PMC:1540739 / 21903-30152 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":"Figures and Tables\nFigure 1 Rationale for a RMCE-ASAP. Using homologous recombination, the gene of interest (GOI, open boxes: exons), is targeted with a construct introducing upstream of coding regions one loxP (blue arrowhead) and downstream, a positive/negative selection cassette (red box) and a second inverted heterologous loxP (purple arrowhead) to create RMCE-ready ES cells. An exchange is performed in these cells by co-transfecting a Cre expression plasmid and a marker-free plasmid with a floxed mutant GOI (green box: mutated exon), to produce a mutant mouse (path A). Importantly RMCE-ASAP incorporates two major improvements over classical RMCE (path B): (i) the positive/negative selection cassette does not prevent germline transmission, so that RMCE-ready mice can be obtained; (ii) the selection cassette does not replace, but rather lies downstream of the GOI. This is a crucial requirement for accelerated phenotyping in somatic cells, as maintaining a functional GOI ensures that the RMCE-ready locus still behaves like a WT locus. Hence, after breeding the RMCE-ready mouse with mice heterozygotes for the GOI, somatic cells with an RMCE-ready locus and a WT or KO allele can be recovered [e.g. RMCE/+ and RMCE/− mouse embryonic fibroblasts (MEFs)]. Such cells, phenotypically similar to +/+ and +/− cells, can then be used for phenotypic analyses of dominant or recessive mutations after a single exchange.\nFigure 2 Generating ES cells with a p53 RMCE-ready locus. The p53 gene is contained in a 17 kb-long EcoRI (RI) fragment (black boxes: coding sequences, white boxes: UTRs). The Flox targeting construct (below), the sequence which was verified before use (Materials and Methods), contains (i) a 3.4 kb-long 5′ homology region; (ii) 0.2 kb upstream of coding sequences, an EcoRI site and L3, a mutant loxP [loxP257, (14)]; (iii) p53 exons; (iv) 0.4 kb downstream, a puroΔTK fusion gene (puDTK) for positive/negative selection (21) and an inverted WT loxP (1L); (v) a 1.2 kb-long 3′ homology region and (vi) the diphteria α-toxin (DTA) gene for targeting enrichment. The recombinants resulting from the depicted crossing-overs are identified by a 6.5 kb band in Southern blot with probe A and a 3 kb band by PCR with primers a and b. A representative Southern, and PCR of one positive (β) and two negative clones, are shown.\nFigure 3 Performing RMCE in ES cells. (A) RMCE with a p53GFP plasmid. The exchange plasmid, the sequence which was verified before use, contains p53GFP coding sequences flanked by L3 and 1L sites. It was electroporated with a Cre expression plasmid. FIAU-resistant clones were analyzed by PCR with primers b and c and Southern blot with probe B. Both approaches led to identical results and identified 54/65 RMCE recombinants. Representative clones (P–Z) are shown (left), analyzed by PCR (top) and Southern (bottom): all clones but Q and T are positive with both assays. All positive clones produced a band of the expected size by PCR, indicating correct recombination at 1L, and displayed only the expected 12 and 5 kb bands by Southern, indicating correct recombination at L3. The absence of bands of aberrant size in Southerns also indicated that the exchange plasmid was neither rearranged nor inserted at ectopic sites. Thus RMCE was efficient and accurate. Recombinant clones were analyzed by western blot with an antibody to p53. In the representative western (right), cells from two independent p53+/GFP ES clones were left untreated or treated with adriamycin (ADR) at 0.5 μg/ml for 24 h, and protein extracts were prepared. p53GFP migrated at the expected size of 80 kDa and