Klf4在维持胚胎干细胞干性方面的研究.ppt

However, in each of the GFP-positive clones we observed partial or complete loss of visible DsRed expression (Fig. 3D,E), although qRT-PCR analysis revealed that the transgenes were not completely silenced (Fig. 3F). (G) Chimeric mouse produced from the K4C12 Epi-iPS clone and agouti germline offspring. This confirms that the developmental capacity has been fully derestricted and the authentic pluripotent state established. These cells should therefore be considered as EpiSC-derived iPS cells, or Epi-iPS cells. Fig. 4. Retention of ground state pluripotency after transgene excision. (A) Splinkerette-PCR reveals 1-3 PB insertions in each iPS clone. We chose two DsRed-positive clones and transfected each with a Cre expression plasmid. After 5 days, cells that no longer expressed DsRed were isolated using flow cytometry with single-cell deposition into 96-well plates (Fig. 4B). (B) Flow cytometry showing the DsRed-negative population in the K4C3 line before and after Cre transfection. (C)Genomic PCR showing loss of the Klf4 transgene and gain of the PB-LTR fragment in two revertant clones. (D) RT-PCR analysis showing the lack of Klf4 transgene and DsRed expression in expanded Cre-reverted cells. Two thirds of the expanded clones retained only the PB terminal repeats. RT-PCR analysis failed to detect expression of the Klf4 transgene or DsRed from these revertants (Fig. 4D). They retained ES cell morphology, Oct4-GFP expression and ES cell marker profile(Fig. 4E,F). (F)Maintained morphology and Oct4-GFP expression in a Cre-reverted Epi-iPS cell line. (G)me3H3K27 staining of Klf4 transgene-deleted iPS cells as compared with parental EpiSCs. (H) Chimeric mouse made with revertant K4C3-A3 cells, and agouti offspring denoting germline transmission. Female chimaeras from two out of three clones produced agouti offspring in their first litter (Fig. 4H),indicative of transmission of iPS cell-derived oocytes. Therefore,complete removal of the Klf4 transgene does not de