Genome-wide maps of Six2 and b-catenin in mesenchymal nephron progenitors. Mus musculus

Self-renewing undifferentiated nephron progenitors express Six2, a transcription factor that is required for their maintenance as undifferentiated progenitors. Differentiation of nephron progenitors is triggered by Wnt/b-catenin signaling. In order to understand how Six2 and Wnt signaling counteract each other, we performed ChIP-seq of Six2 and b-catenin in mesenchymal nephron progenitor cells. Overall design: Nephron progenitors were FACS-isolated from BAC transgenic Six2GFPcre-positive embryonic kidneys at E16.5. For Six2 ChIP, freshly FACS isolated Six2+ cells were used. For b-catenin ChIP, FACS isolated Six2+ cells were aggregated by centrifugation at 850g for 5min and incubated in 10%FBS/DMEM containing 4uM BIO for 24hrs.

可自我更新的未分化肾单位祖细胞表达Six2转录因子，该因子是维持这类细胞未分化状态的必需条件。肾单位祖细胞的分化由Wnt/β-连环蛋白（Wnt/β-catenin）信号通路触发。为阐明Six2与Wnt信号通路的相互拮抗机制，我们在间充质肾单位祖细胞中开展了针对Six2及β-连环蛋白的染色质免疫共沉淀测序（ChIP-seq）实验。

实验整体设计：
肾单位祖细胞通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）从胚胎发育第16.5天（E16.5）的细菌人工染色体（Bacterial Artificial Chromosome, BAC）转基因Six2GFPcre阳性胚胎肾脏中分离获得。针对Six2的ChIP-seq实验，使用新鲜分离得到的Six2阳性细胞即可。针对β-连环蛋白的ChIP-seq实验，则先将分选得到的Six2阳性细胞以850g离心5分钟进行细胞聚团，随后置于含4μM BIO的10%胎牛血清（Fetal Bovine Serum, FBS）/达尔伯克改良伊格尔培养基（Dulbecco's Modified Eagle Medium, DMEM）中培养24小时。