Compared to other viral approaches for in vivo gene manipulation in macrophages, the use of lentiviral vectors offers a stable integration of the transgenic tissue macrophages, efficient transgene expression, and the largest vector size limit. When employing in vivo lentiviral manipulation of metabolic enzymes, we discovered notable functional changes in tissue-resident macrophages. Therefore, we're currently investigating immunometabolism and its role in preserving tissue-specific functions, including the epigenetics and immune function.
In the future, we would like to focus on defining how tissue macrophages affect other local cell populations during homeostasis and in disease models and how those interactions can be employed for disease prevention and resolution. To begin, add the lentiviral components in a 15-milliliter tube and make the volume to 600 microliters with EC buffer. Add 36 microliters of enhancer solution and, using a one-milliliter pipette, mix repeatedly, drawing up and releasing a portion of the liquid back into the solution.
After five minutes, add 120 microliters of transfection reagent and mix approximately 20 times, as demonstrated previously. Leave the tube at room temperature for 10 minutes. Then, add 5.2 milliliters of cDMEM to the tube.
Using a disposable transfer pipette, dropwise add the transfection mixture onto the HEK293T cells monolayer, cultured in a T175 flask. Distribute the plasmid evenly through a gentle side-to-side rocking motion of the flask. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide for 48 hours.
After incubation, under a fluorescent microscope, confirm the effective transfection of HEK293T cells by observing GFP fluorescence. Now, tilt the flask and, using a 25-milliliter serological stripette, collect the medium without disturbing the cell monolayer. Transfer the medium to a pre-labeled 50-milliliter tube and close the lid.
Add 25 milliliters of warm cDMEM along the walls of the flask without disturbing the monolayer, and return the flask to the incubator. After 24 hours, collect the medium, add the decontamination solution into the flask, and place it horizontally for the next 24 hours. To begin, remove the plunger from a 50-milliliter syringe and insert a low-protein binding polyethersulfone or polyvinylidene fluoride 0.45 micrometer sterile filter into the syringe end.
Using a 25-milliliter serological stripette, add the lentivirus transfected HEK293T cell suspension into the syringe and gently return the plunger. Push the plunger slowly and collect the filtrate in a new 50-milliliter tube. Once done, remove the filter from the syringe and dispose of it in the decontamination solution.
Add three milliliters of a 20%sucrose solution into the bottom of the ultracentrifuge tube. Next, set the PIPETBOY to the lowest speed. Hold the ultracentrifuge tube at approximately 45 degrees angle and slowly add the filtered lentivirus-containing medium onto the tube wall.
Observe the clear separation of layers from the tube. If the total tube volume is less than 29 milliliters, add fresh cDMEM to avoid the tube collapsing during the spin. Wipe the ultracentrifuge bucket with 70%alcohol and put the tube adapter in the bottom of the bucket.
Then, place the ultracentrifuge tube into the bucket. Close the bucket and place it into the rack. Pre-cool the ultracentrifuge to four degrees Celsius.
Carefully place the bucket into the rotor and turn on the vacuum. Adjust the acceleration and deceleration rate to the lowest setting and centrifuge at 26, 000 RPM for 90 minutes at four degrees Celsius. After centrifugation, turn off the vacuum and carefully remove the rotor without disturbing the samples.
Observe the ultracentrifuge to confirm there are no spills and leaks from the buckets. Pour the content of an ultracentrifuge tube in one smooth motion into the waste container. Invert the tube onto a double-layer tissue paper for 10 minutes.
Meanwhile, wipe the insides of the bucket with 70%ethanol-soaked tissue paper and air dry it upside down. Using tissue paper, carefully dry any remaining liquid from the rim of the ultracentrifuge tube. Resuspend the pellet in one milliliter of appropriate serum-free media and leave it for 15 minutes.
Using a one-milliliter pipette, gently mix the lentivirus preparations and aliquot them into 1.5-milliliter screw-top tubes. Store the lentiviral preparations at 80 degrees Celsius. Successful lentivirus preparation achieved an infection rate of over 95%with a five microliter dose.
The mean fluorescent intensity of infected cells increases with higher doses. The intraperitoneal injection of 100 microliters of lentivirus preparation yielded the highest percentage and intensity of the GFP signal in mice peritoneal cells. Time course experiments with 100 microliter lentivirus injection revealed a significant percentage of GFP-expressing resident cells at days three and seven, followed by the disappearance of the infected population at day 14.
