The overall goal of this procedure is to produce active adeno-associated virus serotype three vectors which can efficiently transduce human liver cancer cells. This is accomplished by first transecting, three different plasmids into 2 9 3 cells simultaneously. The second step of the procedure is to purify AAV three varion assembled in 2 9 3 cells.
The third step of the procedure is to determine the tighter of the purified AAV three vector stock. The final step of the procedure is to transduce human liver cancer cells using purified AAV three vectors. Ultimately, results can be obtained that show highly expressed EGFP protein in human liver cancer cells through fluorescence microscopy.
The main advantage of this technique over existing method is to achieve high efficiency of gene transfer and expression in human liver cancer cells. Though This method can provide insight into the production of AAV three vectors, it can also be applied to other systems such a more widely used a V two vectors. Generally, individuals new to this method will struggle because of the involvement of a number of steps which can be tedious, Demonstrating the procedure will be a biological scientist from our laboratory.
The experimental manipulations in this section of the protocol are performed inside a tissue culture hood 48 hours before beginning this procedure. Seed 10 15 centimeter tissue culture dishes with human embryonic kidney cell line 2, 9 3 so that the cells are 70 to 80%cofluent on the day of transfection on the day of transfection. Use the website www.
ge shae. com/calc to calculate the amount of plasmid DA to be used for transfection. Once the correct volumes of plasmid DNA have been calculated, proceed to transfection pipette a suitable volume of cell culture media without FBS and antibiotics into a sterile 15 milliliter tube.
Then pipette the required volumes of P helper, PACG two, C3, and PDSA AAV C-B-E-G-F-P into the media. The total volume should be 8, 750 microliters for 10 15 centimeter plates pipette 1, 250 microliters of PEI into the medium containing the plasmid DNA vortex, the D-N-A-P-E-I solution for a few seconds, then incubate the D-N-A-P-E-I solution for five minutes at room temperature to allow D-N-A-P-E-I complexes to form. Retrieve the 10 15 centimeter plates of HEC 2 9 3 cells from the tissue culture incubator.
Then pipette one microliter of D-N-A-P-E-I solution into each of the plates. Incubate the plates in a tissue culture incubator at 37 degrees Celsius and 5%carbon dioxide for four to six hours. After four to six hours, carefully aspirate the medium from the plates.
Then pipette 25 milliliters of complete medium with 10%FBS into each plate. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for around 72 hours. Once 72 hours have passed, remove the plates from the incubator and return to the tissue culture hood.
Then without aspirating the media from the plate, use a cell scraper to scrape the cell layer from the bottom of the plate into the cell culture media. Pour the media containing the cells into a 250 milliliter conical centrifuge tube. Repeat this procedure for each of the transfected plates, pouring the contents of each plate into the same 250 milliliter conical centrifuge tube.
Spin the tube containing the cells and media in a chilled centrifuge. Four degrees Celsius for 10 minutes at 3000 rotations per minute Following centrifugation, discard the supinate, then pipette five milliliters of RB TMS buffer onto the pellet and resus bend. Transfer the cell solution to a clean 15 milliliter conical centrifuge tube.
Place the 15 milliliter tube into a dry ice ethanol bath to freeze for 10 minutes. After the incubation time has elapsed, remove the tube and place it 37 degrees Celsius for 10 minutes. To thaw, repeat this freeze thaw cycle of further two times.
Vortex the tube for a few seconds. After each thaw after the final thawing period, pipette one microliter of 4.8 molar magnesium chloride and two microliters of 25 units per microliter. Benon nase into the cell solution vortex to mix.
Then incubate a 37 degrees Celsius for 40 minutes following incubation. Place the tube into a chilled centrifuge at four degrees Celsius and spin up 4, 000 rotations per minute for 40 minutes towards the end of the centrifugation period, prepare the IAL gradient to be used for purifying the virus. To do this pipette two milliliters of 15%two milliliters of 25%1.5 milliliters of 40%and 1.5 milliliters of 60%IAL into a 13 milliliter seal centrifuge tube.
After centrifugation, pipette the S supernatant from the centrifuge tube, taking care not to disturb the cell pellet, pipette the snat onto the IAL gradient, then pipette enough RB TMS buffer into the tube to completely fill it. Place the tube into a Beckman 90 TI rotor, taking care to ensure that the rotor is properly balanced. Centrifuge at 75, 000 rotations per minute for one hour at 16 degrees Celsius.