was expressed at unexpectedly high levels regardless of stress. (B) RMCE with a p53ΔPGFP plasmid. The p53ΔPGFP exchange construct (which sequence was verified before use) differed from the p53GFP construct only in that it contains a mutated exon 4 (4*) encoding a PRD deletion. RMCE was again very efficient, with 10/12 FIAU-resistant clones producing a 3 kb band by PCR with primers b and c. A western analysis of four FIAU-resistant clones is shown below (with low/high exposures: Lo X/Hi X). As expected, all except clone x expressed p53ΔPGFP. p53ΔPGFP migrated at the expected size of 75 kDa and accumulated after stress, but at lower levels than p53WT. (C) Germline transmission of the p53ΔPGFP mutation. DNA of seven littermates (U42–U48), obtained from mating a p53ΔPGFP chimera with a WT mouse, was analyzed by PCR with primers d and e (see B), with DNA from WT, p53+/ΔP and p53ΔP/ΔP MEFs (5) as controls. U45, a p53+/ΔPGFP mouse, demonstrated transmission of the mutation.\nFigure 4 Germline transmission of the p53 RMCE-ready locus. p53RMCE/+ ES cells were injected into blastocysts to generate chimeric mice. Chimeras (\u003e80%) were then mated with p53+/− mice (Taconic) and MEFs were prepared. MEFs were first genotyped by PCR with primers a and b (see Figure 2) to detect the PuroΔTK marker of the RMCE allele (top). This revealed germline transmission of the p53 RMCE-ready locus in MEFs 1, 2 and 6. Each of these three MEF clones was further analyzed (bottom) with primers f and g (left lanes) and h and i (right lanes), routinely used to genotype p53+/− mice (sequences in Materials and Methods). Primers f and g amplify a 320 bp product from a WT or RMCE allele, while primers h and i specifically amplify a 150 bp product from the Neo marker in the KO allele. MEF 1 are p53RMCE/+ and MEFs 2 and 6 are p53RMCE/− cells.\nFigure 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.\nTable 1 Summary of targeting experiments\nCells Electroporated plasmids Selection drug Targeting method Targeting efficiency Comments\nWT ES Flox Puromycin Homologous recombination 12/300 (4%) Germline transmission of the RMCE locus\np53RMCE/+ ES p53GFP + Cre FIAU RMCE 54/65 (83%) No pregnancy: p53GFP toxicity\np53RMCE/+ ES p53ΔPGFP + Cre FIAU RMCE 10/12 (83%) Germline transmission of the mutation\np53RMCE/− MEF p53GFP + Cre ganciclovir RMCE — No clone: p53GFP toxicity\np53RMCE/− MEF p53GFP + Cre FIAU RMCE — No clone: p53GFP toxicity\np53RMCE/− MEF p53GFP + Cre — RMCE Few fluorescent cells Transiently observed (48h): p53GFP toxicity\np53RMCE/− MEF p53GFP — — No fluorescent cells\np53RMCE/− MEF p53ΔPGFP + Cre Ganciclovir RMCE 9/22 (41%) Phenotypic read-out after a single exchange (loss of cell cycle control)\nIn ES cells, targeting p53ΔPGFP or p53GFP by RMCE was ∼20 times more efficient than targeting Flox by homologous recombination. In MEFs, due to p53GFP toxicity, only the targeting efficiency of RMCE with p53ΔPGFP could be evaluated. Importantly, a phenotypic read-out of the p53ΔPGFP mutation in MEFs, if it had been performed after targeting in fibroblasts by homologous recombination, would have required two rounds of inefficient targeting to target both p53 