FIGURE 4.
A3 mRNA expression profile in hESCs and characterization of hESCs with diminished A3 expression. A, endogenous expression of A3 mRNAs in H9 and H13 hESCs. mRNA copy numbers were determined by real-time RT-PCR. RNA levels were normalized to GAPDH. Values are mean ± S.E. of three independent experiments. B, efficiency of shRNA-mediated knockdown of A3 mRNAs. H9 cells were transduced with lentiviruses carrying pSicoR-MS2 scrambled shRNA or shRNA against one of the A3 mRNAs. Successfully transduced cells were selected with puromycin. RNA was analyzed by real-time RT-PCR to determine expression levels of A3 mRNAs. Results were calculated by the ΔΔCT method. Values are mean ± S.E. of three independent experiments. C, shRNA knockdown of each A3 gene product does not compromise pluripotency. Epifluorescence microscopy of mCherry and immunostaining of Oct3/4 with Alexa 488 secondary antibody of H9 hESCs transduced with lentiviruses carrying pSicoR-MS1 scrambled shRNA or shRNA against one of the A3 mRNAs. Nuclei were stained with Hoechst. D, microscopy of embryoid bodies derived from H9 hESCs transduced with the same lentiviruses after 17 days in culture.