Hek293a cell line

An E1- and E3-deficient human adenovirus serotype 5, the ChAdOx2 derivative of the chimpanzee adenovirus (ChAd) 68 (34 (link)), and Modified vaccinia virus-Ankara (MVA (35 (link)) were used as vaccine vectors. DNA sequences corresponding to vaccine antigen designs were synthesized and cloned into plasmids containing attR1 and attR2 recombination sites (Gateway^® Life technologies, CA, USA) under control of a CMV promotor. Antigen-encoding inserts were then transferred into the vaccine vectors by recombination as previously described (36 (link)–38 (link)). The adenoviral vaccine candidates were produced as previously described using the HEK293A cell line (Invitrogen), which contains a stably integrated copy of the E1 gene that supplies the E1 proteins (E1a and E1b) required to generate recombinant adenovirus (39 (link)), by the Viral Vector Core Facility of the Jenner Institute, University of Oxford, UK. MVA vaccines were produced using the DF-1 cell line and purified using sucrose cushion centrifugation (40 (link)). Virally vectored vaccines were formulated in endotoxin-free PBS. All constructs were verified by sequencing. Vaccine constructs generated are summarized in Table 1 and described further in the Results.

HepG2 cells were obtained from ATCC. The HEK293A cell line was obtained from Invitrogen. Mouse primary hepatocytes were isolated from male C57BL/6J mice at San Francisco General Hospital Liver Center and were cultured as we previously described (14 (link)). HepG2 and Huh7 human hepatic cells were cultured in MEM with 10% FBS.

Plasmid DNA with cloned genes, turboGFP mouse monoclonal antibody (cat. TA150041, clone OTI2H8) was purchased from OriGene Technologies, Inc, USA. The following vectors were used: SLC17A1 (cat. RG211006), SLC22A11 (cat. RG206469), and SLC22A13 (cat.RG222116) cloned in pCMV6-AC-GFP with C-terminal turbo GFP tag. The HEK293A cell line was purchased from ThermoFisher Scientific, USA (cat. R70507). DMEM, fetal bovine serum (FBS), and other cultivation media and supplements were also from the ThermoFisher Scientific. The secondary antibody conjugated with HRP (cat.A90-117P) was purchased from Bethyl-Fortis Life Science, USA. The β-actin primary antibody (clone 8H10D10) was purchased from Cell Signaling, USA. Cultivation plastic for cells was purchased from VWR. Radiolabeled uric acid (MC-1394) was purchased from the Hartman Analytic GmbH, Germany. Western blot material and all common chemicals were from Merck KGaA, Germany, or Penta s.r.o, Czech Republic. Primers for sequencing and mutagenesis were synthesized by Generi biotech, Czech Republic. For measurement of radioactivity, we used liquid scintillant Ultima Gold and a TriCarb 2900TR scintillation counter (USA).

The HEK293A cell line was provided by Thermo Fisher Scientific (MA, USA) and maintained in DMEM (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% (v/v) foetal bovine serum (Thermo Fisher Scientific). The Hap1 cell line was provided by Horizon Genomics (Vienna, Austria) and maintained in IMDM (GE Healthcare, IL, USA) supplemented with 10% (v/v) foetal bovine serum. Cells were cultured at 37°C in a humidified incubator continuously flushed with a mixture of 5% CO₂ and 95% air. Cells were authenticated with a morphology check by microscopy and using growth curve analysis with an Operetta CLS instrument (PerkinElmer, MA, USA). All cell lines were confirmed to be mycoplasma-free.
Tandem ER stress response element-2 (ERSE2), unfolded protein response element (UPRE), amino acid response element (AARE) and heat-shock element (HSE) were assembled by Golden Gate cloning with the Multiplex CRISPR/Cas9 Assembly System Kit (Addgene Kit # 1000000055, MA, USA). 10X ERSE2-10X UPRE-5X AARE (EUA), 25X ERSE2, 25X UPRE, 25X AARE and 5X HSE-EGFP reporter vectors were constructed based on the lenti-Cas9-blast vector (gift from Feng Zhang [Addgene plasmid # 52962]). The DNA sequence of each construct was verified on an ABI 3130 DNA sequencer (Thermo Fisher Scientific). HEK293A and Hap1 cells stably expressing the reporter gene were sorted with an S3e Cell Sorter (Bio-Rad, CA, USA).