AAV-Production-293T-2

AAV stock production

1. Thaw a vial of 293T cells (each vial contains ~3 × 10^6 cells) and plate cells in a 10-cm dish containing 10 ml D10 (DMEM with 10% FBS). Maintain in a 37 °C incubator with 5% CO2 atmosphere.

2. After 3 days, aspirate the medium and wash the dish with 2 ml of 1× PBS. Add 2 ml of trypsin-EDTA solution and incubate at 37 °C for 2 min or until cells detach from culture dish.

3. Add 4 ml of D10 to neutralize trypsin-EDTA solution and transfer the cell suspension to a 15-ml conical tube.

4. Centrifuge at 250g for 5 min at 20 °C in a tabletop centrifuge.

5. Resuspend the cells in 1 ml of D10. Take a 10-ÎŒl aliquot and count the cells using a hemocytometer.

6. Adjust cell concentration to 10^6 cells per ml with D10 and plate 1 ml of cell suspension in eight 10-cm dishes, each contains 10 ml D10. Culture for 72 h in a 37 °C incubator with 5% CO2 atmosphere.

7. After the 3-d incubation, harvest cells as described in Steps 2–4. Resuspend in 5 ml of D10 and take a 10-ÎŒl aliquot for counting with a hemocytometer. Adjust cell concentration to 4 × 10^6 cells per ml and plate 1 ml of cell suspension in 30 10-cm dishes, each containing 10 ml D10. Incubate in a 37 °C incubator overnight.

8. The next day, prepare solution A by combining 15.5 ml of 2× HEPES-saline and 155 ÎŒl of 0.15 M phosphate buffer in a 50-ml tube and, mixing well.

9. In another 50-ml tube, prepare solution B by combining 1.938 ml of 2 M CaCl2, 387.5 ÎŒg of plasmid.

10. Add solution B to solution A, mix well and quickly add 1.0 ml of the transfection mix to each of the 30 dishes of 293T cells.
Use a 10-ml pipette and tilt the dish so that the 1.0 ml of transfection mix is added to a greater depth of medium to prevent ‘blasting’ cells off the dish.

11. Agitate the dishes gently to evenly distribute the precipitate and return the dishes to the 37 °C incubator.

12. Observe a dish 1 h later under a microscope. The precipitate should be visible as small grains about the size of bacteria. Return the dishes to the 37 °C incubator.

13. After 48 h, prepare dry ice/ethanol bath by placing a centrifuge tube rack in a styrofoam container. Add ~2 lbs of dry ice, pour 2 liters of ethanol over it and cover the container.

14. Collect the cells and medium by scraping the cells off the dish with a rubber policeman and transferring it to a 50-ml conical tube. Use Corning 50-ml conical tubes, as they are more resistant to the subsequent freeze-thaw cycles. Rinse dishes with 5 ml of 1× PBS and transfer it to the same conical tube. Harvest four dishes at a time into the same 50-ml tube.

15. Centrifuge at 250g for 5 min at 20 °C in a tabletop centrifuge.

16. Aspirate the medium from the conical tube and repeat Steps 14 ~ 15 until cells are pelleted from all 30 dishes. The same tube can be used for pelleting cells from all the dishes.

17. Resuspend the final pellet from the 30 10-cm dishes in 8 ml of cell lysis buffer.

18. Freeze the pellet in the dry ice/ethanol bath and thaw in a 37 °C water bath.

19. Repeat Step 18 twice and store at − 80 °C.

The pellet can be stored at − 80 °C indefinitely and thawed at your convenience.

20. Thaw cell lysate in a 37 °C water bath.

21. Add Benzonase to the cell lysate at a final concentration of 50 U ml − 1. Benzonase is added to eliminate cellular DNAs/RNAs as well as excess plasmid DNAs present during transfection.

22. Incubate at 37 °C in a water bath for 30 min. Prepare iodixanol gradient during the incubation.

23. Centrifuge at 5,000g for 30 min at 4 °C in a Sorvall high-speed centrifuge using an HS-4 rotor.

24. Collect vector-containing supernatant. The volume of the supernatant is approximately 5 ml.

25. Load the 5 ml of vector-containing supernatant over the iodixanol density gradient prepared in Step 22. Top off the tube with cell lysis buffer. This density gradient purification step can remove contaminants that may influence transduction and targeting frequencies.

26. Centrifuge at 461,300g for 1 h at 18 °C with maximum acceleration and deceleration in a Beckman Ti70 rotor on a Beckman ultracentrifuge; use proper spacers for tubes.

27. Puncture the tube on the side slightly below (3–5 mm) the 60–40% interface with an 18-gauge needle (bevel up) attached to a 10 ml syringe.
28. Collect 3–4 ml from each centrifuge tube by aspiration using the same needle.

CRITICAL STEP Avoid the proteinaceous material near the 40–25% interface.

29. To use the vector without removing iodixanol, proceed directly with Step 30; iodixanol is inert and non-toxic to cells. Alternatively, further purify Serotype 2 vector stocks over a HiTrap heparin affinity column, followed by desalting over a HiTrap desalting column as described in Box 4, before proceeding with Step 30.

AAV titering using neutral gel electrophoresis and Southern blotting

30. To prepare plasmid standards for quantitation, digest the same vector plasmid used to prepare the stock, such that the vector genome is released from the surrounding inverted terminal repeats.
Set up the restriction digest by adding the following to a microcentrifuge tube:
restriction buffer (10×, 2 ÎŒl);
plasmid DNA (1,000 ng);
restriction enzyme (5 U);
and dH2O to a final volume of 20 ÎŒl.

