Adeasier 1 cells

HCMV UL7 containing an HiBiT sequence (5′-GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC-3′) after the signal peptide (nucleotide 142) or the regions approximately 100 bp up- and downstream of miR-US5-1 and miR-UL112 was cloned into the shuttle vector pAdTrack-CMV and transformed into TOP10 cells. After 24 h of incubation at 37°C under kanamycin selection, colonies were selected and screened for the insert by restriction enzyme digest using KpnI and XhoI. The pAdTrack plasmids were then linearized by digesting with restriction endonuclease PmeI and subsequently recombined into Escherichia coli BJ5183 cells containing the adenoviral backbone plasmid pAdEasy-1 (AdEasier-1 cells). pAdTrack-CMV (Addgene plasmid catalog no. 16405) and AdEasier-1 cells (Addgene catalog no. 16399) were a gift from Bert Vogelstein (56 (link)). Recombinants were selected for kanamycin resistance, and the recombination was confirmed by restriction endonuclease analyses. Finally, the recombinant plasmids were linearized with PacI before transfection with Lipofectamine 2000 (ThermoFisher) into the adenovirus packaging cell line HEK293. The control vector Ad GFP, Ad miR-UL112-3p, Ad miR-US5-1, and Ad UL7 were produced and purified and their titers were determined in HEK293 cells, as previously described (27 (link)). The Ad-GFP-U6-h-FOXO3a-shRNA (shADV-209273) was purchased at Vector Biosystems, Inc.

pAdTrack-CMV (Addgene plasmid #16405) and AdEasier-1 cells (Addgene #16399) were gifts from Bert Vogelstein (52 (link)). The pAdTrack plasmids were linearized by digesting with restriction endonuclease Pme I and subsequently recombined into E. coli BJ5183 cells containing the adenoviral backbone plasmid pAdEasy-1 (AdEasier-1 cells). Recombinants were selected for kanamycin resistance, and the recombination confirmed by restriction endonuclease analyses. Finally, the recombinant plasmids were linearized with PacI before transfection with Lipofectamine 2000 (Thermo Fisher) into the adenovirus packaging cell lines HEK293 cells. The control vector Ad-GFP, Ad-HiBiTUL7, Ad-HiBiTUL8-V5, and Ad-HiBiTUL8-mutDTEL-V5 were produced, purified, and titered in HEK293, as previously described (48 (link)).