The goal of this procedure is to produce recombinant adeno-associated viruses or r AAVs for effective in vitro or in vivo transgene delivery. This is accomplished by first transecting HEC 2 93 cells with DNA plasmids encoding the trans gene and viral genes. The transfected cells are then scraped and lies to harvest the assembled RAAV particles.
Next, the R AAVs are purified from the cell lysate through heparin column purification. The final step is to tighter the viral stocks. Ultimately, the results show strong and persistent virus mediated trans gene expression through immunofluorescence microscopy.
At this point, the RA AAVs are ready for in vitro or in vivo applications. The main advantage of using our technique over other purification methods, such as the cesium chloride gradient purification, is that we do not require to use an ultracentrifuge or toxic materials. We also yield higher and pure viral vectors.
Prior to the start of this protocol, prepare high quality plasmid, DNA stocks and scream for integrity as described in the written protocol. To begin plate two 80%COFLUENT 150 centimeter squared flasks of HEC 2 9 3 cells into five 15 centimeter diameter NOC tissue culture dishes, culture cells. In standard DMEM with low glucose containing 10%fetal calf serum, approximately 48 hours after plating on the day of transfection cells should be 70 to 80%confluence three hours before transfection.
Remove the DMEM and replace with ice cove's modified dobe CHO'S medium or IMDM containing 5%fetal calf serum. Prepare the transfection mixture for a single batch of virus as outlined in the written protocol In a class two tissue culture hood sterile filter the transfection mixture into a 50 milliliter tube whilst vortexing the solution quickly add 13 milliliters of two times heap. He's buffered saline.
Replace the lid of the 50 milliliter tube and continue to vortex for 15 seconds. Leave to stand for one minute and 45 seconds. A white precipitate should form Gently add five milliliters of the transfection solution to each 15 centimeter tissue culture dish.
Swirl the plates to mix after incubating for 16 hours. Remove IMDM replace with DMEM and continue to incubate the cells. To harvest the R AAVs.
Remove media from cell culture plates. 72 hours after transfection and discard always should be treated with vercon solution or other suitable disinfectant. Gently wash the cells in warm one times phosphate buffered saline.
Add 25 milliliters of warm PBS to each plate and gently remove the cells with a cell scraper. After collecting the suspension in 50 milliliter tubes, pellet the cells at 800 times G for 10 minutes. Discard the supinate and resuspend the pellet.
In 10 milliliters of 150 millimolar sodium chloride buffer per tissue culture plate, split the suspension into two 50 milliliter tubes. Next lies cells with a freshly prepared solution of 10%Sodium deoxy coate. Then add benzoate nuclease for removal of nucleic acids.
After mixing the tubes thoroughly incubated 37 degrees Celsius for one hour. Following incubation, remove cellular debris by centrifugation at 3000 times G for 15 minutes. Transfer the supinate to a fresh 50 milliliter tube ensuring that all cell debris has been removed to prevent blocking of heparin.
Columns for purification of r AAVs. A 10 milliliters of sodium chloride buffer to a syringe and set up the high trap heparin columns using a peristaltic pump so that solutions flow through the column at one milliliter per minute. It is important to ensure that no air bubbles are introduced into the heparin column.
Equilibrate the column with 10 milliliters of 150 millimolar sodium chloride buffer. Apply 50 milliliters of virus solution to the column and allow the solution to flow through. Then wash the column with 20 milliliters of 100 millimolar sodium chloride buffer.
Using a five milliliter syringe. Continue to wash the column with one milliliter of 20 millimolar sodium chloride buffer followed by one milliliter of 300 millimolar buffer. Discarding the flow through.
Employ five milliliter syringes and gentle pressure to elute the virus from the column by applying increasing high suru elution buffers. Collect the eluate in a 15 milliliter centrifuge tube. Concentrate the vector using AmCon Ultra four centrifugal filter units with a 100, 000 molecular weight cutoff.
Load four milliliters of the column eluate into the concentrator and centrifuge at 2000 times G for two minutes. After repeating the centrifugation, the concentrated volume should be approximately 250 microliters. Discard the flow through and reload the concentrator with the remaining virus solution.
Add 250 microliters of PBS to the virus for a final volume of 500 microliters and removed from the concentrator. As a final step, filter the vector through a 13 millimeter diameter 0.2 micron syringe filter to tighter the stock virus. Prepare 18 polyol lysine coated glass cover slips by seeding HEC 2 9 3 cells so that they're 40 to 50%cofluent for titration of Cree dependent R AAVs.
Use HEC 2 9 3 cells that stably express Cree recombinase. Infect each well with serial dilution of the RAAV vector as described in the written protocol using three wells per dilution after 72 hours, fix HEC 2 9 3 cells by adding an equal volume of 4%power of formaldehyde to the medium for 10 minutes at room temperature following incubation, rinse the cells three times in PBS and once with distilled water. Then using mounting media, place the cover slips onto the slides.
Cell side down. Count the number of trenched cells from the three wells that have the highest dilution factor but still contain infected cells. Find the average of these and multiply by the dilution factor to give the number of infectious units per microliter HEC 2 9 3 cells transduced with a virus encoding enhanced green fluorescent protein are shown.
Consistent titers of around six times 10 to the six infectious particles per microliter are generally achieved. Shown here are examples of stereotaxic injections of Cree dependent R AAVs encoding green fluorescent protein into different brain regions of adult par albumin Cree transgenic mice. Viral green fluorescent protein expression is restricted to Cree expressing neurons in different target regions.
In this example, green fluorescent protein immunoreactivity is shown in the reticular thalamus and globus palus, the dentate gyrus and the ca one region of the hippocampus Once mastered. This technique can be performed in seven to eight days for virus production and three days for tittering if performed properly.