31. Incubate the restriction digest in a 37 °C water bath for 1 h. Stop the reaction by adding 2 ÎŒl of 0.5 M EDTA.

32. Run 2 ÎŒl of the restriction digest on a 1% (wt/vol) agarose gel to confirm that the plasmid is completely digested.

33. Accurately quantify the digested DNA content by using a fluorometer and make serial dilutions as follows: 100, 30, 10 and 3 pg/ÎŒl.

34. Prepare 1% (wt/vol) agarose gel in 1× TAE. Allow it to set at room temperature for 1 h.

35. Label four microcentrifuge tubes as 1,000, 300, 100 and 30 pg. Pipette out 10 ÎŒl from each standard dilution into the appropriate tube.

36. Take the AAV stock out of the − 80 °C freezer and thaw at room temperature.

37. Prepare two microcentrifuge tubes, each containing 3 ÎŒl of TE and 1 ÎŒl of 6× loading dye. Add 2 ÎŒl of the 1:10 vector stock dilution to the first tube and 2 ÎŒl of the 1:100 stock dilution to the other (dilution preparation described in step 19, Box 4).

38. Clip the AAV stock tubes tightly.

39. Boil them for 2.5 min, snap-cool quickly on ice.
CRITICAL STEP The vector is boiled to disrupt its capsid and to release the single-stranded DNA genome before loading it onto a neutral gel.

40. Load the standards and AAV stock dilutions along with λ/HindIII marker on the 1% (wt/vol) agarose gel and run at 120 V for 4 h.

41. Stain the gel in 1× TAE containing 0.5 ÎŒg ml − 1 of ethidium bromide for 30 min. The gel can be destained in dH2O for 20 min if required. Photograph on a UV transilluminator with a fluorescent ruler by the side.

42. Transfer the gel to a tray and acid-depurinate with 500 ml of 0.25 M HCl for 10 min, twice. Rock the gel gently.

43. Remove the HCl solution and neutralize with 500 ml of 0.4 M NaOH for 15 min, twice. Rock the gel gently.

44. Transfer to a Hybond-XL membrane with 1 liter of 0.4 M NaOH overnight using standard Southern blot wicking methods63.

45. The next day, briefly rinse the membrane in 2× SSC.

46. UV crosslink the membrane on a UV Stratalinker (auto-crosslink setting).
PAUSE POINT The membrane can be stored at 4 °C, wrapped in plastic wrap, for several months.

47. Prehybridize the membrane with 14 ml of Church buffer containing 150 ÎŒg ml − 1 of salmon sperm DNA (boiled for 5 min and snap-chilled on ice) for 3–4 h in a hybridization cylinder rotating in a hybridization incubator at 65 °C.

48. Synthesize a radiolabeled probe by random priming with a Rediprime II kit according to the manufacturer’s instructions.
CAUTION Follow all relevant institutional guidelines while working with radioactivity. Radioactive probe must be prepared in a fume hood. Wear protective clothing including gloves, plastic shield and safety glasses.

49. Purify the radiolabeled probe with a ProbeQuant G-50 microcolumn. Denature it by boiling for 5 min and snap-cool on ice.
CRITICAL STEP Use screw cap tubes for boiling to prevent leakage of radioactive probe.

50. Add the denatured probe to the prehybridization solution and incubate at 65 °C overnight in the hybridization oven.

51. The following day, remove the radioactive hybridization solution and dispose of it properly. Wash the hybridized membrane twice with 45 ml of Southern wash solution 1 for 15 min at 65 °C in a hybridization cylinder.

CAUTION Follow institutional guidelines while working with radioactivity. The radioactive waste must be discarded in the designated radioactive waste disposal container. Wear protective clothing including gloves, plastic shield and safety glasses.

52. Wash the membrane twice with 45 ml of Southern wash solution 2 for 15 min at 65 °C in a hybridization cylinder.

53. Expose the membrane to X-ray film at − 80 °C overnight. Develop the X-ray film on a Kodak 480 RA processor. This provides a visual record of the stock titer.

54. Measure the radioactive signal by placing the membrane in a PhosphorImager cassette for 30–60 min and analyze on a PhosphorImager using ImageQUANT densitometry software.

55. Generate a standard curve based on the radioactive signal of the standard dilutions and determine the vector titer. The radioactive signal from the vector sample(s) should fall within the standard curve and can be used to calculate the number of genome-containing vector particles per ml. Accurate titering of the vector stock is critical for performing infections at controlled multiplicity of infections (MOIs), which is known to influence targeting frequencies.

Transduction of cells with AAV vector stock

56. To transduce somatic cell lines (MSCs, fibroblasts, transformed cell lines) follow option A; the protocol works efficiently for primary or transformed cell lines but MSCs have been used here as an example. To transduce pluripotent cells, follow option B.

(A) Transduction of somatic cell lines ● TIMING 14 d

(i) Harvest MSCs by trypsin treatment, as described in Steps 2–4, except use MSC medium instead of D10.

(ii) Resuspend the cell pellet in 1 ml of MSC medium. Take a 10-ÎŒl aliquot and determine the cell number using a hemocytometer.

(iii) Plate 2.5 to 5 × 104 cells per well of a 24-well plate in 0.5 ml of MSC medium. Culture in a 37 °C incubator overnight.

