Virapower adenoviral gateway expression kit

Plasmids containing IR-ECD/IGF1R-ICD or IGF1R-ECD/IR-ICD chimeric receptor were kindly provided by Dr. C. Ronald Kahn (Joslin Diabetes Center, Boston, MA, USA). The chimeric receptors were cloned into adenovirus backbone plasmid using ViraPower Adenoviral Gateway Expression kit (Invitrogen, Carlsbad, CA, USA). The VSMCs were infected with adenovirus at 100 MOI.

To generate recombinant adenoviruses expressing Cdc25A-wild type (wt) or mutants, DNA encoding wt or mutant Cdc25A (S76A or S82A) was, respectively, excised from pCDNA4/TO-Cdc25A-wt, S76A, or S82A [51 (link)] (gifts from Dr. Xianghong Zou, Ohio State University, Columbus, OH, USA) using BamH1 and EcoR1. The recombinant adenoviruses and the control virus expressing green fluorescence protein (GFP) were generated and amplified using ViraPower^™ Adenoviral Gateway^™ Expression Kit (Invitrogen, Carlsbad, CA, USA) following the manufacture's instruction. All adenoviruses were amplified, titrated and used as described previously [52 (link), 53 (link)].

To generate adenovirus expressing USP7 or LacZ (negative control) with C-terminal sequential peptide affinity (SPA)-tag^{98 (link)}, the coding sequence for the SPA tag was first inserted between the EcoRI and NotI sites of the multicloning site of pENTR4 (Life Technologies). The USP7 or LacZ coding sequences were then individually inserted between the BamHI and KpnI sites of the multicloning sites to generate constructs expressing C-terminally tagged USP7 or β-Gal. USP7-SPA and LacZ-SPA were transferred to the pAd⁄CMV⁄V5-DEST adenovirus expression vector using the ViraPower™ Adenoviral Gateway™ expression kit (Invitrogen). Adenovirus was generated and amplified in HEK293A cells as per the manufacturer’s instructions. The virus was titred onto AGS cells, followed by immunofluorescence microscopy and Western blotting using antibody against the FLAG epitope in the SPA tag (F1804 from Sigma; 1:800 dilution) to verify expression of the full length proteins and determine the minimum amount of virus that could be used to infect most of the cells.

The shuttle vector for making recombinant adenoviruses containing the GADD45G gene (Ad-GADD45G) was synthesized based on Gateway technology using ViraPower Adenoviral Gateway Expression Kit (Invitrogen) as per the manufacturer’s instructions. The human GADD45G full-length open reading frame (ORF) clone (Ultimate ORF clone, Invitrogen) was provided in an entry vector (Gateway pENTR221, Invitrogen). The GADD45G ORF was cloned into the destination vector pAd/CMV/V5-DEST by using LR Clonase II enzyme (Invitrogen) to synthesize pAd/CMV/V5-GADD45G. pAd/CMV/V5-GADD45G was then digested with Pac I enzyme and transfected into HEK293A cells using Lipofectamine 2000 (Invitrogen) to produce recombinant adenoviruses (Ad-GADD45G). For use as controls, pAd/CMV/V5/lacZ for β-galactosidase expression was provided with the kit and recombinant adenoviruses containing the lacZ gene (Ad-LacZ) were generated. The shuttle vector for making recombinant adenoviruses expressing hemagglutinin (HA)-tagged MAGEH1 proteins (Ad-HA-MAGEH1) was synthesized by Applied Biological Materials (Ferndale, WA) For purification and concentration, the Adeno-X maxi purification kit (Takara, Mountain View, CA) was used. For titration, HEK293A cells infected with recombinant adenoviruses were detected using an antibody specific for the adenovirus hexon protein with the Adeno-X rapid titer kit (Takara).

HEK293T and Plat-E cells were transfected using ExtremeGENE9 (SIGMA) as described in the manufacturer’s protocol. Splenic macrophages were infected with retroviruses using a Platinum Retrovirus Expression System (Cell Biolab). Infected cells were selected in MEMα containing 2 μg/mL puromycin for 2 days, cultured for a further 2 days in the absence of the antibiotic and then incubated with 200 ng/mL RANKL for 6 days. Infected macrophages were incubated with RANKL for longer than non-infected macrophages because osteoclast differentiation occurred more slowly after retrovirus infection and selection with puromycin. Recombinant adenoviruses were constructed using the ViraPower Adenoviral Gateway Expression Kit (Invitrogen). The obtained adenoviruses were used to express EGFP-Rab7 variants in osteoclasts induced from macrophages.

Primary mouse hepatocytes were cultured in William’s medium E (Thermo Fisher Scientific) supplemented with 10% foetal bovine serum at 37 °C in 5% CO₂. Cells were treated with either an adenovirus (ViraPower Adenoviral Gateway Expression Kit, Thermo Fisher Scientific) expressing mouse Nrg1α or siRNA (Supplemental Table 1, Integrated DNA Technologies KK, Tokyo, Japan) targeting for mouse Nrg1 24 h before stimulation with 500 μM 8-bromo-cAMP (Cayman Chemical, Ann Arbor, MI, USA) and 100 nM dexamethasone (Sigma) for 3 h. Viruses encoding β-galactosidase were used as a control. A multiplicity of infection of 50 for each virus was used for the infection. In some experiments, cells were pretreated with rNRG1α (1 or 10 nM) 30 min, or inhibitors such as Ly294002 (Wako Pure Chemical Industries), PD98059 (Cayman Chemical) or lapatinib (BioVision, Inc, Milpitas, CA, USA) 1 h before the stimulation. The conditioned media (24 h incubation) of isolated hepatocytes were collected to determine whether the N-terminal region of NRG1 endogenously expressed in hepatocytes was cleaved and released.

The recombinant adenoviruses expressing the selected ASFV antigens, as well as the negative control Ad-Luc (Ad-Luciferase), were generated, scaled up, and titrated as previously described (infectious focus units [IFU]) [17 (link),18 (link),19 (link)]. Thirty-five antigens were used to generate adenovirus expression constructs. Due to its large size, the pp220 polyprotein (Georgia 2007/1 isolate; GenBank Accession FR682468) sequence was split into two parts designated p220.1 and p220.2 (Table 1). Briefly, the polypeptide sequences of the ASFV antigens were modified to add in-frame, HA, and FLAG tags at their N- and C-termini, respectively. The modified protein sequences were used to generate synthetic genes (GenScript) which were codon-optimized for expression in swine. All the genes were then used to generate recombinant replication-incompetent adenoviruses encoding the ASFV genes (designated Ad5-ASFV 1 to 10) using the ViraPower™ Adenoviral Gateway™ Expression Kit (Thermo Fisher Scientific K493000). Antigen expression by the adenoviruses was confirmed as previously described [17 (link),18 (link),19 (link)] (Figure 1).