Rapad cmv adenoviral expression system

Adenoviruses were generated using the RAPAd CMV adenoviral expression system (Cellbiolabs, San Diego, CA). High-throughput screening was performed at the University of Illinois High-Throughput Screening Facility. For each group, 1x 10⁷ C33a cells were plated in 150 mm² dishes in 20 ml cDMEM. For p53xTE6 group, 1 x 10⁸ GFU/mL of p53-Luc adenovirus and 1 x 10⁹ GFU/mL TE6 adenovirus were plated. For RbxTE7 group, 1 x 10⁸ GFU/mL of p60RbRen adenovirus and 1 x 10⁹ GFU/mL of βTrCP-E7 adenovirus were added. For control group, Ad-Red was substituted for TE6 or TE7 adenovirus in the p53 control and Rb control, respectively. 12-24h later, 10 μl cells were collected and plated in 384 well plates containing 10 μl DMEM with 20 μM compounds, making the final screening compound concentration of 10 μM, which is a concentration commonly used in cell-based high-throughput screens [13 (link), 14 (link)]. Experimental wells were plated with 2000 p53-TE6 cells and 2000 Rb-TE7 cells per well. Control wells were plated with 2000 p53-Red cells and 2000 Rb-Red cells per well. After 8h, plates were stored at -20 C for 1–3 days and assayed using Dual Glo Luciferase Assay (Promega). p53-Luc counts were normalized based to the control Rb-Ren activity within individual wells. Positive hits were scored as ≥ 2-fold above vehicle treated p53-Luc wells.

The preparation of recombinant adenoviruses expressing FLAG-Drp1K38A, FLAG-Drp1S637A, and GFP from the CMV promoter were as previously described [28 (link)] by using the RAPAd CMV Adenoviral Expression system (Cell Biolabs, Inc. San Diego, CA, USA.), following the manufacturer's instructions. To produce vectors expressing these genes under the GFAP promoter, the CMV promoter gene was removed from the pacAD5 CMV shuttle vectors and replaced with a rat GFAP promoter gene. Cloning was conducted by using standard protocols, and DNA was extracted and purified by using Zymo Research DNA kits (Cambridge Bioscience). The recombinant adenoviruses were amplified in HEK293 AD cells and then purified by using a sucrose-based method [35 (link)]. Briefly, virus-containing media was centrifuged at 3000 rpm for 5 min and passed through a 0.4 μm filter. The media was then loaded onto 10% Sucrose/TENS (50 mM Tris–Hcl pH 7.4, 100 mMNacl, 0.5 mM EDTA) in a 4:1v/v ratio and centrifuged at 10,000 rpm for 4 h at 4 °C. The resulting virus pellet was resuspended in PBS at 4 °C overnight and the purified virus was titrated using a DAB assay (Adenovirus Monoclonal Antibody (E28H) MA17001 Invitrogen, Vector Lab SK-4100 DAB kit). The titres of the adenoviruses injected in the animals were between 9.5 × 10⁸ pfu/ml and 1.5 × 10⁹ pfu/ml.