(iv) The following day, thaw a vial of AAV vector stock quickly in a 37 °C water bath. Pipette up and down vigorously with a pipette, vortex for 30 s, and spin in a microcentrifuge for 30 s at maximum speed to suspend the vector and pellet debris.

(v) Add the desired amount of vector stock. Typically, an MOI of 2,000–20,000 genome-containing vector particles per cell is adequate. When a particular AAV stock is being used for the first time, cells should be transduced at three MOIs,each differing by a log, to determine the optimal dose for transduction.

(vi) Expose cells to the vector overnight. Leave one well untransduced as a control for selection.

(vii) The following day, harvest the cells by trypsin treatment and plate three 10 cm dishes, each with 33% of cells (other dilutions can be plated instead, in order to obtain the desired number of transduced colonies per dish). In addition, plate 0.5% of the cells to calculate plating efficiency in a 10-cm dish. The untransduced cells should also be plated
the same way to serve as a control for selection.

(viii) Add antibiotic to the MSC medium at the appropriate concentration. The concentration should be empirically determined beforehand using a kill curve. Add antibiotic-containing medium 48 h post transduction to all dishes, except to the 0.5% plating dish reserved for determining plating efficiency.

(ix) Change the medium every third day.

(x) Maintain the dishes in selection medium for the next 8–10 d or until the cells in the uninfected control dish are dead.

(xi) If clones are not to be picked, stain the dishes with Coomassie blue. Stain the unselected dish as well. Otherwise, proceed to Step 57A.

(B) Transduction of PSCs ● TIMING 20 d

(i) Add 3.5 ml of 0.1% (wt/vol) gelatin to a 6 cm dish. Place the dish in a 37 °C incubator for 30 min.

(ii) Aspirate the gelatin and seed the dish with 3.3 × 105 irradiated (mitotically inactivated) MEF cells in MEF medium (prepared as described in Box 1).
CRITICAL STEP As selection is used in gene targeting experiments, we use irradiated MEFs prepared from the DR-4 mouse strain bearing resistance genes for four different drugs: G418, 6-thioguanine, puromycin and hygromycin.

(iii) Culture in a 37 °C incubator with 5% CO2 atmosphere overnight.

(iv) The following day, wash the MEF dish with 1× PBS and replenish with 3 ml of ESC medium. Return the dish to the 37 °C incubator.

(v) Fill a 15-ml conical tube with 7 ml of prewarmed ESC wash medium.

(vi) Take a frozen straw of ESCs/iPSCs from liquid N2 and thaw in a beaker of lukewarm tap water. We freeze ESCs and iPSCs in Cassou straws64, with about 2 to 4 × 105 cells per straw; one straw can be used to seed one 6-cm dish preseeded with MEFs. To thaw, cut the bottom off the straw with sterile scissors over a waste bin.

(vii) Cut the top off the straw over the 15-ml conical tube and let the cells drain into the tube.

(viii) Centrifuge the cells at 250g for 3 min at 20 °C, resuspend in 1 ml of ESC medium and plate in the pre-seeded MEF dish containing 3 ml ESC medium (from Step 56B(iv)). Culture in a 37 °C incubator with 5% CO2.

(ix) After 3 d, prepare a six-well plate and a 6-cm dish of irradiated MEFs. Briefly, a six-well plate is coated with 1.5 ml of 0.1% (wt/vol) gelatin per well and a 6-cm dish with 3.5 ml for 30 min at 37 °C. The gelatin is aspirated and the six-well plate is seeded with 1.8 × 105 MEFs per well and the 6-cm dish with 3.3 × 105 MEFs in MEF medium. Incubate in a 37 °C incubator overnight.

(x) Four days after thawing ESC/iPSCs, remove the medium from the dish. Add 2 ml of 0.1% (wt/vol) Dispase solution to the 6 cm dish to enzymatically disaggregate the cells.

(xi) Incubate at room temperature for 3 min. The dish should be observed under an inverted microscope. The ESC/iPSC colonies should begin to curl up along their edges.

(xii) Aspirate 0.1% (wt/vol) Dispase solution.

(xiii) Add 2 ml of ESC wash media to the dish and aspirate it.

(xiv) Add 2 ml of ESC wash medium again and pipette with a plugged Pasteur pipette to remove cells, working from the bottom of the dish up.

(xv) Collect all the cells into a 15-ml conical tube containing 5 ml of ESC wash medium.

(xvi) Rinse the dish with 3 ml of ESC wash medium and transfer into the 15-ml conical tube.

(xvii) Centrifuge at 250g for 3 min at 20 °C in a tabletop centrifuge.

(xviii) Discard the supernatant and resuspend the pellet in 3 ml of ESC medium.

(xix) Take a 100-ÎŒl aliquot for counting cells on an automated cell counter (Nucleo Counter) according to the manufacturer’s instructions. The cell counts can also be determined using a hemocytometer after disaggregating the 100-ÎŒl aliquot into a single cell suspension with trypsin-EDTA solution; however, the cells are very small and difficult to observe.

(xx) Plate 105 cells per well of a six-well plate preseeded with irradiated MEFs (from Step 56B(ix)) and place in a 37 °C incubator with 5% CO2. In addition, plate 5 × 103 cells over a 6-cm dish (from Step 56B(ix)) for calculating the plating efficiency.

(xxi) The following day, change the medium with fresh ESC medium. Infect the cells with the AAV stock by adding stock directly to the dish at an MOI of 2,000–20,000 genome-containing particles per ESC/iPSC cell, after resuspending vector particles as described in Step 56A(iv). Leave one well untransduced as a control for selection. Place in a 37 °C incubator overnight.

