PMC:1540739 / 27376-28826 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1540739","sourcedb":"PMC","sourceid":"1540739","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1540739","text":"Figure 5 Performing RMCE in MEFs. (A) RMCE with the p53GFP plasmid leads to the transient observation of cells with intense nuclear fluorescence. p53RMCE/− MEFs, electroporated with a Cre expression plasmid and the p53GFP exchange plasmid, were analyzed 48 h later by fluorescence microscopy. A typical field (left to right: fluorescence, phase contrast, merged) with a fluorescent cell (arrow) is shown. The fluorescent cell is enlarged (extreme right). (B) RMCE with the p53ΔPGFP plasmid. p53RMCE/− MEFs, electroporated with a Cre expression plasmid and the p53ΔPGFP plasmid, were selected with ganciclovir. PCR with primers d and e (Figure 3B) indicated that 9/22 ganciclovir-resistant clones integrated the ΔP mutation [top row, a representative analysis of 10 clones (Q–Z) is shown]. PCR with primers b and c next verified that the detected PRD deletions resulted from RMCE at the p53 locus, not random integration (middle row, as expected clones R, S, T and W are positive, but not Q). Western analysis of positive clones (bottom row) showed that p53ΔPGFP accumulated after ADR, but at lower levels than p53WT. (C) Phenotypic assay of p53ΔPGFP: loss of cell cycle control. Asynchronous p53RMCE/− and p53ΔPGFP/− MEFs left untreated, or irradiated with doses of 6 or 12 Gy, were analyzed (top shows a typical experiment; bottom plots results from ≥4 independent experiments and ≥3 independent MEFs). Note that the p53RMCE locus encodes a WT p53.","divisions":[{"label":"label","span":{"begin":0,"end":8}}],"tracks":[]}