RNA-seq of H9 hESC line carrying shRB expression cassette, and H9 hESC line carrying shRB expression cassette in addition to CRISPR/Cas9-mediated nonsense mutations on RBL1 and RBL2. RNA-seq of H9 hESC line carrying shRB expression cassette, and H9 hESC line carrying shRB expression cassette in addition to CRISPR/Cas9-mediated nonsense mutations on RBL1 and RBL2

In order to investigate the roles of retinoblastoma family genes during early human development, mutant H9 human embryonic stem cells were generated. Frameshift mutations were introduced in RBL1 and RBL2 genes using the CRISPR/Cas9 technology, and then the shRNA-expression cassette to knockdown RB upon tetracycline treatment was integrated. These cells were cultured in definitive endoderm differentiation conditions for 3 or 6 days with/without tetracycline.

为探究视网膜母细胞瘤家族基因在人类早期发育过程中的作用，研究人员构建了突变型H9人类胚胎干细胞（human embryonic stem cells）。借助CRISPR/Cas9技术在RBL1与RBL2基因中引入移码突变，随后整合了可在四环素处理下敲低RB基因表达的短发卡RNA（shRNA）表达盒。将上述细胞置于定形内胚层（definitive endoderm）分化培养条件下，分别培养3天或6天，并设置有无四环素处理的对照组别。