(xxii) The next day, change the medium with ESC medium. Place in a 37 °C incubator overnight.

(xxiii) After 48 h, add ESC medium containing the appropriate antibiotic in all wells except in the plating efficiency dish, which should not receive the antibiotic.

(xxiv) Change the medium every alternate day. At 7–8 d after plating, stain the 6-cm plating efficiency dish with Coomassie blue.

(xxv) On the seventh day, spike the dishes with irradiated MEFs. Briefly, thaw irradiated MEFs in 7 ml of ESC wash medium, centrifuge at 250g for 5 min at 20 °C in a tabletop centrifuge at room temperature.

(xxvi) As the cells are spinning, remove the medium from the six-well plate, wash once with 1× PBS and replenish with 1 ml of fresh ESC medium per well.

(xxvii) Aspirate the medium from the conical tube, resuspend the MEFs in 6 ml of ESC medium and add 1 ml per well of the six-well plate. When spiking, we plate half the number of MEFs plated initially. For example, for spiking the six-well plate, 5.5 × 105 (9.1 × 104 per well) MEFs are required. Place in a 37 °C incubator.

(xxviii) Maintain the dishes in selection media for the next 8–10 d or until the cells in the uninfected control well are dead. The medium should be changed daily. Proceed to step 57B for picking drug-resistant foci.

Picking drug-resistant clones

57. Follow option A to pick somatic cell clones; the protocol works efficiently for primary or transformed cell lines. MSCs have been used here as an example. Follow option B to pick pluripotent cell clones.

(A) Picking drug-resistant clones: somatic cell clones ● TIMING 0.5 h
(i) Identify drug-resistant colonies by scanning the dishes (from Step 56A(xi)) with an inverted microscope and circle them using a permanent ink pen on the bottom of the dish.

(ii) Prepare a 24-well plate with 0.5 ml of MSC medium per well planning on one well per colony to be picked.

(iii) Aspirate medium from the dish containing drug-resistant colonies and wash once with 1× PBS.

(iv) With forceps, place cloning cylinders (see EQUIPMENT SETUP) over all the foci to be picked. Ensure that the cloning cylinders are firmly attached to the dish and that they encircle the colonies.

(v) Pipette 20 ÎŒl of trypsin-EDTA solution into the cloning ring and incubate for 2 min at 37 °C. Add 30 ÎŒl of MSC medium to neutralize the trypsin-EDTA.

(vi) Collect the detached cells from the cloning cylinder with a pipette and plate each colony into one well of the 24-well plate prepared in Step 57A(ii).

(vii) After picking the clones, stain the dishes with Coomassie blue to determine the number of CFUs.

(viii) If required, further culture and passage the clones to bigger dishes for freezing and harvesting DNA. We generally transfer somatic cells to 10-cm dishes for DNA extraction.

(B) Picking drug-resistant clones: PSCs ● TIMING 1 h

(i) Seed a 24-well plate with 3.7 × 104 irradiated MEFs per well, 1 d before picking resistant colonies. Place in a 37 °C incubator overnight.

(ii) The next day, wash the 24-well plate with 1× PBS and add 0.5 ml of ESC medium.

(iii) Identify drug-resistant colonies by scanning the dishes (from Step 56B(xxviii)) with an inverted microscope and circle the resistant colonies underneath the dish with a permanent marker.

(iv) Prepare calibrated pipettes by pulling them over a flame to refine the tip. The new ends should be shaped in the form of a small needle with an opening.

(v) Set up a stereomicroscope in the tissue culture hood. It can be wiped with a 70% (vol/vol) ethanol solution to improve sterility.

(vi) Assemble the mouth pipette by attaching rubber tubing on either side of a Millex GS filter unit (0.22 ÎŒm). Attach the small red mouthpiece to one end (the rubber tubing and the mouthpiece are supplied along with the calibrated pipettes).

(vii) Spray the calibrated pipette with 70% (vol/vol) ethanol and wipe off with a Kimwipe.

(viii) Attach the calibrated pipette to the other end of the mouth pipette.

(ix) While viewing with the dissecting microscope, break up each colony into tiny fragments with the calibrated pipette and aspirate the fragments by suction.

(x) Plate the fragments from each colony into a separate well of the 24-well MEF plate prepared in Step 57B(i).

CRITICAL STEP Use a separate calibrated pipette for each clone to avoid contamination.

(xi) After picking the clones, stain the dishes with Coomassie blue to determine the number of CFUs. This will identify the colonies that were not picked.

(xii) If required, carry out further passaging and expansion of the clones for freezing and DNA extraction using 0.1% (wt/vol) Dispase solution, as described in Step 56B(x–xviii), using larger wells and dishes as needed. ESCs/iPSCs should be passaged twice a week to prevent them from differentiating. We typically passage ESCs/iPSCs to a 6-cm dish
for DNA extraction.

Genomic DNA isolation ● TIMING Overnight incubation and 2 h

58. Remove the medium from the tissue culture dishes from Steps 57A(viii) or 57B(xii), rinse once with 1× PBS and aspirate. Let the dishes air-dry. One dense 6-cm ESC/iPSC dish containing approximately 1 X 10^6 cells yields ~20 ÎŒg of genomic DNA. A 10-cm MSC dish containing approximately 1 X 10^6 yields up to 20 ÎŒg of DNA.

