Isolation and Characterization of Embryonic Stem Cells from Usp16 Mutant Mice

Blastocysts were flushed out from the uterus of Usp16^+/− or Usp16^2lox/+:CAGG-Cre E3.5 female mice with a 27.5 gauge needle connected to 1 ml syringe filled with 1 ml 2i medium^{45 (link)}. Blastocysts were then transferred to feeder cell plates with 200 μl sterile tip and cultured with 2i medium^{46 (link)} or regular ESC medium for 5–8 days. ESC-like colonies formed from ICM outgrowths were handpicked using sterile pipette tips and dissociated by 0.25% trypsin and expanded in ESC medium. For proliferation rate assay, 1 × 10⁴ ESCs were seeded in 12 well plates and cell numbers were counted and the same seeding procedure was repeated every other day. Western blot assay and qPCR were performed using standard protocol included in the method section^{47 (link)}. Antibodies include anti-Usp16 (1:1000)^{26 (link)}, anti-ubH2A (Millipore, 05-678, 1:1000), anti-ubH2B (Millipore, 05-1312, 1:1000), anti-H3 (Abcam, ab100938, 1:5000), anti-GAPDH (Sigma, G9545, 1:5000), Anti-Nanog (R&D Systems, AF2729, 1: 2000), Anti-Oct4 (Santa Cruz Biotechnology, SC9081, 1:2000), and anti-Sox2 (Cell signaling, 3579, 1:2000). Alkaline phosphatase substrate kit was from Vector laboratory (SK-5100).