Protocol for Transduction of HepG2 Cells with Non-AAV2 Vectors Note: when perform in vitro cell-based assay, please keep in mind that Non-AAV2 vectors generally give lower level expression than AAV2 vectors. In addition, non-AAV2 vectors are sensitive to fetal bovine serum or any other type of serum. This protocol can be adapted to other type of mammalian cells, provided that the concentration of etoposide needs to be empirically determined. 1. Culture HepG2 cells until they are 70% ~ 90% confluent. 2. Trypsinize the cells and prepare cell suspension at concentration of 3 x 10 5 cells/ml. 3. Transfer 500μl to each well of 24-well plates and culture overnight. 4. In the next morning prepare 10-fold serial dilutions of AAV vectors in serum-free culture media containing 20μM etoposide* (etoposide makes transduction more robust). Prepare 2 ml for each dilution. (Suggested dilutions: 2 x 1010vg/ml, 2 x 109vg/ml, 2 x 108vg/ml, 2 x 107vg/ml, 2 x 106vg/ml) 5. Remove the media from the cells and gently rinse the cells with 0.5ml of serum-free media and remove again. 6. Add 500μl of the diluted AAV to each well. Prepare duplicates or triplicates for each dilution. Add just the media to last well as negative control. 7. The next morning, add 500μl of culture media containing 20% FBS. 8. Culture for 2 to 3 days and examine under fluorescence microscope if GFP or RFP is the marker gene. Or prepare for antibody staining of the cells with specific antibody.
Prepare stock solution of Etoposide (A.G. Scientific, San Diego, CA) Dissolve etoposide powder in DMSO to prepare 20mM (1000x) stock solution and store it at -20°C. *Note: the etoposide concentration for each mammalian cell line will be different and need to be empirically determined. For HepG2 and 293 cells, 20μl is tolerated and gives good expression.
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