PAUSE POINT The dishes can be wrapped in laboratory film and frozen at − 20 °C for up to 6 months, if desired. Dishes should be thawed at room temperature on a bench before use.

59. Add 750 ÎŒl of genomic DNA lysis buffer per dish. Incubate for 10 min with gentle, occasional swirling.

60. Scrape cells with a rubber policeman, pour into a 2-ml microcentrifuge tube. The solution should be very viscous and mostly clear.

61. Incubate at room temperature overnight on a rotating shaker (ten rotations per min).

62. The next day, add 4.5 ÎŒl of RNAse A (4 mg ml − 1 stock) once the DNA is in solution. Mix samples by inverting 25 times and incubate at 37 °C for 60 min.

63. Cool samples to room temperature. Add 250 ÎŒl of protein precipitation solution to the RNAse-treated cell lysate.

64. Vortex to mix the cell lysate with the protein precipitation solution.

65. Centrifuge at 17,900g (typically full speed) for 10 min at room temperature in a microcentrifuge. The precipitated proteins will form a tight pellet.

66. Gently pour the supernatant into a fresh microcentrifuge tube. If the protein pellet is not tight, then vortex, incubate on ice for 3 min and centrifuge again at 17,900g for 5 min at room temperature in a microcentrifuge.

67. Pour the supernatant into a fresh microcentrifuge tube containing 750 ÎŒl of 100% isopropanol, leaving behind the precipitated protein. Mix the samples by inverting gently 50 times.

68. Centrifuge at 17,900g for 5 min at room temperature in a microcentrifuge. The DNA will be visible as a small white pellet.

69. Add 500 ÎŒl of 70% (vol/vol) ethanol and invert the tube several times to wash the pellet.

70. Centrifuge at 17,900g for 5 min in a microcentrifuge at room temperature. Pour off the ethanol carefully.

71. Invert and drain the tube on a clean absorbent paper and allow to air-dry for 10 min.

CRITICAL STEP The pellet may not be very tight; drain the ethanol gently, taking care not to disturb the pellet at the bottom.

72. Add 100 ÎŒl of TE buffer. Hydrate the DNA by incubating at 65 °C for 1 h and overnight at room temperature.

PAUSE POINT For long-term storage, store at − 20 °C.

Screening for targeted clones by Southern blotting ● TIMING 5 d

73. Digest 5–10 ÎŒg of genomic DNA samples with 50 U (5 U ÎŒg–1 of DNA) of restriction enzyme for 8 h. Half of the restriction enzyme can be added at the beginning and the other half can be added after 4 h.

PAUSE POINT The restriction digests can be stored at − 80 °C and heated to 56 °C for 3 min before loading.

74. Pour an 0.8% (wt/vol) agarose gel in 1× TAE, and load 5 ÎŒg of the digested samples after mixing with the loading dye.

CRITICAL STEP High-molecular-weight genomic DNA is sometimes not uniformly distributed in TE, which can lead to inaccurate measurement before digestion. Thus, the digested DNA samples need to be requantitated on a fluorometer before loading onto the gel. λ/HindIII DNA can be used as size markers.

75. Run the gel at 120 V for 4–5 h in 1× TAE buffer.

76. Perform Southern blotting as described in Steps 41–54. A representative Southern blot is shown in Figure 2.

PREPARATION AND LOADING OF IODIXANOL GRADIENT

Preformed iodixanol step gradients are used to expedite the centrifugation step. The 40% and 25% iodixanol steps are used to remove contaminants with lower densities including empty capsids, and a 60% step serves as a cushion for genome-containing recombinant AAV virions. To clearly see the steps, phenol red is included in the upper 25% and lower 60% steps. The first step (15%) has 1 M NaCl to destabilize ionic interactions between macromolecules. The following volumes are for two gradients using OptiSeal tubes. The 1.27 mm x 89 mm spinal needle holds ~0.4 ml.
1. Prepare the following iodixanol solutions:
15% Iodixanol step: Mix 4.5 ml of 60% iodixanol and 13.5 ml of 1 M NaCl/PBS-MK buffer.
25% Iodixanol step: Mix 5 ml of 60% iodixanol, 7 ml of PBS-MK buffer and 30 ÎŒl of Phenol red.
40% Iodixanol step: Mix 6.7 ml of 60% iodixanol and 3.3 ml of PBS-MK buffer.
60% Iodixanol step: Mix 10 ml of 60% iodixanol and 45 ÎŒl of Phenol red.
2. Overlay each centrifuge tube with these solutions in the order below using a 10-ml syringe and 1.27 mm × 89 mm spinal needle, taking care to avoid bubbles. The same needle can be used for loading all steps.
5 ml of 60% iodixanol step
5 ml of 40% iodixanol step
6 ml of 25% iodixanol step
9 ml of 15% iodixanol step
3. Continue from Step 25 of PROCEDURE.

PURIFICATION OF AAV2 STOCKS BY HEPARIN AFFINITY COLUMN FOLLOWED BY DESALTING

1. Remove the top cap from the HiTrap heparin affinity column and add a few drops (~200 ÎŒl) of PBS-MK buffer to the top of the column to avoid air bubbles.

2. Connect the HiTrap Luer adaptor to the top of the column. Some of the buffer will overflow; absorb this with Kimwipes.

