Calcium phosphate

To produce the AAV serotype 9 vectors, AAV-293 cells were cotransfected using calcium phosphate (Invitrogen, Carlsbad, CA, USA) with the AAV plasmid of interest, a chimeric packaging construct, and an adenoviral helper plasmid, as described previously.³³ (link) Cells were harvested two days post-transfection and lysed by successive freeze/thaw cycles and sonication, followed by Benzonase (Novagen, Madison, WI, USA) and deoxycholic acid (Sigma Aldrich, St. Louis, MO, USA) treatments and 3 consecutive rounds of cesium chloride (Invitrogen, Carlsbad, CA, USA) density gradient ultracentrifugation. Fractions containing the AAV vector particles were collected and dialyzed in Dulbecco’s PBS (GIBCO, BRL) containing 1 mM MgCl₂. Quantitative real-time PCR with SYBR Green and primers for the HS-CRM8-TTRmin (Cas9 vector) or U6 promoter (gRNAs) were used to determine vector titers. The HS-CRM8-TTRmin forward primer sequence was 5′-GGAGGCTGCTGGTGAATATT-3′ and the reverse primer sequence was 5′- TCCAAACCTGCTGATTCTG-3′. The U6 forward primer sequence was 5′-GCAGGCTTTAAAGGAACCAA-3′ and the reverse primer sequence was 5′- ACTGCAAACTACCCAAGAAAT-3′. To generate the standard curves, known copy numbers of the corresponding vector plasmids were used.

We expressed IFNλ4 in a Drosophila S2 cell expression system (Invitrogen). The cells were stably cotransfected with plasmids encoding IFNL4-Histag and a blasticidin resistance gene in a ratio of 19:1 using calcium phosphate (Invitrogen). Cells were then passed into ExpressFive serum-free medium (Invitrogen) containing 25 µg/ml blasticidin and scaled up under constant selection to 1 liter suspension cultures at 125 rpm in spinner flasks. At a density of 5.0 × 10⁶ cells/ml, cells were induced by 0.8 mM CuSO₄ to produce IFNλ4 for 8 d. Recombinant IFNλ4 protein was isolated from the supernatant by affinity chromatography, eluted in an imidazole gradient on a Ni²⁺-IDA-based His60 resin (Takara Bio Inc.). The eluate was analyzed by a Coomassie gel to identify enriched fractions, which were subsequently concentrated by ultracentrifugation columns and desalted in PBS using PD-10 columns (GE Healthcare). To remove nonspecifically bound proteins, we performed size exclusion chromatography using an ÄKTA 9 high pressure liquid chromatography system with a Superdex 200 analytical column (GE Healthcare). Finally, we added 0.1% BSA as a carrier protein and froze single-use aliquots in 20% glycerol.

S2 cells were maintained at 25°C in Schneider’s Drosophila medium (S9895, Sigma) supplemented with 10% FBS (F0718, Gibco), 100 U/ml penicillin (Life Technologies), and 100 μg/ml streptomycin (Life Technologies). Transfection of S2 cells was performed using calcium phosphate according to the manufacturer’s instructions (Invitrogen). For S2 cell immunostaining, 48 h after transfected, cells were harvested and washed with PBS. Cells were fixed in 4% formaldehyde in PBS buffer for 20 min at room temperature. Then, cells were treated with PBT for 20 min, washed with PBS for 20 min three times. To mark the nucleus, cells were stained with DAPI for 20 min after secondary antibody incubation. Images of cells were acquired under FV10-ASW Olympus confocal microscope. The following antibodies were used for immunoprecipitation and western blot: mouse anti-HA (F-7) (1:2500; Santa Cruz); mouse anti-Myc (9E10) (1:2500; Santa Cruz); mouse anti-Fg (M2) (1:5000; Sigma); LysoTracterRed (1:500; Sigma); MitoTracterRed (1:500; Sigma); ERTracterGreen (1:500; Sigma); mouse anti-Actin (1:5000; ABclonal Technology); goat anti-mouse HRP (1:10000; Abmax). The blots were visualized using a chemiluminescent detection kit (Millipore).