The overall goal of the following experiment is to dissect key host innate immune molecules regulating antiviral cytokine production upon gamma herpes virus 68 or gamma HV 68 injection. This is achieved by comparing antiviral cytokines, secreted by gamma HV 68 injected Mavs wild type and knockout mice. The second step is to quantify cytokines produced by Mavs Wild type and Mavs knockout MEF cells after gamma HV 68 infection, which confirms Mavs is utilized to suppress antiviral cytokine secretion.
The next step is to restore Mavs and Mavs knockout MEF cells in order to assess whether cytokine expression levels are rescued by exogenous Mavs. Results are obtained that show Gamma HV 68 hijacks Mavs, a key innate immune signaling component to abrogate antiviral cytokine production in vivo and ex vivo based on western blotting analysis. Eliza and Q-R-T-P-C-R in Mavs Wild type and knockout mice and corresponding MEF cells.
This measure can help to answer the key questions in the innate field, such as host innate immune response upon gum hepworth infection demonstrating the procedure will be leaning to a postdoc in Dr.P'S lab. Intranasally inoculate the anesthetized C 57 black six mice with 40 microliters of 40 plaque forming units of the natural mirroring pathogen gamma herpes virus 68. After sacrificing the mice at experimental time points post-infection, open the chest with a left lateral cut along the sternum, cut through the ribs with sharp scissors and collect the lung tissues.
Place the left lobe into a sterile 1.5 milliliter screw caps tube containing 500 microliters of 1.0 millimeters. Zirconia silica beads. Add one milliliter of cold serum free DMEM and homogenize the lung tissue by bead beading for 30 seconds.
Chill the tubes on ice for two minutes and repeat the bead beading process. Once swans, then centrifuge the tubes at four degrees Celsius and collect the supernatant. Now measure the desired cytokines using commercially available cytokine ELIZA kits according to the manufacturer's instructions.
Next, place the right lobe of the lung tissue into another bead containing tube and add one milliliter of triol homogenize and collect the supernat as described earlier. Then extract the RNA according to the manufacturer's instructions. To determine the RNA concentration measure absorbance readings at 260 and 280 nanometers.
Next, perform a reverse transcription reaction with one microgram of total RNA templates in a total final volume of 20 microliters. Now for the quantitative real-time PCR dilute the resulting CD NA 50 times with DNAs and RNAs free water for each reaction at 0.5 microliters of a pair of gene specific or control primers. Five microliters of the cyber master mix and four microliters of the diluted CD NA.Calculate the threshold cycles to determine the relative quantity of mRNA.
Transcripts of select antiviral cytokines grow the wild type and knockout mouse embryonic fibroblasts to 80%co fluency. Split the MEF cells into 12 well plates at 100, 000 cells per well to synchronize and maximize cytokine induction. Prepare a working stock of gamma herpes virus for infecting the cells in triplicate with a multiplicity of infection of five to 10.
Now remove the medium and add gamma HV 68 containing suspension to the MEF cells. Place the plates in a tissue culture incubator rocking every 30 minutes over the course of a two hour incubation. Next, remove the gamma HV 68 containing medium.
Wash the cells once with PBS and then add fresh complete DMEM at various time points. Post-infection, harvest a medium into 1.5 milliliter einor tubes. Wash the cells with cold PBS and harvest the infected cells by trypsin digestion.
Measure the antiviral cytokines in medium by Eliza as shown earlier. Also extract RNA, prepare CDNA and perform Q-R-T-P-C-R to determine the antiviral cytokine gene expression profile as shown earlier. Split confluent 2 9 3 T cells one day before transfection and allow the cells to reach 40%co fluency at the time of transfection.
Transfect the 2 9 3 T cells with the plasmids using the calcium phosphate precipitation method. After six to eight hours post transfection, replace the media with fresh complete DMEM at 72 hours post transfection. Collect the medium containing the lentivirus centrifuge at 4, 000 GS for 15 minutes and filter the super names through a 0.22 micrometer membrane.
Then centrifuge the containing medium at 110, 000 Gs for two and a half hours at four degrees Celsius. Carefully resuspend the viral pellets in a small volume of the medium of interest. Next, infect knockout MEF cells with one milliliter of lentivirus and two milliliters of fresh complete DMEM supplemented with poly brain centrifuge.
The plates at 500 GS at 30 degrees Celsius for 30 minutes. Then transfer the cells to a tissue culture incubator at six hours post infection. Replace the medium with fresh complete DMEM, split the infected MEF cells at 24 hours post infection and at 48 hours post infection, add pur mycin to a final concentration of one microgram per milliliter to select for cells that stably express the viral construct.
Maintain the MEF cells in pur mycin containing media and verify the protein expression by immuno blotting with corresponding antibodies. These cells can now be used for viral infection, cytokine gene expression, and secretion experiments. As shown earlier, the MAVS gene codes for a mitochondrial antiviral signaling protein that plays a key role in viral immunity.
These results show that gamma HV 68 infection of Mavs knockout mice leads to significantly higher production of CCL five in vivo. MA embryonic fibroblasts were then used to investigate the mechanism of regulated cytokine production. Ex vivo.
Here the Mavs knockout cells produced more antiviral cytokine than wild type during gamma HV 68 infection, which recapitulates the en vivo phenotype, reconstituted expression of Mavs and Mavs knockout MEFs decreased CCL five production. Collectively, these EISA and quantitative PCR results demonstrate that Mavs is necessary for gamma HV 68 to suppress antiviral cytokine production, both in vivo and ex vivo After its development. This technique paved the way for researchers in the field of innate immunity to explore host antiviral response in transgenic mice infected by herpes virus.
