The overall goal of this procedure is to incorporate gag genes derived from subtype CHIV one infected individuals into a replication competent HIV one plasmid. In order to assess the effect of gag sequence polymorphisms on HIV one replication capacity in vitro, this is accomplished by first amplifying the gag gene from the patient's plasma. The second step is to clone the patient derived gag sequence into the replication competent infectious molecular clone MJ four.
Next, the gag MJ four chimera plasmid is transfected in order to generate infectious viral stocks and titered to assess infectivity on an indicator cell line. The final step is to measure in vitro replication capacity in the G XR 25 T-cell line. Ultimately, a radio labeled reverse transcriptase assay is used to quantify virion production over time in order to visualize viral replication kinetics.
The method we're going to describe today can help answer key questions in the HIV field. These include defining the determinants of HIV replicative fitness and understanding how HIV fitness affects long-term pathogenesis. Describing the methods today will be Jessica Prince and Dan Klaban, two of my graduate students.
To begin this procedure, reverse transcribe CDNA from RNA and amplify the first round DNA products using reverse transcriptase and a thermo stable DNA polymerase in a one step RT PCR. Then using one microliter of the first round PCR amplification as the DNA template, perform a nested second round PCR amplification. Next, add three microliters of five x loading dye to five microliters of the second round reaction volume.
Run it at 120 volts on a 1%aros, TAE gel containing a UV fluorescent DNA stain until the bands are resolved. Then visualize the 1.6 KB bands on a blue light illuminator. After that, run the remaining 45 microliter reaction volume on a 1%aros, TAE gel containing the DNA stain.
Excise the appropriate bands with a clean razor blade and combine three positive reactions per individual. Subsequently extract DNA from the gel slice using a gel purification kit and ute it in the nuclease free water. Freeze the product at minus 20 degrees Celsius for use later in this procedure.
After amplifying the long terminal repeat five prime UTR region of the MJ four plasmid. Visualize the 1.3 KB PCR products on the illuminator, excise the positive amplicons, then purify and freeze them as previously described. Next, create the fused MJ four LTR gag amplicons via splice overlap extension PCR.
Visualize, excise, purify and freeze the 3.2 kb long amplicon. Now digest 1.5 micrograms of MJ four plasmid and 1.5 micrograms of purified LTR gag PCR product with BCL one restriction endonuclease for 1.5 hours at 50 degrees Celsius. Subsequently, add one microliter of NGOM four to the digestion reaction and incubate it at 37 degrees Celsius for one hour.
After that, add five x loading dye to the restriction digest reactions. Slowly electrophoresis the total volume on a 1%aros, TAE gel containing A DNA stain for one to two hours at 100 volts. Visualize, excise and purify the indicated bands for cloning as previously described.
Next, prepare the ligation reactions using purified LTR gag. Insert an MJ four vector DNA at a three to one insert to vector ratio. Incubate the ligation reactions overnight at four degrees Celsius.
Then transform JM 1 0 9 chemically competent bacteria with ligation products spread on LB auger plates with 100 micrograms per milliliter. Ampicillin grow transformed bacteria at 30 degrees Celsius for at least 22 hours. To confirm cloning fidelity, cut the mini prep DNA with NGO M four and HPA one restriction enzymes in a double digest at 37 degrees Celsius for two hours.
Afterward, analyze it on a 1%aros. TAE gel. Calculate the volume of virus needed from each stock based on the IU per microliter titer from a TZM BL cell assay in order to infect five times 10 to the fifth G XR two five cells at a multiplicity of infection of 0.05.
Be sure to wear proper personal protective equipment for working with replication competent HIV, which includes a full gown, face mask and double gloves on the day of infection. Remove virus stocks from the minus 80 degrees Celsius freezer and thaw. Next, dilute the virus in C-R-P-M-I media to a volume of 100 microliters.
