Zhang Jun-Zheng, Gao Wei, Yang Hong-Bo, Zhang Bo, Zhu Zuo-Yan, Xue You-Fang
Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education, College of Life Sciences, Peking University, Beijing, China.
Stem Cells. 2006 Dec;24(12):2661-8. doi: 10.1634/stemcells.2006-0017. Epub 2006 Sep 7.
The pluripotency of mouse embryonic stem (ES) cells is maintained by self-renewal. To screen for genes essential for this process, we constructed an RNA interference (RNAi) library by inserting subtracted ES cell cDNA fragments into plasmid containing two opposing cytomegalovirus promoters. ES cells were transfected with individual RNAi plasmids and levels of the pluripotency marker Oct-4 were monitored 48 hours later by real time RT-PCR. Of the first 89 RNAi plasmids characterized, 12 downregulated Oct-4 expression to less than 50% of the normal level and 7 of them upregulated Oct-4 expression to more than 150% of the normal level. To investigate their long-term effect on self-renewal, ES cells were transfected by these 19 RNAi plasmids individually and G418-resistant colonies were subjected to alkaline phosphatase (AP) staining after 7 days selection. Except for 4 plasmids that caused cell death, the ratio of AP positive colonies was repressed to less than 60% of the control group by the other 15 plasmids and even below 20% by 10 plasmids. The cDNA fragments in these 10 plasmids correspond to eight genes, including Zfp42/Rex-1, which was chosen for further functional analysis. RNAi knockdown of Zfp42 induced ES cells differentiate to endoderm and mesoderm lineages, and overexpression of Zfp42 also caused ES cells to lose the capacity of self-renewal. Our results indicate that RNAi screen is a feasible and efficient approach to identify genes involved in ES cells self-renewal. Further functional characterization of these genes will promote our understanding of the complex regulatory networks in ES cells.
小鼠胚胎干细胞（ES细胞）的多能性通过自我更新得以维持。为了筛选这一过程所必需的基因，我们构建了一个RNA干扰（RNAi）文库，方法是将扣除后的ES细胞cDNA片段插入含有两个反向巨细胞病毒启动子的质粒中。将单个RNAi质粒转染ES细胞，48小时后通过实时RT-PCR监测多能性标志物Oct-4的水平。在所鉴定的前89个RNAi质粒中，有12个将Oct-4表达下调至正常水平的50%以下，其中7个将Oct-4表达上调至正常水平的150%以上。为了研究它们对自我更新的长期影响，将ES细胞分别用这19个RNAi质粒转染，经过7天的选择后，对G418抗性菌落进行碱性磷酸酶（AP）染色。除了4个导致细胞死亡的质粒外，其他15个质粒将AP阳性菌落的比例抑制至对照组的60%以下，10个质粒甚至将其抑制至20%以下。这10个质粒中的cDNA片段对应于8个基因，包括Zfp42/Rex-1，我们选择该基因进行进一步的功能分析。Zfp42的RNAi敲低诱导ES细胞向内胚层和中胚层谱系分化，Zfp42的过表达也导致ES细胞丧失自我更新能力。我们的结果表明，RNAi筛选是鉴定参与ES细胞自我更新的基因的一种可行且有效的方法。对这些基因的进一步功能表征将促进我们对ES细胞中复杂调控网络的理解。
