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

=== (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.