In order to achieve a 0.05 MOI add it to the vbo tissue culture treated 96 well plate, including both a positive and a negative control in each set of replication experiments and set up the plate such that every other column is blank to limit cross-contamination between wells. Afterward, count the GXR two five cells using an automated cell counter and calculate the number of cells needed in total for all infections. Aliquot the required volume into a 50 milliliter conical tube and centrifuge to pellet the cells.
Then aspirate the media carefully and resuspend in C-R-P-M-I at a concentration of five times 10 to the fifth cells per 100 microliters, pipette the cells into a sterile trough and mix thoroughly. Subsequently using a multi-channel pipette, add 100 microliters of cells into each well of the 96 well plate containing the diluted virus and mixed thoroughly. After that, add two microliters of five milligrams per milliliter solution of poly brain to each well and mix the cells thoroughly.
Then incubate at 37 degrees Celsius in a tissue culture incubator with 5%CO2 for three hours in order to wash the infected cells centrifuge. The 96 well V bottom plate to pellet the cells. Then carefully remove 150 microliters of the medium and replace with a fresh 150 microliters of C-R-P-M-I without disturbing the cell pellet.
Repeat the procedure two more times to sufficiently wash the cells after the last centrifugation and the addition of 150 microliters of C-R-P-M-I resuspend the cell pellet with a multi-channel pipette. Add the mixture from each well to 800 microliters of C-R-P-M-I in a well of a 24 well tissue culture plate. Then place the plate in a 5%CO2 tissue culture incubator at 37 degrees Celsius every two days.
Transfer 100 microliters of supernatant from the surface of the culture well to a well of a 96 well you bottom plate and freeze for subsequent analysis using the RT assay, then resuspend the cells thoroughly and remove half of the remaining volume to prevent cell overgrowth. After that, restore the original volume by adding 550 microliters of fresh C-R-P-M-I to each well in this procedure, add one to two microliters of 10 millicuries per milliliter of DTTP and four microliters of one molar DIO three etol to each one milliliter aliquot of ourt master mix. Carefully mix each 1.5 milliliter micro centrifuge tube of RT master mix DTT and DTTP with a 1000 microliter pipette and transfer to a sterile trough.
Next dispense 25 microliters of labeled RT.Mix into each well of a 96 well thin walled PCR plate. Add five microliters of each supernatant to the PCR plate containing the RT master mix. Then seal the PCR plate with an adhesive foil cover and incubate for two hours at 37 degrees Celsius in a thermocycler after incubation, make small holes in the foil cover using a 200 microliter multichannel pipette.
Mix the samples five times. Transfer five microliters of each sample to DE 81 paper and allow all the samples to air dry for 10 minutes. Place the blots in separate washing containers.
Subsequently wash the blots five times with one XSSC. Then two times with 90%ethanol for five minutes per wash before letting them air dry. Once dry, carefully wrap the blot in Saran wrap and expose it to the phospho screen in a tightly sealed cassette overnight at room temperature.
Once ready, analyze the phospho screens with a phospho imager and quantify the radioactive transcripts.Shown. Here are the representative gel images depicting the electrophoretic separation of the various PCR amplicons necessary to generate a patient derived gag amplicon capable of being cloned into the MJ four plasmid backbone. This representative gel image depicts the electrophoretic separation of restriction digests for cloning patient gag genes into MJ four.
And this representative gel image depicts the electrophoretic separation of restriction digests a purified gag MJ four chimera plasmid DNA. This graph shows the reproducibility of the replication assay over time in the G XR two five cell line. The same gag MJ four chimeric viruses were used to infect G XR two five cells in two independent experiments performed approximately one year apart.
Replication scores were generated by calculating the slope of log transformed DLU values and normalizing that slope to wild type MJ four. The two independent measurements are strongly correlated and highlight the reproducibility of assays performed at different times and with cells at different passages. This technique paved the way for researchers in the field of hiv aids to explore the viral determinants of pathogenesis.
In acute subtype C infection, the most prevalent subtype worldwide. Working with replication competent HIV one can be extremely hazardous. All steps involving live virus should be performed in a BSL two plus facility.
All surfaces should be regularly decontaminated and double gloves worn at all times.