3. Add a few drops (~100 ÎŒl) of PBS-MK buffer to the top Luer adaptor.

4. Place the 10 ml syringe with 10 ml PBS-MK buffer into the Luer adaptor.

5. Remove the bottom and snap it off.

6. Wash the column with PBS-MK buffer. It takes ~8 min to deliver 10 ml PBS-MK.

CRITICAL STEP Air bubbles must be removed from the liquid in the syringe at all steps before pushing it through the column, as they tend to impede flow. Do not exceed a flow rate of 4 ml/min.

CRITICAL STEP Each time a step is completed, replace the bottom cap before you remove the syringe from the column, then remove the syringe and add 100 ÎŒl of PBS-MK buffer to the Luer adaptor. When the syringe is placed in the Luer adaptor for the next step,remove the bottom cap.

7. Load the column using the 10-ml syringe containing the iodixanol fraction (from Step 29 of PROCEDURE). Use a syringe pump and rate of 20 ml/h. Reserve approximately 25–30 ÎŒl before loading on the column for quantitation.

8. Collect the flow-through in a 14-ml tube.

9. Wash column with 5 ml of PBS-MK buffer using a syringe pump at the rate of 20 ml/h.

10. Collect the flow-through in a 14-ml tube.

11. Elute vector from the column with 2.4 ml of 1 M NaCl/PBS-MK buffer.

12. Collect the first 0.4 ml in a microcentrifuge tube. This is the ‘void volume’.

13. After this initial 0.4-ml void volume, collect the following fractions: fraction 1 is 1 ml, fraction 2 is 0.5 ml and fraction 3 is 0.5 ml. The vector should come off the column in the first 1.5 ml (fractions 1 and 2).

14. Desalt after the heparin affinity column purification with a HiTrap desalting column. Wash the HiTrap desalting column with 25 ml of DMEM (or other medium/buffer) in a 30-ml syringe.
CRITICAL STEP Do not exceed a flow rate of 5 ml/min.

15. Prepare the following:
A 3-ml syringe containing 1.5 ml of fractions 1 and 2.
A 3-ml syringe containing 3 ml of DMEM high glucose (or other medium/buffer to be used when transducing cells).

16. Position the column to collect fractions into microcentrifuge tubes: the first tube for the 1.5 ml of void flow-through, then ‘exchange’ fractions X1–X3 (1 ml each).

17. Load the 1.5 ml of vector into the 3-ml syringe.

18. Quickly load the 3 ml of DMEM high glucose or other medium in the second 3-ml syringe.

19. The vector should be in fractions X1 and X2. Pool fractions X1 and X2 and make 10 aliquots of 200 ÎŒl each (or other aliquot sizes) in microcentrifuge tubes. Prepare 1:10 and 1:100 dilutions of the AAV stock in TE buffer for determining titer later.

The vector stock and dilutions can be stored at –80 °C indefinitely. If necessary, store stocks in small aliquots and
avoid repeated freezing and thawing.

PURIFICATION OF AAV2 STOCKS BY HEPARIN AFFINITY COLUMN FOLLOWED BY DESALTING-2

1. Equilibrate HiTrap heparin affinity column with 10 ml PBS-MK at 1 ml/min.

2. Load the column with AAV at 0.3 ml/min.

3. Collect the flow-through in a 14-ml tube.

4. Wash column with 5 ml of PBS-MK buffer using a syringe pump at the rate of 0.3 ml/min.

5. Collect the flow-through in a 14-ml tube.

6. Elute AAV from the column with 2.4 ml of 1 M NaCl/PBS-MK buffer.

7. Collect the first 0.4 ml in a microcentrifuge tube. This is the ‘void volume’.

8. After this initial 0.4-ml void volume, collect the following fractions: fraction 1 is 1 ml, fraction 2 is 0.5 ml and fraction 3 is 0.5 ml. The AAV should come off the column in the first 1.5 ml (fractions 1 and 2).

9. Desalt using a HiTrap desalting column. Wash the HiTrap desalting column with 25 ml of PBS in a 30-ml syringe.

Do not exceed a flow rate of 5 ml/min.

10. Prepare the following:
A 3-ml syringe containing 1.5 ml of fractions 1 and 2.
A 3-ml syringe containing 3 ml of PBS.

11. Position the column to collect fractions into microcentrifuge tubes: the first tube for the 1.5 ml of void flow-through, then ‘exchange’ fractions X1–X3 (1 ml each).

12. Load the 1.5 ml of AAV into the 3-ml syringe.

13. Quickly load the 3 ml of PBS in the second 3-ml syringe.

14. The AAV should be in fractions X1 and X2. Pool fractions X1 and X2 and make 10 aliquots of 200 ÎŒl each (or other aliquot sizes) in microcentrifuge tubes. Prepare 1:10 and 1:100 dilutions of the AAV stock in TE buffer for determining titer later.

The vector stock and dilutions can be stored at –80 °C indefinitely. If necessary, store stocks in small aliquots and avoid repeated freezing and thawing.

