Isolation of erythropoietin-producing cells using embryonic stem cells

1. 设计了EGFP整合促红细胞生成素（EPO）基因的靶向载体。EGFP-IRES-puroR基因被敲入EPO基因的起始密码子。将该载体电沉积于C57BL/6衍生的胚胎干细胞中，构建EPO-EGFP胚胎干细胞。将这些胚胎干细胞分别培养于外胚层、中胚层和内胚层等不同条件下，用流式细胞法测定EGFP。将EPO-EGFP ES细胞注入小鼠胚泡，建立EPO-EGFP+/-小鼠。用荧光显微镜分析肾EPO-EGFP+/-切片。用流式细胞仪对EPO-EGFP+/-小鼠分离细胞进行分析。

1. Targeting vector was designed to integrate EGFP in erythropoietin(EPO) gene. EGFP-IRES-puroR gene was knocked-in into the start codon of EPO gene. This vector was electroprated into C57BL/6 derived ES cells and then EPO-EGFP ES cells were established. These ES cells were cultured in various condition such as ectoderm, mesoderm and endoderm and EGFP was evaluated by FACS.2. EPO-EGFP ES cells were injected into mouse blastcyst and then EPO-EGFP+/-mouse was established. Section of kidney from EPO-EGFP+/-was analyzed by fluorescent microscopy. Isolated cells from EPO-EGFP+/-mouse were analyzed by FACS.