WAe009-A-14

We have generated a homozygous ATP2A2 knockout human embryonic stem cell line H9-ATP2A2-/- .H9 was transfected with Cas9 vector（Px459，Addgene,62988） which expresses a specific sgRNA sequence targeting for the exon1 of ATP2A2. After electroporation, cells were seeded on a 6-well, Matrigel precoated overnight plate and treated with 0.3ug/mL puromycin in E8 medium. Anti-puromycin clones were emerged after 4 days of electroporation. 15 clones were picked and maintained in E8 medium. DNA was extracted for genotype confirming using PCR sequencing. Clone number 4 was confirmed by homozygous deleting 1 “A” base pair. For further characterization, this clone was expanded. H9-ATP2A2-/- grows as a clone size and expresses stem cell pluripotency hallmarks like OCT-4, SSEA4, processing a normal karyotype and the ability to differentiate into all three germ layers in vitro.