Nonessential amino acid stock

mAG-haESC derivation and culture were conducted as previously described.¹¹ Briefly, all mAG-haESCs were derived in 15% KSR knockout DMEM (Gibco) supplemented with 0.1 mM mercaptoethanol (Merck Millipore), 1 mM L-glutamine (Merck Millipore), nucleosides (100×, Merck Millipore), 1% nonessential amino acid stock (Merck Millipore), penicillin/streptomycin (100×, Merck Millipore), 2i (1 μM PD0325901 and 3 μM CHIR99021), and 1,000 U/mL mouse LIF (Merck Millipore). Haploid cells were sorted after outgrowth cells expanded to one 12-well plate. Then, mAG-haESCs were propagated in 15% FBS ES medium supplemented with 1 mM L-glutamine, 0.1 mM mercaptoethanol, nucleosides (100×), 1% nonessential amino acid stock, penicillin/streptomycin (100×), 2i, and 1,000 U/mL mouse LIF. Periodic enrichment (every 3∼5 weeks, depending on the cell lines) of haploid cells treated with staining buffer (15 μg/mL Hoechst 33342 and 2.5 μM verapamil) was conducted with a BD FACS Aria II cell sorter.

The injection of sperm into enucleated oocytes was conducted as previously described (Yang et al., 2012 (link)). The blastocysts were planted on feeders in 15% KSR Knockout DMEM supplemented with 2i: 1 μM PD0325901 (Selleck) and 3 μM CHIR99021 (Selleck). After 6–8 days in culture, the outgrowths were passaged and cultured on feeders using 2i containing ES medium. ES medium contains DMEM (Merck Millipore) supplemented with 15% (v/v) fetal bovine serum (HyClone), 1 mM L-glutamine (Merck Millipore), 0.1 mM mercaptoethanol (Merck Millipore), 1% nonessential amino acid stock (Merck Millipore), penicillin/streptomycin (100×, Merck Millipore), nucleosides (100×, Merck Millipore), and 1,000 U/mL LIF (Merck Millipore).