Chicken serum

The avian cell lines DF-1 and QT6 were both obtained from the American Type Culture Collection. DF-1 cells were cultured at 39°C in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, The Netherlands) supplemented with 100 U ml^-1 penicillin, 100 U ml^-1 streptomycin, 2mM L-glutamine (PSG) and 10% Hyclone Characterized Fetal Bovine serum (FBS HC, Thermo Fischer Scientific, The Netherlands). QT6 cells were cultured at 37°C in Medium 119 (Lonza) supplemented with PSG, 5% FBS HC, 1% chicken serum (Sigma-Aldrich, Germany) and 5% tryptose phosphate broth (MP Biomedicals, Belgium). BSR-T7 (kind gift of K. Conzelmann) were cultured in DMEM supplemented with PSG and 10% FBS FC at 37°C. A549 were cultured in HAM’s F-12 (GIBCO, Life Technologies, The Netherlands). Vero cells and human pancreatic adenocarcinoma cell lines were cultured as previously described [31 (link)]. In case of virus infection experiments, 2% FBS was used in all media (infection media).

The 3T3-L1 fibroblasts were differentiated into mature adipocytes, as described above, and shock-frozen by ice-cold acetone (ROTH, Karlsruhe, Germany) for immunocytochemistry. The air-dried cells were incubated in PBS for rehydration. The endogenous peroxidase activity was blocked with 3% H₂O₂ (ROTH, Karlsruhe, Germany). To avoid non-specific protein binding, the cells were incubated in 10% bovine serum albumin (BSA, ROTH, Karlsruhe, Germany), 10% fetal calf serum (FCS, Sigma-Aldrich, Steinheim, Germany) and 10% chicken serum (Sigma-Aldrich, Steinheim, Germany), followed by 3 h incubation in a moist chamber with a polyclonal anti-TLR7 antibody from rabbit (2 µg/mL in 1% BSA; Invitrogen, Carlsbad, CA, USA). The cells were then stained with peroxidase-conjugated goat anti-rabbit IgG (1.6 µg/mL in 1% BSA; Jackson Immuno Research, West Grove, Pennsylvania, USA) for 90 min. The color development with 3-amino-9-ethylcarbazole (AEC) substrate (Vector Laboratories, Burlingame, CA, USA) at room temperature was stopped after microscopic examination. The rabbit isotype-matched IgG sera (ab37415; Abcam, Cambridge, UK) served as an isotype control. Parallel experiments without primary antibodies were carried out as negative controls.

Two independent biological replicates of micromass cultures were prepared from limb buds of embryonic day (E) 4.5 chick embryos, infected with RCAS-BP(A) retroviruses carrying no recombinant protein and cultivated for 5 days as described previously (Solursh et al., 1978 (link); Ibrahim et al., 2013 (link)). Briefly, ectoderm was dissociated by using a Dispase solution (Gibco) at 3 mg/ml and limb mesenchyme was digested by using a solution composed of 0.1% Collagenase type Ia (Sigma-Aldrich), 0.1% Trypsin (Gibco) and 5% FBS (Biochrom, Berlin, Germany) in DPBS (Gibco). Prior to seeding, mesenchymal cells were mixed with retroviruses (1:1) and maintained in culture for 5 days at 37°C in DMEM/Ham's F-12 (1:1) medium (Biochrom) supplemented with 10% FBS, 0.2% chicken serum (Sigma-Aldrich), 1% L-glutamine (Lonza, Basel, Switzerland) and 1% penicillin/streptomycin (Lonza).

Chicken PGCs were cultured in accordance with our standard procedure [2 (link)]. Briefly, PGCs from White Leghorn embryonic gonads at 6 days old (Hamburger-Hamilton stage 28) were maintained in knockout Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (FBS) (Hyclone, South Logan, UT, USA), 2% chicken serum (Sigma-Aldrich, St. Louis, MO, USA), 1× nucleosides (Millipore, Billerica, MA, USA), 2 mmol/L L-glutamine (Gibco), 1× nonessential amino acids (Gibco), β-mercaptoethanol (Gibco), 1 mmol/L sodium pyruvate (Gibco), and 1× antibiotic-antimycotic (Gibco). Human basic fibroblast growth factor (bFGF) (Koma Biotech, Seoul, Korea) at 10 ng/mL was used for PGC self-renewal. The cultured PGCs were subcultured onto mitomycin-inactivated mouse embryonic fibroblasts at 5- to 6-day intervals by gentle pipetting without any enzyme treatment. For DF-1, the cells were maintained in DMEM with high glucose (Hyclone), 10% FBS, and 1× antibiotic-antimycotic. Cultured cells were grown at 37 °C in a 5% CO₂ incubator.

DF-1 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS). The PGCs used were derived from a domestic chicken species (three-yellow chicken) and stored in our lab [5 (link), 36 (link)]. Chicken PGCs were cultured in a cKO medium composed of KO-DMEM, supplemented with 7.5% fetal bovine serum (FBS; Hyclone, USA), 20% BRL conditioned medium, 2.5% chicken serum (Sigma, St. Louis, MO, USA), 2 mM glutamine, 1 mM pyruvate, 1× nucleosides, 1× non-essential amino acids, 0.1 mM β-mercaptoethanol and 4 ng/mL human recombinant FGF. All cells were cultured at 37 °C in a 5% CO₂ environment.
DF-1 cells and PGCs were transfected in the presence of Lipofectamine 3000 Reagent according to the manufacturer’s instructions. Briefly, a total 8 μg of plasmids (Cas9: donor = 1: 1) were used for each well of a six-well plate, and the transfection solution was removed 12 h after transfection. Positive cells were sorted 3 days after transfection by FACS for further culture and analysis. For stable PGC line establishment, a second FACS was performed 14 days after the first sorting. All components without special statements were bought from ThermoFisher (ThermoFisher Scientific, Waltham, MA, USA).