivity. Confocal microscopy enables three-dimensional detection and allocation of flourescent molecules inside the cells. This technique was used to determine the location of CD4 fused to mCerulean or mCherry inside the cells. Additionally, microscopy of cells after transfection with CD4 and CCR5 receptor constructs fused with split protein systems or HIV-protease cleavage site was used to show that after cell activation (with gp120, pseudoviruses or HIV protease) cleavage of mCerulean or mCherry from the receptor occured. Thus, we can expect that the same occurs when T7 polymerase is fused instead of mCerulean or mCherry.<br>  

In our experiments, cell membranes were stained with SynaptoRed C2 (5 mM) for 5 minutes at 25 μM concentration, endoplasmic reticula with ER-Tracker Red (1 mM) for 10 minutes at 2,5 μM and lysosomes with LysoTracker Green (1 mM) for 5 minutes at 1 μM. Cells were viewed using a confocal microscope with an oil-immersion objective. The 458, 488, 514 (all multi-line Argon laser) and 543 (He-Ne laser) wavelengths were used to excite mCerulean, LysoTracker Green, SynaptoRed C2 and mCherry (or ER-Tracker Red), respectively. </p><br>

       <p class="p1">Flow cytometry is a method which allows monitoring of cells in a flow stream on the basis of light scattering and fluorescence on a single cell level. This method allows measurment of more parameters in a time and determination of a fraction cells with a specific characteristic. There is the possibility of a wide range of applications, however, we used two: annexin V and 7-AAD staining for detection of apoptotic cells and antibody staining for detection of surface expression of transmembrane proteins.<br>

Apoptotic cells are characterized by certain morphological changes, one of them being translocation of phosphatidylserine (PS) from the inner to the outer layer of plasma membrane bilayer. This change occurs early in the process. Annexin V is a protein, which binds specifically to PS. As this translocation of PS occurs early in the process, Annexin V is a sensitive probe to detect early apoptotic cells. Annexin V can be conjugated to a fluorochrome and thus be detected by means of flow cytometry.<br>  

Antibody staining was used for detection of transmembrane receptors CD4, CCR5 and CXCR4 on cell surfaces. For this purpose composite parts with receptors and Myc or HA tags which flutter on the cell surface were designed. After transfection with plasmids expressing these receptors, cells were incubated with primary anti-tag antibodies, and then with secondary antibodies, which bind to constant, species-specific region of primary antibodies. Secondary antibodies were conjugated with fluorophores (PE or DyeMer), which emit light at 575 nm and 610 nm, respectively. While using  specific anti-Myc or anti-HA tag antibodies only cells transfected with our receptor constructs and not endogenous receptors were detected. </p><br>

       <p class="p1">ELISA (enzyme-linked immunosorbent assay) is an immunological technique which enables semiquantitative determination of the amount of molecules of interest in the cell lysate using specific antibodies. Antibodies are bound to the surface of a microtiter plate and molecules of interest present in the cell lysate bind to them. The amount of bound molecules is detected using another primary antibody conjugated with biotin. Streptavidin conjugated with horseradish peroxidase specifically interacts with biotin. Peroxidase enzymatically cleaves a chromogenic substrate and the absorbance is measured. ELISA was used to determine the amount of caspase-3 protein after the activation of the engineered system with HIV protease, gp120 or pseudoviruses.</p><br>