Zeocine

HEK293 cells (ATCC, Manassas, VA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin in a 5% CO₂ atmosphere in a humidified incubator at 37°C. To stably express ADAMTS17S-A, the ADAMTS17S-A plasmid was transfected in HEK293 cells using Lipofectamine 3000 (ThermoFisher Scientific). After 24h DMEM medium was replaced with fresh DMEM supplemented with 0.5 mg/mL zeocine (ThermoFisher Scientific) for selection. The stable cells were expanded on 150 mm cell culture dishes, rinsed once with 20 mL PBS when confluent, and maintained in serum-free DMEM, which was collected every 48–72h. Medium was cleared of cell debris, pooled, and stored at −20°C. 1 L conditioned medium was concentrated to ~25 mL using an Amicon Ultrafiltration Unit (MilliporeSigma) with a 10 kDa molecular-weight cut-off membrane. The concentrated medium was cleared by centrifugation for 10 minutes at 10 000 rpm in a Beckman J17 rotor and applied to a Capturem Maxiprep Nickel Column (Takara Bio USA) according to the manufacturer’s protocol. The filtrate was reapplied once. The resin was washed with 20 mmol/L imidazole diluted in the supplied wash buffer, and bound protein was eluted with 2 × 750 μL elution buffer (eluate 1 and 2). The quality of the eluted protein was evaluated by SDS-PAGE using a 10% polyacrylamide gel.

HEK-293 cells stably expressing SLO3 and LRRC52 were provided by Jerod Denton (Vanderbilt University) and maintained in DMEM complete, composed of Dulbecco’s Modified Eagle Medium supplemented with 700 μg/mL Zeocine, 10% FBS, and 1% Penicillin-Streptomycin (Thermo Fisher Scientific). Cells were grown on plastic Petri dishes and flasks at 37 °C and 5% CO₂ in a Galaxy 170 S incubator (Eppendorf). Prior to patching, flasks at ~80% confluence were rinsed with Hank’s balanced salt solution followed by disassociation with trypsin-EDTA (Thermo Fisher Scientific). Suspended cells were diluted in DMEM complete and distributed to 9.2 cm² Petri dishes coated with 1 μL 2% Matrigel® Basement Membrane Matrix (Corning) in Minimum Essential Medium (Thermo Fisher Scientific).

ESC clones carrying both the Nanog transgene and a CAG monomeric Kusabira-orange (mKO) fluorescence reporter were selected by neomycin (Sigma-Aldrich) and zeocine (Life Technologies). D4 PGCLCs were induced from D2 EpiLCs with Nanog and used for derivation of EGCLC. For ESC or D4 PGCLC injections, GOF-GFP ESCs were co-transfected with a vector, which enabled inducible expression of Nanog and constitutive expression of Venus, a variant of EGFP. For D4 PGCLC, after induction of PGCLCs with Nanog, cells were stained with PE conjugated-CD61 antibody (1:10, Biolegend, 104308) and Alexa660 conjugated-SSEA-1 antibody (2.5 μl/ 10⁵ cells, eBioscience, clone eBioMC-480, 50-8813) according to the manufacturer’s instructions. Double positive PGCLC cells were collected by using a S3 cell sorter (Biorad). Embryos for chimera experiments were obtained from CBA/C57BL/6 F1 crossed with C57BL/6 mice. Blind tests or randomization methods were not used. The sex of embryos was not determined. Manipulations of embryos were performed as described previously³⁷ . Briefly, five cells were injected into a morula, which were subsequently cultured in KSOM (Millipore). At the following day, the embryos were transferred into the uteri of pseudopregnant mice. All embryos were analysed one week after embryo transfer, which corresponded to embryonic day 9.5.