Following centrifugation. Collect the 40%IAL fraction by inserting a five milliliter syringe fitted with an 18 gauge needle into the idal gradient at the 40 to 60%boundary. Carefully aspirate the syringe and collect the 40%idal fraction.
Expel the 40%IAL fraction from the syringe into a 50 milliliter conical tube. Then fill the tube to a final volume of 40 milliliters with buffer a. Assemble the filtration apparatus using a high trap Q HP column according to the manufacturer's instructions.
Then wash the column with the following solutions. 25 milliliters of buffer, A 25 milliliters of buffer B 50 milliliters of buffer, A 40 milliliters of the prepared idal virus sample in buffer, A 50 milliliters of buffer A and finally 20 milliliters of buffer C.Collect buffer C in a 20 milliliter Apollo concentrator. Then centrifuge the sample in a chilled centrifuge set at four degrees Celsius for 10 minutes at 3000 rotations per minute after centrifugation, discard the flow through.
Then add 20 milliliters of chilled PBS to the Apollo concentrator and centrifuge gain in a chilled centrifuge at four degrees Celsius for 10 minutes at 3000 rotations per minute following centrifugation. Once again, discard the flow through pipette 500 microliters of chilled PBS onto the membrane to resuspend the virus from the membrane. Then pipette 10 microliter aliquots of the viral vector into silicone treated eend orph tubes and store at minus 80 degrees Celsius until use thaw one of the 10 microliter aliquots of purified virus on ice.
Pipette 40 microliters of double distilled water into the tube to dilute the virus. Then add 0.2 microliters of 25 units per microliter, Ben Nase and incubate the mixture at 37 degrees Celsius for one hour. Meanwhile, pipette 50 microliters of 0.2 nanograms per microliter plasmid standard into a clean eend orph tube.
Following incubation, add 50 microliters of 100 millimolar sodium hydroxide to the tube containing the virus. Bens aase mix and to the tube containing the plasmid standard. Incubate both tubes at 65 degrees Celsius for 30 minutes.
After the 30 minutes have elapsed immediately chill both tubes on ice for at least five minutes. Then cut a piece of transfer membrane to the same size as a piece of bio dot filter paper. Place the transfer membrane and three pieces of bio filter paper into a shallow tray.
Fill the tray with 10 times SSC and soak for 20 minutes while the membrane is soaking. Set up the slot block SF apparatus according to the manufacturer's instructions. Connect the apparatus to a vacuum flask, then pipette 100 microliters of 10 times SSC into each of the slots to be loaded with dilution of the virus sample and plasmid standard.
Remove the tubes from the ice centrifusion chilled centrifuge set at four degrees Celsius for one minute at 13, 000 rotations per minute. Then add 100 microliters of 20 times SSC and one microliter of six times loading dye to each tube. Then pipette an additional 100 microliters of 10 times SSC into each tube and load 100 microliters of each sample into adjacent slots.
Repeat the dilution and loading procedure until the total volume of each sample has been loaded into the slot block apparatus and further additions of 10 times SSC, your volume of 100 microliters or less. Once all of the samples have passed through the membrane, disassemble the slot loss apparatus and remove the membrane with forceps. Take care to handle the membrane at the edges and place the membrane into the chamber of the inspector linker SL 1000 UV crosslinker.
Then select energy optimal cross-link cross-linking should take approximately 30 seconds. Now prepare for hybridization by boiling one milliliter of salmon sperm DNA for five minutes. After boiling for five minutes, the salmon sperm DNA should be immediately chilled on ice for at least five minutes.
Then prepare the pre hybridization solution according to the written protocol. Pipette 25 milliliters of the pre hybridization solution containing salmon sperm DNA into a hybridization tube. Carefully transfer the cross-linked membrane to the hybridization tube and then place the cap on the tube, then ensuring that the pre hybridization solution thoroughly covers the membrane.
When in the horizontal position, incubate in a hybridization chamber at 65 degrees Celsius overnight. The next day, begin probe preparation by pipetting 50 nanograms of DNA template into an einor tube and adding 10 microliters of double distilled water to dilute, boil the diluted DNA for five minutes and then immediately chill on ice for at least five minutes after chilling. Centrifuge the diluted DNA template at 13, 000 rotations per minute for one minute of room temperature.
