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

=== (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.