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

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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.