Leukemia inhibiting factor

For experimental validation of the enhancer function of TEs in mouse ESCs, we used the RW4 cell line, and cultured it as previously described66 (link). RW4 cells were cultured in 0.1% gelatin-coated Petri dishes in culture medium containing DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco), 10% neonatal calf serum (Hyclone), nucleosides (Sigma), 1,000 U ml⁻¹ leukemia-inhibiting factor (Sigma) and 0.1 mM β-mercaptoethanol (Gibco).
To estimate the regulatory potential of TE and non-TE sequences in mouse ESCs, we used the Dual-Glo luciferase assay (Promega). For the luciferase assay, we cloned TEs into the pGl4.23 vector (containing firefly luciferase; Supplementary Tables 5A–C and 9). We co-transfected the TE-plasmid with pRL-TK (containing renilla luciferase) using lipofectamine (X-GENE-HD at 1 μl for 1 μg of plasmid DNA) into RW4 cells plated in 0.1% gelatin-coated 96-well plate. After 24 h, we assayed the luciferase levels according to the Dual-Glo reporter assay system (Promega).
We performed each luciferase experiment in triplicate, and repeated each experiment three times. The luciferase fold change is estimated as the ratio of firefly and renilla luciferase for each TE, and then normalized by the empty vector (pGl4.23 with no insert).

Wild-type male mESCs (E14.wt) were cultivated on 0.1% gelatine (Sigma, France) and CD1 feeder cells (37°C, 5% CO₂) in DMEM (4.5 g/l glucose) w-Glutamax-I, 15% foetal calf serum ESC-tested, leukemia inhibiting factor (5 μg) (Sigma), 50 mM ß-Mercaptoethanol (Invitrogen, France), penicillin/streptomycin (Invitrogen), 200 mM L-glutamine (Invitrogen), and non-essential amino acids (GIBCO, France). To work under feeder-free conditions cells were treated with 1 mg/ml Collagenase (GIBCO) and 2 mg/ml Dispase (GIBCO) and cultivated for one passage without feeder cells on 0.1% gelatine (Sigma) coated plates. Experiments were conducted at passage 26–29. Mouse embryonic fibroblasts (3T3 ATCC) were cultivated in DMEM (4.5 g/l glucose), 10% newborn calf serum and gentamycin (Invitrogen).
For NPC generation, we followed the protocol of Bibel et al. (2007) (link). Briefly, 6 × 10⁶ mESC were cultured in DMEM (4.5 g/l glucose) w-Glutamax-I, 10% foetal calf serum ESC-tested, 50 mM ß-Mercaptoethanol (Invitrogen), penicillin/streptomycin (Invitrogen), 200 mM L-glutamine (Invitrogen), and non-essential amino acids on bacteriological Petri dishes (37°C, 5% CO₂) to start differentiation. After 4 days retinoic acid (5 μm) (Sigma) was added to induce NPC formation. Experiments were conducted 8 days after differentiation.