Expression of hKlf4 in the mES cells was induced in the absence of Dox under two different conditions, one in the undifferentiated state maintained by LIF and the second in the RA induced differentiation condition treated for 1.5 days. The protein expression in cell extracts was detected by Western blot analysis. The avidin-HRP conjugate detected strong bands at the size corresponding to the biotinylated hKlf4 protein in the absence of Dox in both the LIF condition and the RA condition. Also, in all the samples, the avidin-HRP conjugate detected endogenous biotinylated proteins, the expression levels of which were lower than those of the induced biotinylated hKlf4 proteins. Western blot analysis using the antiBirA antibody detected the BirA bands in the absence of Dox in both the undifferentiated LIF condition and the differentiated RA condition. When Dox was present, the biotinylated hKlf4 protein and the BirA protein were almost undetectable, indicating that this regulatable in vivo biotinylation expression system was relatively tight. It has been reported that Klf4 expression is regulated by the LIF signaling pathway, thus the hKlf4 ES cell line was cultured in the presence or absence of LIF. As expected, in the presence of LIF, cells showed compact and undifferentiated morphology. In the absence of LIF, cells grew more flattened. After inducing forced expression of hKlf4, within 12 hours, obvious differences in cell morphology were observed compared with the un-induced control. After two days of induction, the ES cells expressing hKlf4 grew less than the control cells. After four than the cells without induction of hKlf4. Cell proliferation assays showed that after one day of induction the cell numbers of the hKlf4 expressing cells were around two-fold lower than those of the Dox+ control cells, and after three days of induction the cell numbers of the hKlf4 expressing cells were around 10-fold fewer than those of the Dox+ control cells. Therefore, hKlf4 induction strongly affected cell growth and viability in the presence or absence of LIF. The effects of expression of hKlf4 in mES cells on gene expression were analyzed. Gene expression was quantified by realtime RT-qPCR. Cells were harvested after induction for 1.5 days. Recombinant hKlf4 mRNA expression was detected using specific hKlf4 qPCR doppler echocardiography evaluating aortic velocity primers. In the absence of Dox, hKlf4 mRNA was highly induced, and in contrast, in the presence of Dox, hKlf4 mRNA was almost undetectable. Total Klf4 mRNA, including transgene hKlf4 mRNA and endogenous mKlf4 mRNA, was detected by specific conserved total Klf4 primers. As expected, in the control Dox+ ES cells, total Klf4 was expressed at high levels in the presence of LIF, and expressed at low levels in the absence of LIF. In the Dox2 hKlf4 expressing ES cells, total Klf4 was expressed significantly higher than total Klf4 in the Dox+ control cells. In addition, endogenous mKlf4 mRNA was detected by specific mKlf4 primers. In the Dox2 hKlf4 expressing ES cells, endogenous mKlf4 was expressed significantly lower than in the Dox+ control cells, suggesting that endogenous mKlf4 was repressed by the induction of hKlf4, and that endogenous mKlf4 was auto-regulated to prevent its overexpression. Several Klf family members, Klf2, Klf4 and Klf5, are expressed in mES cells and down-regulated during mES cell differentiation. Klf2, Klf4 and Klf5 cooperatively bind to target genes and regulate their expression. In the Dox2 hKlf4 expressing ES cells, Klf2 and Klf5 were expressed significantly lower than those in the Dox+ control cells, suggesting that they were repressed by the induction of hKlf4. It has been demonstrated that Esrrb can replace Klf4 in OSKM mediated somatic cell reprogramming. Esrrb and Klf4 have many common targets.