TetR-based inducible short hairpin RNA interference system. (A): Graphical overview of the system (based on tetracycline [Tet] systems manual; http://www.clonetech.com). Constitutively expressed TetR protein binds to the TO sequence within the H1 promoter, blocking expression of the target sequence hairpin. On addition, doxycycline binds to TetR protein, resulting in a conformational change that renders the TetR unable to bind the TO element, and so hairpin transcription is uninhibited. Following removal of doxycycline from the system, the process is reversed, and inhibition of hairpin transcription is restored. (B): Immunoblot analysis of eight Shef4 TetR subclones for Tetr expression. OCT4 protein was also stained as a marker of the undifferentiated state. The TetR5 clone was chosen as it demonstrated high expression of both TetR and OCT4. Abbreviations: TetR, tet repressor; TO, tet operator.

OCT4 target downregulation is dose- and time dependant. (A): Reverse transcription-polymerase chain reaction (RT-PCR) analysis of OCT4 and HPRT (loading control) was performed on cells cultured for 3 days with varying concentrations of dox. (B): These data are represented graphically and show a steady decrease in OCT4 expression with increasing concentrations of dox. (C): Time trial analysis of OCT4 expression by RT-PCR following treatment with dox. After 3-days time point dox was removed form the culture. (D): Graphical representation of the RT-PCR data showing maximal knockdown between 2 and 3 days of induction. Following removal of dox, OCT4 expression does not revive due to irreversible differentiation of cell cultures. (E): Real time RT-PCR analysis of β2M expression in β2M-shRNAi clone cells, using HPRT as the reference gene control. Cells were cultured for up to 3 days with dox and a further 6 days in the absence of dox, showing recovery of β2M expression. t test analysis was performed against dox untreated cells. *, p > .05; **, p > .005. Abbreviations: β2M, β2-microglobulin; d, day; dox, doxycycline; HPRT, hypoxanthine-guanine phosphoribosyl transferase; hr, hour.

Induction of OCT4-specific hairpin transcription results in OCT4 knockdown. (A): Northern blot showing expression of OCT4-specific RNA hairpins following addition of dox to the culture. (B): Whole cell lysates of Shef4 TetR5 OCT4-shRNAi cells were stained for OCT4 and β-actin (loading control) following 3 days −/+ dox. (C): OCT4-shRNAi interference cells were cultured for 5 days in the absence (top panels) or presence (lower panels) of 5 ng/ml dox. Expression of OCT4 (right panels) is severely reduced by culture with dox. Scale bar: ∼250 μm. Abbreviations: dox, doxycycline; shRNAi, short hairpin RNA; tRNA, transfer RNA.

OCT4 downregulation results in cell differentiation and a dose-dependant increase in transcription of lineage specific genes. (A): Phase contrast images of Shef4Tetr5 OCT4-shRNAi colonies showing morphological differentiation following doxycyline-induced downregulation of OCT4. Images were taken of colonies 3 days (I, II) and 7 days (III, IV) postseeding in the absence (I, III) or presence (II, IV) of doxycyline. White bars: ∼500 μm. (B): Flow cytometric analysis of day 7 cultures from noninduced and dox-induced cells shows OCT4 knockdown results in loss of stem cell surface marker expression. The percentage of cells expressing surface markers of the undifferentiated state SSEA3 and TRA-1-60 was significantly reduced (*, p > .05; **, p > .005) following dox-induced OCT4 downregulation. Results are taken from three separate experiments. (C): Real-time reverse transcription-polymerase chain reaction analysis showing reduced transcription of pluripotency associated genes in OCT4 knockdown cells. (D): β2M-shRNAi Shef4 cells were cultured for 7 days in absence or presence of dox at 1 ng/ml and OCT-shRNAi Shef4 cells in the presence of doxcycyline from 0 to 1 ng/ml. RNA samples prepared from the cells were used for real time PCR analysis against a panel of genes expressed in differentiated cells types. β2M knockdown was also confirmed in the β2M-shRNAi cells. Data are relative to the samples from 0 ng dox, using hypoxanthine-guanine phosphoribosyl transferase as a reference gene. t test analysis was performed against relevant dox untreated cells. *, p > .05; **, p > .005. Abbreviations: dox, doxycycline; shRNAi, short hairpin RNA interference; SSEA3, stage-specific embryonic antigen-3.