Editing AAV-Production-293T-2 (section)

== <br>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 â TIMING 14 d<br>  ===

<br> (i) Harvest MSCs by trypsin treatment, as described in Steps 2â4, 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 Ã 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 2,000â20,000 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<br>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 8â10 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 â TIMING 20 d<br>  ===

<br> (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 Ã 105 irradiated (mitotically inactivated) MEF cells in MEF medium (prepared as described in Box 1).<br> 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 1Ã PBS and replenish with 3 ml of ESC medium. Return the dish to the 37 °C incubator.<br> <br> 

(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 Ã 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.<br> 

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

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(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. 

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(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 Ã 105 MEFs per well and the 6-cm dish with 3.3 Ã 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.<br> <br> (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 manufacturerâs 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 Ã 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 2,000â20,000 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 7â8 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.<br> <br> (xxvi) As the cells are spinning, remove the medium from the six-well plate, wash once with 1Ã PBS and replenish with 1 ml of fresh ESC medium per well.<br> <br> (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 Ã 105 (9.1 Ã 104 per well) MEFs are required. Place in a 37 °C incubator.<br> <br> (xxviii) Maintain the dishes in selection media for the next 8â10 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 â TIMING 0.5 h<br> (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 1Ã 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 â TIMING 1 h  ===

<br> (i) Seed a 24-well plate with 3.7 Ã 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 1Ã 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).<br> <br>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(xâxviii), 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<br>for DNA extraction. 

=== Genomic DNA isolation â 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 1Ã 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 â 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 â 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 â 20 °C. 

Screening for targeted clones by Southern blotting â TIMING 5 d 

73. Digest 5â10 ÎŒg of genomic DNA samples with 50 U (5 U ÎŒgâ1 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 â 80 °C and heated to 56 °C for 3 min before loading. 

74. Pour an 0.8% (wt/vol) agarose gel in 1Ã 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. Î»/HindIII DNA can be used as size markers. 

75. Run the gel at 120 V for 4â5 h in 1Ã TAE buffer. 

76. Perform Southern blotting as described in Steps 41â54. A representative Southern blot is shown in Figure 2.<br> 

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