Buffers and Reagents

Glass Pasteur pipettes (sterile, cotton plugged)
• Hemocytometer (Brightline Hemacytometer, Hausser Scientific)
• High-speed centrifuge (RC 5B Plus, Sorvall)
• HiTrap Desalting column (GE Healthcare, cat. no. 17-1408-01)
• HiTrap heparin affinity column (1.0 ml; GE Healthcare,cat. no. 17-0406-01)
• H2O bath at 37 °C (183, Precision Scientific)
• Hybridization cylinders (Robbins Scientific)
• Hybridization incubator (400, Robbins Scientific)
• Inverted microscope (DMLS, Leica)
• Irradiator with a Cesium-137 source (J.L. Shepherd and Associates,model no. 143-5A)
• Kodak 480 RA processor (X-OMAT, KODAK)
• Liquid nitrogen tank (CryoMed, Forma Scientific)
• Luer adaptor, Union Luer female (GE Healthcare, cat. no. 18-1112-51)
• Microcentrifuge (5415C, Eppendorf)
• Millex GS filter unit (0.22 ÎŒm; Millipore, cat. no. SLGS033SS)
• Mouth pipette (supplied along with the calibrated pipette except for the 0.2-ÎŒm Millex GS filter unit; VWR International, cat. no. 53432-783)
• Needles (18- and 20-gauge)
• PhosphorImager (Storm 820, Amersham)
• Rotating shaker (Labquaker shaker 400110, Barnstead/Thermolyne)
• Rubber policeman
• Screw-cap micro tubes (Sarstedt, cat. no. 72.692.005)
• Spinal needle with blunt end (1.27 mm × 89 mm)
• Stereomicroscope (MZ6, Leica)
• Stericup and Steritop vacuum-driven filtration system (0.22-ÎŒm pore,GP Millipore Express PLUS membrane; Millipore, cat. no. SCGPT01RE)
• Syringe pump (Legato 200, KD Scientific)
• Syringes (10 ml)
• Tabletop centrifuge, ventilated and refrigerated (model no. 5677,Forma Scientific)
• Tissue culture incubator (37 °C with 5% CO2 atmosphere; Steri-Cult 200,Forma Scientific)
• Ultracentrifuge (XL-80, Beckman)
• UV Stratalinker (2400, Stratagene)
• UV transilluminator (FBT1V-88, Fisher)
• Water bath shaker (C76, New Brunswick Scientific)
• RepeatMasker software (http://www.repeatmasker.org/)
• ImageQuant software (GE Healthcare)
• Kimwipes

REAGENT SETUP

CRITICAL Unless stated otherwise, reagents can be stored indefinitely at the specified temperature.

CaCl2 (2.0 M) Dissolve 29.4 g of CaCl2·6H2O in a final volume of 100 ml dH2O. Sterilize by passing through a 0.22-ÎŒm filter and store in 2-ml aliquots at 4 °C. Cell lysis buffer (for AAV stock preparation) Add 3 ml of 5 M NaCl and 5 ml of 1 M Tris-HCl (pH 8.5) to 80 ml of dH2O. Adjust the pH to 8.5 with NaOH and adjust the volume to 100 ml with dH2O. Sterilize by passing through a 0.22-ÎŒm filter and store at 4 °C.

Church buffer Combine 500 ml of 1 M phosphate buffer (pH 7.2), 2 ml of 0.5 M EDTA, 10 g BSA and 70 g SDS in a final volume of 1 liter with dH2O. Store at room temperature (25 °C).

Coomassie blue stain Combine 1.5 g Coomassie Brilliant Blue G, 300 ml methanol and 100 ml glacial acetic acid in a final volume of 1 liter with dH2O. Store at room temperature.
D10 (DMEM with 10% FBS (vol/vol)) Combine 500 ml of DMEM high glucose with 50 ml of FBS, 5 ml of penicillin/streptomycin and 2.5 ml of amphotericin B (final concentration, 1.25 ÎŒg ml − 1). There is no need to filter-sterilize the solution as all components are sterile. Store at 4 °C and use within 1 month.

Dispase solution (0.1% (wt/vol)) Dissolve 100 mg of Dispase in 100 ml of ESC wash medium. Sterilize by passing through a 0.22-ÎŒm filter and store at 4 °C. Use within 1 week.

EDTA solution 0.5 M (pH 8.0) Dissolve 186.1 g of disodium EDTA·2H2O in 700 ml of H2O. Adjust the pH to 8.0 with NaOH and adjust volume to 1 liter with dH2O. Sterilize by autoclaving and store at room temperature.

ESC medium Combine 500 ml of F12/DMEM, 100 ml of KSR, 6 ml of penicillin/streptomycin, 6 ml of sodium pyruvate, 6 ml of nonessential amino acids, 0.6 ml of 0.1 M β-mercaptoethanol and 0.3 ml of a 4-ÎŒg/ml bFGF solution. Sterilize by passing through a 0.22-ÎŒm filter and store at 4 °C. Use within 2 weeks.

ESC wash medium Add 50 ml of KSR to 500 ml of DMEM high glucose. There is no need to filter sterilize as both components are sterile. Store at 4 °C for up to 2 months.

Gelatin (0.1% (wt/vol)) Dissolve 5 g of gelatin in 1 liter of dH2O to prepare 0.5% stock solution, sterilize by autoclaving and store at room temperature. Prepare a 0.1% gelatin working solution as a 1:5 dilution of the 0.5% stock in sterile dH2O. Store at room temperature.

Genomic DNA lysis solution Combine 1 ml of 1 M Tris-HCl (pH 8.0), 0.2 ml of 0.5 M EDTA (pH 8.0) and 0.1 g of SDS in a final volume of 100 ml with dH2O. Store at room temperature.

HEPES-saline (2×) Dissolve 8.18 g of NaCl and 5.96 g of HEPES in 400 ml dH2O. Adjust pH to 7.1 with 0.5 N NaOH. Adjust the volume to 500 ml with dH2O. Sterilize by passing through a 0.22-ÎŒm filter and store in aliquots at 4 °C.

