Mouse embryonic stem cells (R1) were purchased from ATCC and routinely cultured (37 °C 5% CO2) on 0.1% gelatin coated dishes in mES medium (MES-M), which contains Knockout DMEM supplemented with 20% FBS, 1 × GlutaMAX™-I, 1 × non-essential amino acid, 1 mM sodium pyruvate, 0.1 mM β-mercaptoethanol and 1000 U/mL human leukemia inhibitory factor (LIF). All reagents were from Life Technologies (Grand Island, NY, USA) except for human LIF (Peprotech, Rocky Hill, NJ, USA).
Transfection of mESCs with the mouse embryonic stem cell NucleofectorTM Kit (LONZA, Walkersville, MD, USA) was performed according to the manufacturer’s manual. Briefly, mESCs were dissociated into single cells with Accutase (Merk Millipore, Watford, UK) and counted. 2 × 106 cells were gathered for nucleofection with 2.5 µg of each CRIPSR/Cas9n plasmid and 5 µg targeting vector pMrnF9 and then plated on 10 cm dishes coated with mouse embryonic fibroblast (MEF) feeding cells. Ten µM of Y27632 was added to the medium to improve the viability of transfected cells. Two days later, G418 was used for selection of resistant clones at the final concentration of 200 µg/mL. About 9 to 11 days after transfection, resistant clones were picked and expanded. Candidate targeted clones were screened by PCR with F1/R1.
Transfection of mESCs with the mouse embryonic stem cell NucleofectorTM Kit (LONZA, Walkersville, MD, USA) was performed according to the manufacturer’s manual. Briefly, mESCs were dissociated into single cells with Accutase (Merk Millipore, Watford, UK) and counted. 2 × 106 cells were gathered for nucleofection with 2.5 µg of each CRIPSR/Cas9n plasmid and 5 µg targeting vector pMrnF9 and then plated on 10 cm dishes coated with mouse embryonic fibroblast (MEF) feeding cells. Ten µM of Y27632 was added to the medium to improve the viability of transfected cells. Two days later, G418 was used for selection of resistant clones at the final concentration of 200 µg/mL. About 9 to 11 days after transfection, resistant clones were picked and expanded. Candidate targeted clones were screened by PCR with F1/R1.