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{"project":"2_test","denotations":[{"id":"16870721-9443911-76660904","span":{"begin":2019,"end":2021},"obj":"9443911"},{"id":"16870721-3048409-76660905","span":{"begin":2113,"end":2115},"obj":"3048409"},{"id":"16870721-16616333-76660906","span":{"begin":2686,"end":2687},"obj":"16616333"},{"id":"16870721-1552940-76660907","span":{"begin":3887,"end":3889},"obj":"1552940"},{"id":"16870721-9443911-76660908","span":{"begin":4471,"end":4473},"obj":"9443911"},{"id":"16870721-16616333-76660909","span":{"begin":5870,"end":5871},"obj":"16616333"}],"text":"RESULTS AND DISCUSSION\nThe rationale for RMCE-ASAP is detailed in Figure 1. The first step requires generating a floxed allele in ES cells that will serve as the substrate for subsequent exchanges (RMCE-ready ES cell, Figure 1). The targeting strategy is detailed in Figure 2. The frequency of targeting was 4% (12/300 puromycin-resistant clones, analyzed by Southern blot and long-range PCR, Figure 2).\nWe next tested the efficiency of RMCE in ES cells, using a replacement construct encoding p53 fused to GFP (p53GFP) to enable tracking p53 in individual live cells. Importantly however, GFP fluorescence was not used to screen cells with targeted events, as we wanted to develop a general method to isolate marker-free recombinants. The exchange strategy is detailed in Figure 3A. We picked 65 ES cell clones resistant to 1-(-2-deoxy-2-fluoro-1-b-d-arabino-furanosyl)-5-iodouracil (FIAU) due to TK loss and analyzed their DNA by PCR and Southern blot. Strikingly, 54 proper recombinants were identified, indicating very high RMCE efficiency (83%). RMCE also proved to be precise, as no aberrant bands were detected in PCR and Southern blots (Figure 3A). p53+/GFP ES clones were analyzed by western blot with an antibody against p53, and found to express an additional band at the expected size (ca. 80 kDa). Surprisingly, the fusion of GFP to p53 apparently altered p53 stability: steady-state levels of p53GFP were much higher than those of wild-type p53 (p53WT) in unstressed cells, and did not vary significantly after DNA damage, so that the levels for both p53WT and p53GFP were similar after adriamycin treatment (Figure 3A).\nSix independent p53+/GFP clones were injected into blastocysts and transferred to pseudo-pregnant females using standard procedures. Strikingly, no pregnancies were obtained. It has been shown that the p53 pathway is regulated very differently in ES and somatic cells: ES cells contain relatively high p53 levels and lack the p53-mediated DNA damage responses found in somatic cells (18). This, together with the observation that p53 levels decrease during mouse embryogenesis (19), suggested an explanation for the observed lack of pregnancies: we speculate that the high levels of p53GFP in the ES cells injected into blastocysts might have prevented normal embryonic development once these cells began to differentiate and the p53 pathway became functional.\nTo test this possibility, we performed RMCE with a p53 fusion gene in which the p53 proline-rich domain (PRD) was deleted (p53ΔP), and the p53ΔP was fused to GFP. We used this mutant because deleting the proline-rich domain decreases stability and compromises DNA-damage responses in vivo (5). According to our hypothesis, this hypomorphic mutant should not prevent embryonic development. RMCE with a p53ΔPGFP replacement plasmid was again very efficient and western blots revealed an additional band of the predicted molecular weight only in p53+/ΔPGFP ES cells (Figure 3B). As expected, the PRD deletion correlated with lower expression levels: p53ΔPGFP was much less abundant than p53WT in all p53+/ΔPGFP clones (Figure 3B). We next determined whether p53+/ΔPGFP ES cells could generate chimeric mice and transmit the modified allele through the germline. Two p53+/ΔPGFP ES cell clones were injected into blastocysts and highly chimeric (\u003e80%) mice were obtained. Heterozygote pups were recovered from the mating the chimeras with WT mice (Figure 3C). These data demonstrate that marker-free RMCE is very efficient in ES cells and allows germline transmission of a targeted mutation (see Figure 1, path A).\nWe next determined whether the RMCE approach could be used to target mutations at the p53 allele in somatic cells (Figure 1, path B). We first verified that the RMCE-ready p53 locus (p53RMCE) could be transmitted through the germline by mating p53RMCE/+ chimeras with p53+/− mice (20) (Figure 4). Importantly, this allowed us to generate p53RMCE/− MEFs, which were used to test RMCE at the p53 locus in somatic cells. We first attempted RMCE in MEFs by electroporating p53RMCE/− MEFs with a Cre-expression plasmid and the p53GFP plasmid, followed by selection with FIAU or ganciclovir. Strikingly, no clones with an exchanged allele were identified (data not shown). RMCE with p53GFP in ES cells showed that p53GFP is expressed at high levels (Figure 3A), and as mentioned before, the p53 pathway that can be activated in MEFs is not readily activated in ES cells (18). The results above suggest that high levels of p53GFP could be tolerated by ES cells but toxic to MEFs, so that MEFs in which an RMCE had occurred failed to proliferate. To test this possibility, p53RMCE/− MEFs were electroporated with the p53GFP replacement construct with or without a Cre-expression plasmid, then analyzed by fluorescence microscopy 48 h after electroporation. The experiment was done without selection to enable observation of cells under conditions where a failure to proliferate would not derive from FIAU or ganciclovir toxicity but rather solely from the effects of p53GFP. We observed a few fluorescent cells only when the Cre expression plasmid was co-electroporated, suggesting that such cells resulted from RMCE. Importantly, the rare fluorescent cells had a flat, ‘fried-egg’ appearance typical of senescent cells (Figure 5A), and when plates were observed 5 days later, the cells had detached. Altogether, the results suggest that RMCE can give rise to p53GFP-expressing MEFs, but they die rapidly owing to p53GFP toxicity.\nWe also performed RMCE in p53RMCE/− MEFs with the p53ΔPGFP construct. p53ΔPGFP-expressing MEFs were viable, recovered with an efficiency of ∼40%, and, as expected from ES cell experiments, expressed a p53ΔPGFP protein at much lower levels than p53WT (Figure 5B). Unlike WT MEFs, p53ΔP/ΔP MEFs are unable to arrest cycling after irradiation (5). Likewise, we found that irradiation doses that arrested p53RMCE/− MEFs (which express a wild-type p53 from the RMCE-ready locus, see Figure 2) did not arrest p53ΔPGFP/− MEFs (Figure 5C). These data show that a single RMCE-ASAP reaction in heterozygous MEFs enables detection of a recessive phenotype. The results confirm that deleting the proline rich domain leads to less active p53 with impaired cell cycle control, and also indicate that a GFP C-terminal fusion can dramatically alter p53 regulation. A summary of our results is presented in Table 1.\nThese data report the development and implementation of an improved RMCE approach that enables efficient allele modification in ES cells to generate mice and in heterozygous MEFs to accelerate phenotypic analyses. The success of RMCE-ASAP relied on the integrated use of inverted heterologous loxP sites, a positive/negative selection marker that preserves the germline capacity of ES cells, and, for somatic cells, the existence of a knock-out allele of the gene of interest. These characteristics should make RMCE-ASAP a robust and general technology for analysis of mammalian genes under conditions that preserve normal control mechanisms in different tissues. In addition, RMCE-ASAP could be used to generate fibroblastic cell lines tailored for the repeated targeting of widely studied genes (p53, c-myc, NF-KB, etc.)."}

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PMC:1540739 / 14131-21382 JSON TXT