Irradiated MEFs MEFs are used as feeder cells for the culture of ESCs/iPSCs. DR4 MEFs are used because they are resistant to G418, 6-thioguanine, puromycin and hygromycin, and can be used in experiments that use these selective agents.
CRITICAL Prepare the irradiated MEFs in advance as described in Box 1.

KCl (250 mM) Dissolve 1.86 g of KCl in dH2O in a final volume of 1 liter. Sterilize by autoclaving and store at room temperature.

Loading dye (6×) Combine 5 ml of glycerol, 25 mg of bromophenol blue and 25 mg of xylene cyanol in sterile dH2O in a final volume of 10 ml. Store at 4 °C in 1-ml aliquots.

LB medium (Luria-Bertani medium) Dissolve 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl in dH2O in a final volume of 1 liter. Adjust pH to 7.0 with 5 N NaOH. Autoclave and store at room temperature for up to 3 months.

MEF freezing medium To prepare 15 ml of freezing medium, combine 1.5 ml of FBS, 1.5 ml of DMSO and 12 ml of MEF medium. Prepare fresh before use.

MEF medium Mix 500 ml of DMEM high glucose with 50 ml of FBS and 5.6 ml of penicillin/streptomycin. There is no need to filter-sterilize the medium, as all components are sterile. Store at 4 °C and use within 1 month.

MgCl2 (2 M) Dissolve 19 g of MgCl2 in dH2O in a final volume of 100 ml. Sterilize by autoclaving and store at room temperature.

MSC medium Combine 500 ml of DMEM low glucose with 50 ml of FBS, 5.6 ml of l-glutamine and 5.6 ml of penicillin/streptomycin. There is no need to filter-sterilize the medium as all components are sterile. Store at 4 °C and use within 1 month.

NaCl/PBS-MK buffer (1 M) Dissolve 5.84 g of NaCl, 26.3 mg of MgCl2 and 14.91 mg of KCl in 1× PBS in a final volume of 100 ml. Sterilize by passing through a 0.22-ÎŒm filter and store at 4 °C.

PBS-MK buffer Dissolve 26.3 mg of MgCl2 and 14.91 mg of KCl in 1× PBS in a final volume of 100 ml. Sterilize by passing through a 0.22-ÎŒm filter and store at 4 °C.

Phosphate buffer (0.15 M) Add 4.95 ml of 1.0 M NaH2PO4 and 10.05 ml of 1.0 M Na2HPO4 to dH2O in a final volume of 100 ml. Sterilize by passing through a 0.22-ÎŒm filter and store in aliquots at 4 °C.
Protein precipitation solution Combine 60 ml of 5 M C2H3KO2 and 11.5 ml of glacial acetic acid in a final volume of 100 ml with dH2O. Store at room temperature.

Salmon sperm DNA Dissolve salmon sperm DNA in dH2O at a concentration of 10 mg ml − 1. To dissolve the DNA, stir the solution for at least 2–4 h at room temperature. Adjust the concentration of NaCl to 0.1 M and extract the solution once with phenol and once with phenol/chloroform. Recover the aqueous phase and shear the DNA by passing the solution rapidly 12 times through a 17-gauge needle. Precipitate using ethanol, centrifuge and dissolve at a concentration of 10 mg ml − 1. Denature DNA by boiling for 10 min and store at − 20 °C in 1-ml aliquots. Boil for 5 min and snap-chill on ice before use in hybridization.

Super optimal broth (SOB) medium Dissolve 20 g of tryptone, 5 g of yeast extract and 0.5 g of NaCl in 900 ml of dH2O. Add 10 ml of 250 mM KCl and adjust pH to 7.0 with 5 N NaOH. Adjust the volume to 1 liter with dH2O and sterilize by autoclaving on a liquid cycle. Store at room temperature for up to 3 months. Add 5 ml of sterile 2 M MgCl2 before use.

AAV & Adeno

Day 0

Plate Ad-293 cells at 3e5 per well in 6 well plate (adeno) or 5e5 per 6 cm dish (AAV)

Day 1

Replace media

Adenovirus

A.  1 ug of PacI digested pAdE construct in 250 ul OPTI-MEM without serum

B.  3 ul of Lipofectamine 2000 in 250 ul OPTI-MEM without serum, incubate 5 minutes at RT

Gently mix A and B together and incubate for 20 minutes at RT. May be slightly cloudy.

Add complexes directly to a single well of six well plate.

48 hours post-transfection, trypisinize each well and transfer to a 10 cm plate.

Replace media every 2-3 days until 80% CPE

AAV

A.  1.5 ug of ITR AAV vector construct and 4.5 ug of packaging vector (DF1-6) in 500 ul OPTI-MEM without serum. (3:1 ITR:packaging vector ratio)

B.  15 ul of Lipofectamine 2000 in 500 ul OPTI-MEM without serum, incubate 5 minutes at RT

Gently mix A and B together and incubate for 20 minutes at RT. May be slightly cloudy.

Add complexes directly to a single 6 cm dish.

48 hours post-transfection, check for fluorescence and harvest cells with cell scraper. 80% of virus will be in cell pellet.

Size of culture vessel (surface area)	Approximate cells/plate for transfection	Volume of media/well	Volume of trypsin
24 well plate (2 cm2)	1 x 105	500 ul
6 well plate (35mm, 10 cm2)	3 x 105	2 ml	300 ul
6 cm dish (20 cm2)	5 x 105	5 ml	0.5 ml
10 cm dish (60 cm2)	2 x 106	12 ml	1 ml
15 cm dish (135 cm2)	1 x 107	25 ml	2 ml