Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

The overall goal of this procedure is to measure proteasome activity in living cells using A GFP Deron Fusion Protein first clone the custom oligonucleotides for Deron and Deron frameshift. Enter the P-E-G-F-P-C one vector and continue with the protocols for the gateway system in Vitrogen, then produce viruses for each construct using HEK 2 93 FT cells. After determining the titer transduced cells with the virus and apply antibiotic selection, then expand the resulting stable GFP DRON or GFP Dron frameshift expressing cells.

This technique facilitates experiments that monitor cell responses to given treatments through fluorescence signal measurements. For instance, Proteasome activity can be easily assessed using epi fluorescence or flow cytometry. The main advantage of this technique over existing methods like lumino or fluoro genic assays using whole cell is acids, is that the proteasome activity is measured in living cells For the transfection dilute 36 microliters of lipo 2000 reagent in 1.5 Milliliters of DMEM in another 15 milliliter falcon tube dilute the DNA constructs in 1.5 milliliters of DMEM.

After five minutes, combine the two and incubate for 20 minutes at room Temperature. Now replace the medium from a confluent culture of HEK 2 93 FT cells in a T 75 flask with seven milliliters of DMEM. Then add the DNA lipo mixture to the flask and mix by rocking it back and Forth.

Incubate the flask overnight at 37 degrees Celsius in a ified 5%CO2 incubator. The next day replace The medium with 10 milliliters DMEM. After 48 hours, harvest the supernatant Filter the virus containing supernatant through an oh 0.45 micrometer PVDF filter.

To Concentrate the virus, add one volume PEG solution to four volumes of supernatant and incubate for two hours at four degrees Celsius with periodic mixing by inversion. Harvest the virus by centrifugation after Aspiring the SNA of the white pellet centrifuge again to remove any remaining PEG solution. Resuspend the final pellet in PBS by Peppe, then vigorously vortex for 20 to 30 seconds.

Store 100 microliter aliquots of virus at minus 80 degrees centigrade Seed U2 OS cells in six well plates and incubate overnight. For transfection, replace the culture medium with one milliliter of DMEM containing eight micrograms per milliliter. Poly brain set aside one well as positive control and test a series of viral concentrations in remaining wells.

Incubate overnight the following day. Replace the medium with two milliliters of DMEM and incubate for 24 hours. Begin selection with 10 micrograms per milliliter blastic in, and continue to replace the antibiotic containing medium every second Day.

Approximately six to Seven days later, verify cell death of the un transduced. Then wash the cells three times with PBS to stain the cells. Cover with crystal violet and incubate for five to 10 minutes at room temperature, then aspire the dye, rinse the weld's twice with double distilled water and air dry the plate.

Finally, count the colonies to calculate transfection units of the virus per milliliter Seed. Human diplo fibroblasts on six Well plates with eight micrograms per milliliter. Poly brain as transduction enhancer.

Use a multiplicity of infection of two in a total of one milliliter. Change medium. The next Day when the culture is 70 to 80%confluent.

Begin chronic selection with 10 micrograms per milliliter. Blast aside in Over days, monitor the completed cell Death indicating selection is complete. Expand the stably transduced cells to generate a stable cell line expressing GFP DERON or GFP Deron Frameshift fusion Proteins Start by seeding cells carrying either GFP DRON or GFP DRON frameshift the next day, three hours prior to facts.

Treat control cells with a protosome inhibitor. Wash twice with PBS and harvest the cells using O 0.5 milliliters of trypsin. To Stop the trypsin.

Use 4.5 milliliters of culture medium and transfer the cells to a 15 milliliter Falcon. Harvest cells by centrifugation After one wash with PBS resuspend, the pellet in 400 microliter of fax buffer. Transfer the cells into fax tubes and place on ice.

Now measure samples by flow cytometry. The GFP Deron fusion protein carries a sequence which is targeted to the proteasome for immediate degradation. By contrast, the frameshift mutant GFP Deron FS is not degraded by proteasomes.

Hence, when transduced with GFP Deron, young human deployed fibroblasts with expected high proteasomal activity show a low fluorescent signal under epi fluorescence. The same cells transduced with GFP Deron Frameshift display 39.7%of positive cells. Treatment of the cells with proteasome inhibitor ll and L results in increased signals to a maximum of approximately 63%in both GFP dron and GFP Dron frameshift cells.

Similar measurements in dermal fibroblasts, isolated from young, middle aged, and old donors reveal a clear cut increase in GFP signal between young individuals and middle aged donors, indicating a decrease in protosome activity in samples obtained from aged donors. After watching this video, you should have a good understanding of how to produce lentiviral particles, transfect cells, and determine proteasome activity in living cells.