alleles.","divisions":[{"label":"title","span":{"begin":0,"end":18}},{"label":"figure","span":{"begin":19,"end":1430}},{"label":"label","span":{"begin":19,"end":27}},{"label":"caption","span":{"begin":29,"end":1430}},{"label":"p","span":{"begin":29,"end":1430}},{"label":"figure","span":{"begin":1431,"end":2352}},{"label":"label","span":{"begin":1431,"end":1439}},{"label":"caption","span":{"begin":1441,"end":2352}},{"label":"p","span":{"begin":1441,"end":2352}},{"label":"figure","span":{"begin":2353,"end":4621}},{"label":"label","span":{"begin":2353,"end":2361}},{"label":"caption","span":{"begin":2363,"end":4621}},{"label":"p","span":{"begin":2363,"end":4621}},{"label":"figure","span":{"begin":4622,"end":5472}},{"label":"label","span":{"begin":4622,"end":4630}},{"label":"caption","span":{"begin":4632,"end":5472}},{"label":"p","span":{"begin":4632,"end":5472}},{"label":"figure","span":{"begin":5473,"end":6923}},{"label":"label","span":{"begin":5473,"end":5481}},{"label":"caption","span":{"begin":5483,"end":6923}},{"label":"p","span":{"begin":5483,"end":6923}},{"label":"label","span":{"begin":6924,"end":6931}},{"label":"caption","span":{"begin":6933,"end":6965}},{"label":"p","span":{"begin":6933,"end":6965}},{"label":"table","span":{"begin":6966,"end":7778}},{"label":"tr","span":{"begin":6966,"end":7062}},{"label":"th","span":{"begin":6966,"end":6971}},{"label":"th","span":{"begin":6973,"end":6996}},{"label":"th","span":{"begin":6998,"end":7012}},{"label":"th","span":{"begin":7014,"end":7030}},{"label":"th","span":{"begin":7032,"end":7052}},{"label":"th","span":{"begin":7054,"end":7062}},{"label":"tr","span":{"begin":7063,"end":7165}},{"label":"td","span":{"begin":7063,"end":7068}},{"label":"td","span":{"begin":7070,"end":7074}},{"label":"td","span":{"begin":7076,"end":7085}},{"label":"td","span":{"begin":7087,"end":7111}},{"label":"td","span":{"begin":7113,"end":7124}},{"label":"td","span":{"begin":7126,"end":7165}},{"label":"tr","span":{"begin":7166,"end":7248}},{"label":"td","span":{"begin":7166,"end":7178}},{"label":"td","span":{"begin":7180,"end":7192}},{"label":"td","span":{"begin":7194,"end":7198}},{"label":"td","span":{"begin":7200,"end":7204}},{"label":"td","span":{"begin":7206,"end":7217}},{"label":"td","span":{"begin":7219,"end":7248}},{"label":"tr","span":{"begin":7249,"end":7341}},{"label":"td","span":{"begin":7249,"end":7261}},{"label":"td","span":{"begin":7263,"end":7277}},{"label":"td","span":{"begin":7279,"end":7283}},{"label":"td","span":{"begin":7285,"end":7289}},{"label":"td","span":{"begin":7291,"end":7302}},{"label":"td","span":{"begin":7304,"end":7341}},{"label":"tr","span":{"begin":7342,"end":7418}},{"label":"td","span":{"begin":7342,"end":7355}},{"label":"td","span":{"begin":7357,"end":7369}},{"label":"td","span":{"begin":7371,"end":7382}},{"label":"td","span":{"begin":7384,"end":7388}},{"label":"td","span":{"begin":7390,"end":7391}},{"label":"td","span":{"begin":7393,"end":7418}},{"label":"tr","span":{"begin":7419,"end":7488}},{"label":"td","span":{"begin":7419,"end":7432}},{"label":"td","span":{"begin":7434,"end":7446}},{"label":"td","span":{"begin":7448,"end":7452}},{"label":"td","span":{"begin":7454,"end":7458}},{"label":"td","span":{"begin":7460,"end":7461}},{"label":"td","span":{"begin":7463,"end":7488}},{"label":"tr","span":{"begin":7489,"end":7593}},{"label":"td","span":{"begin":7489,"end":7502}},{"label":"td","span":{"begin":7504,"end":7516}},{"label":"td","span":{"begin":7518,"end":7519}},{"label":"td","span":{"begin":7521,"end":7525}},{"label":"td","span":{"begin":7527,"end":7548}},{"label":"td","span":{"begin":7550,"end":7593}},{"label":"tr","span":{"begin":7594,"end":7643}},{"label":"td","span":{"begin":7594,"end":7607}},{"label":"td","span":{"begin":7609,"end":7615}},{"label":"td","span":{"begin":7617,"end":7618}},{"label":"td","span":{"begin":7620,"end":7621}},{"label":"td","span":{"begin":7623,"end":7643}},{"label":"tr","span":{"begin":7644,"end":7778}},{"label":"td","span":{"begin":7644,"end":7657}},{"label":"td","span":{"begin":7659,"end":7673}},{"label":"td","span":{"begin":7675,"end":7686}},{"label":"td","span":{"begin":7688,"end":7692}},{"label":"td","span":{"begin":7694,"end":7704}},{"label":"td","span":{"begin":7706,"end":7778}}],"tracks":[]}