Then working according to the institution's radioactivity safety guidelines. Prepare the radioactive probe by adding two microliters of 10 millimolar DN TPS without DCTP two microliters of hexa nucleotide. Mix one microliter of AU and five microliters of 32 P-D-C-T-P incubate at 37 degrees Celsius for one hour following incubation.
Prepare G 50 columns by centrifusion at 3000 rotations per minute for two minutes. Then load the probe onto the column and centrifuge your gain at 3000 rotations per minute for two minutes and collect the flow through. At this point, the radioactivity of the flow through is measured using standard procedures to determine the counts per minute per milliliter of the probe.
Next, boil the probe for five minutes. After this, immediately chill the probe on ice for five minutes. The probe is then ready to be used for hybridization without removing the membrane.
Pipet the probe as a concentration of six times center the five counts per minute per milliliter directly into the pre hybridization solution in the hybridization tube. Incubating the hybridization chamber overnight at 65 degrees Celsius the next day. Discard the hybridization solution according to institutional radio activity protocols and pipette 25 milliliters of two times SSC plus 0.1%SDS into the tube and wash the membrane for 15 minutes at room temperature.
Discard the solution and pipette 25 milliliters of 0.1 times SSC plus 0.1%SDS into the tube. Wash the membrane at 65 degrees Celsius for 30 minutes. After this wash, discard the solution and briefly rinse the membrane in 25 milliliters of 0.1 times SSCA room temperature.
Wrap the membrane in kling film and place into an exposure cassette working in the dark room. Place a piece of film on top of the membrane and close the cassette to protect from light. Place the exposure cassette into the minus 80 degrees Celsius freezer for six.
After six hours. Develop the film and determine the titer of the viral stocks by comparing the density of the viral bands to the bands from the plasmid standards. Once the titer has been determined, the virus can be used to transduce cells first seed one times 10 to the four hep 2 93 TT cells.
In each well of a 96 well plate incubate the cells for at least 18 hours in a 37 degrees Celsius 5%carbon dioxide incubator. Following incubation, aspirate the media from each well and carefully pipette 50 microliters of medium without antibiotics and FBS into each. Well aspirate this media and then repeat the process to wash the cells.
Dilute the recombinant a a v vectors in media without FBS and antibiotics to a final concentration of 5, 000 VGS per cell. Add 50 microliters of this solution or 50 microliters of medium without FBS as a negative control to the wells of the 96 well plate and incubate for two hours in a 37 degree Celsius 5%carbon dioxide incubator. Following incubation, remove the virus containing media and wash the cells twice as before, using 50 microliters of complete medium per well.
After discarding the last wash, add 150 microliters of complete medium to each well and incubate the plate in a 37 degree Celsius 5%carbon dioxide incubator for 72 hours. Finally, visualize the EGFP expression using fluorescence microscopy. Fluorescence intensity can be quantified using Image J software with trans gene expression assessed as total area of green fluorescence per visual field.
An example of a quantitative DNA slot block for determining the tituss of RAV three vectors is shown here. This membrane has been probed with P 32 labeled EGFP, specific DNA probe. The top row of the block comprises twofold dilutions of purified viral stocks digested with Ben Nase.
The middle and bottom rows are comprised of A-A-V-E-G-F-P standards diluted from one nanogram or 10 nanograms of plasmid respectively. The titer of the viral stocks is determined by comparison with the plasmid standards. The numbers shown on the blot correspond to the number of plasmid DNA copies.
This is an example of recombinant a A V vector mediated transgene expression. In a human hepatoblastoma cell line cells were visualized 72 hours post transduction using a fluorescence microscope. The first image illustrates the results of a mock infection.
The second image shows HEP 2 93 TT cells transduced with 5, 000 VGS per cell of all AAV two EGFP vectors at 37 degrees Celsius for two hours. The final image shows transduction with 5, 000 VGS per cell of R AAV three EEG FP Under the same conditions. It can be seen that RAAV three is much more efficient at transducing this cell type compared to R AAV two.
This method can help answer key questions in the gene therapy field, such as how to target human liver cancer cells. The implication of this technique extend towards therapy of human liver cancer because a V three has the ability to express therapeutic gene specifically in human liver cancer at a high level.
