Role of ABC transporter Bcrp 1 in maintenance of pluripotency in mouse embryonic stem cells

在这项研究中，我们的目的是确定ABC转运蛋白Bcrpl在小鼠胚胎干细胞多能性维持机制中的作用。用流式细胞术和RTP-CR分析法检测未分化和分化ES细胞中Bcrpl蛋白和mRNA的表达。此外，我们还开发了改良的双荧光素酶报告基因检测系统来评估RNAi的效果. Bcrpl与Oct 3/4在小鼠ES细胞中表达的相关性流式细胞术分析显示，Bcrpl在小鼠ES细胞中的表达与调节未分化状态的转录因子Oct 3/4正相关。ES细胞经诱导分化后，Bcrpl和Oct 3/4的表达下降.小鼠Bcrpl mRNA具有三种同种型（称为同种型A、B和C），并且Bcrpl mRNA同种型的差异表达在干细胞和体细胞中交替调节。用RT-PCR方法分析Bcrpl mRNA亚型在成年小鼠和未分化ES细胞中的表达模式。同种型B的转录本在所有组织和ES细胞中强烈表达。同种型A在大多数组织和ES细胞中也有表达，但同种型C在ES细胞中转录水平很低。体外诱导分化后，Bcrpl各亚型mRNA表达量立即下降，随后出现各种分化标志物.改进的双荧光素酶报告基因检测系统的建立为了建立针对Bcrpl mRNA各亚型的敲低ES细胞系用于功能分析，我们建立了改进的双荧光素酶报告基因检测系统来评价RNAi的效果。在小鼠ES细胞中，基于报告基因的荧光素酶检测系统与Western blot分析结果显示，shRNA表达载体对目的蛋白的抑制作用呈正相关。

In this research, we aimed to define the role of ABC transporter Bcrpl in the mechanisms of maintenance of pluripotency in mouse embryonic stem cells. Expression profile of Bcrpl protein and mRNA in undifferentiated and differentiated ES cells was examined by flowcytometry and RTP-CR analysis. Moreover, we developed modified dual luciferase reporter assay system to evaluate RNAi efficacy.1. Correlation of the expression of Bcrpl with Oct3/4 in mouse ES cells.Flowcytometry analysis showed the expression of Bcrpl positively correlated with Oct3/4, a transcription factor regulating undifferentiated status, in mouse ES cells. Moreover, the expression of Bcrpl as well as Oct3/4 in ES cells decreased after in intro differentiation.2. Expression of Bcrpl mRNA isoforms in ES cells.Mouse Bcrpl mRNA has three isoforms (termed isoform A, B and C) and the differential expression of Bcrpl mRNA isoforms is alternatively regulated in the stem cells and somatic cells. The expression pattern of Bcrpl mRNA isoforms in various tissues in adult mice and undifferentiated ES cells was analyzed by RT-PCR. The transcript of isoform B was strongly expressed in all tissues and ES cells. The isoform A was also expressed in the most tissues and ES cells but isoform C was alternatively transcribed and was quite low in ES cells. After induction of in vitro differentiation, all Bcrpl mRNA isoforms immediately decreased and various differentiation markers appeared thereafter.3. Development of modified dual luciferase reporter assay system to evaluate RNAi efficacy.In order to establish knockdown ES cell lines against each isoforms of Bcrpl mRNA for functional analysis, we developed modified dual luciferase reporter assay system to evaluate RNAi efficacy. Reporter-based luciferase assay system represented positive correlation with Western blot analysis in the reduction of target protein by shRNA expression vector in mouse ES cells.