Premature termination of in vivo reprogramming leads to cancer development through altered epigenetic regulation [array]. Mus musculus

We report a reprogrammable mouse system in which reprogramming factor expression in vivo can be controlled temporally by treatment with doxycycline (Dox). Transient expression of reprogramming factors in vivo results in tumor development in various tissues, consisting of undifferentiated dysplastic cells. We analyzed the kidney tumors developed in reprogrammable mice for global gene expressions and DNA methylations. Overall design: Reprogrammable mice at 4 weeks of age were treated with Dox for 7 days followed by the withdrawal. Seven days after the withdrawal, kidney tumors were analyzed for gene expressions and DNA methylations with microarray and RRBS method, respectively. Normal kidney tissue at the same age and ES cells were analyzed as controls. To examine the early changes of gene expressions, transgene-expressing kidney cells were FACS sorted and they are utilized for microarray analysis. Primary liver tumors in reprogrammable mice and transplanted secondary kidney tumors in the subcutaneous tissues of immnodeficient mice were also analyzed for gene expressions.

本研究报道了一种可重编程小鼠系统，该系统中体内重编程因子的表达可通过强力霉素（doxycycline, Dox）处理实现时间维度的精准调控。体内重编程因子的瞬时表达可诱导小鼠多组织产生肿瘤，肿瘤组织由未分化的发育异常细胞构成。
本研究针对可重编程小鼠体内生成的肾脏肿瘤，开展了全基因组基因表达与DNA甲基化分析。
整体实验设计：选取4周龄的可重编程小鼠，经强力霉素处理7天后停药；停药7天后，分别通过微阵列（microarray）技术与简化代表性亚硫酸氢盐测序（reduced representation bisulfite sequencing, RRBS）分析肾脏肿瘤的基因表达与DNA甲基化水平。以同周龄正常肾脏组织与胚胎干细胞（embryonic stem cells, ES cells）作为对照样本。
为探究基因表达的早期变化，本研究通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）分离了表达转基因的肾脏细胞，并将其用于微阵列分析。
本研究还对可重编程小鼠体内生成的原发性肝脏肿瘤，以及免疫缺陷小鼠皮下移植的继发性肾脏肿瘤开展了基因表达分析。