Harvesting of the 50 plates (15 cm) from the previous day and picking of 50 clones were done, and the collected bacteria were incubated in a 700 mL LB culture for 3 hours with shaking, after which a megaprep (Invitrogen) was done.
mini-preps of the 50 clones were done, and 10 samples were sent for sequencing.
02/10/2010
Transfection of 13 T flasks (150 cm2, Hek 293 cells) was done, with the plasmid DNA purified the previous day (the shuffled cap genes library) and an adeno-helper plasmid, 35 micrograms from each plasmid for each flask, using HBSS for the transfection. A GFP control for transfection was also used.
Analysis of the sequencing results for the 10 samples identified good shuffling of the cap genes. More mini-preps were inoculated for the same 50 clones.
03/10/2010
Mini-preps for the samples from the previous day were done.
05/10/2010
Harvesting of the cells for virus production was done by:
- Washing the flasks with the medium they contain, and collecting the media from 13 flasks in 500 ml corning conical centrifuge tubes.
- The cells were pelleted by centrifuging at 1500 rpm for 15 min at 4 ͦC
- Discard supernatant
- Washing: Resuspend the cells in 1X PBS and transfer to a 50 ml falcon
- Pellet the cells again by centrifuging at 1500 rpm for 15 min at 4 ͦC
- Discard supernatant
Freeze-thaw cylces for cell rupturing:
- Add 20 ml lysis buffer to the cell pellet
- Put in liquid nitrogen for 5 min
- Thaw at 37ͦC
The cycles were repeated for 5 times, and the sample was frozen for next day.
06/10/2010
The preparation for virus purification using iodixanol gradient was initiated according to the following:
- The sample from previous day was sonicated in a sonication bath for 1 min 20 sec.
- 50 units/ml benzonase were added to destroy RNA and DNA from non-AAV sources, which could later interfere with qPCR.
- The sample was incubated at 37 ͦC for 30 min, and vortexed every 10 min.
- The sample was then centrifuged for 15 min at 3270 xg
- The supernatant was transferred into a new 50 ml falcon
Virus purification using iodixanol gradient centrifugation:
- A Pasteur pipette was plugged into a Beckman quick-seal centrifuge tube
- Using a 1000 µl pipette, 20 ml of the virus suspension were added the gradient was poured through the Pasteur pipette in the following order:
- 7 ml 15% Iodixanol solution
- 5 ml 25% Iodixanol solution
- 4 ml 40% Iodixanol solution
- 4 ml 60% Iodixanol solution
- The Pasteur pipette was carefully removed and the tube was sealed. A tare tube was prepared in the same way.
- Ultracentrifugation of the sample was carried out at 50,000 rpm for 2:30 hours at 4ͦC.
- The virus-containing phase is the 40% iodixanol phase, which was sucked out using a syringe and a needle. The AAV sample was divided into aliquotes, one of them stored at 4 ͦC for qPCR the next day, and the rest were frozen in liquid nitrogen then transferred to -20 ͦC.
07/10/2010
qPCR of the sample from the previous day was carried out according to the protocol in the Methods section, and the titre of the viral library was determined to be 1.3*10^11 viral genomes/ml.
The selection rounds of the viruses in the library were initiated on Huh-7 cells, the infection of cells was carried out as follows:
- AAV MOI 100, 10 and 1 were used on Huh-7 cells plated on 6-well plates, and the viruses were allowed to infect cells for 1 hour and 30 min.
- Adeno-virus 5 (MOI 10) was added then in a Biohazard II lab under the superviosion of lab personnel.
- Cells were incubated in the Biohazard II lab.
08/10/2010
- A non-ITR AAV helper vector was obtained and maxi-preped to clone into it the cap genes from the 50 single clones picked.
09/10/2010
- The non-ITR vector was cut with AscI and PacI, and so was the pTR-UF3 containg cap genes from the 50 clones. The bands were purified from the gel.
10/10/2010
The cap genes were ligated into the non-ITR vector, and Hek 293 cells were prepared in 24-well plates for transfection.
The first selection round Huh-7 cells infect with AAV library and Adeno-virus 5 were monitored under the microscope, and the typical cytopathic effect caused by Adeno-5 infection (roundening of cells) was observed. Cells were harvested by washing the wells with the media (in this case, one well of the 6-well plate was used).
11/10/2010
The harvested cells from the previous day were incubated at 56 ͦC for 20 min to inactivate adeno virus 5. Then, freeze-thaw cycles (between liquid nitrogen and 37ͦC, see above) were carried out to lyse the cells and get the AAVs out. A 5-minute centrifugation at full speed was then done, and the supernatant containing the 1st selection round AAVs was collected and aliqueted and stored at -20ͦC (initial freezing with liquid nitrogen), or used immediately to infect the 2nd-selection-round Huh-7 cells as follows:
"
