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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)."}

{"project":"craft-ca-core-ex-dev","denotations":[{"id":"T11419","span":{"begin":41,"end":49},"obj":"CL:0002322"},{"id":"T11420","span":{"begin":44,"end":49},"obj":"CL_GO_EXT:cell"},{"id":"T11421","span":{"begin":59,"end":70},"obj":"SO_EXT:sequence_substitution_entity_or_process"},{"id":"T11422","span":{"begin":71,"end":80},"obj":"SO_EXT:engineered_biological_sequence"},{"id":"T11423","span":{"begin":81,"end":89},"obj":"SO_EXT:sequence_coding_function"},{"id":"T11424","span":{"begin":90,"end":93},"obj":"PR_EXT:000003035"},{"id":"T11425","span":{"begin":94,"end":99},"obj":"SO_EXT:sequence_fusion_process"},{"id":"T11426","span":{"begin":108,"end":111},"obj":"PR_EXT:000003035"},{"id":"T11427","span":{"begin":135,"end":138},"obj":"PR_EXT:000003035"},{"id":"T11428","span":{"begin":158,"end":163},"obj":"CL_GO_EXT:cell"},{"id":"T11429","span":{"begin":226,"end":231},"obj":"CL_GO_EXT:cell"},{"id":"T11430","span":{"begin":306,"end":312},"obj":"CHEBI_SO_EXT:molecular_indicator_or_label_or_marker_or_tag"},{"id":"T11431","span":{"begin":318,"end":330},"obj":"SO_EXT:sequence_recombinant_entity"},{"id":"T11432","span":{"begin":336,"end":344},"obj":"SO_EXT:sequence_substitution_process"},{"id":"T11433","span":{"begin":393,"end":400},"obj":"CL:0002322"},{"id":"T11434","span":{"begin":396,"end":400},"obj":"CL_GO_EXT:cell"},{"id":"T11435","span":{"begin":401,"end":407},"obj":"SO_EXT:sequence_cloned_entity"},{"id":"T11436","span":{"begin":494,"end":496},"obj":"GO_PR_EXT:thymidine_kinase"},{"id":"T11437","span":{"begin":521,"end":524},"obj":"CHEBI_SO_EXT:DNA"},{"id":"T11438","span":{"begin":573,"end":585},"obj":"SO_EXT:sequence_recombinant_entity"},{"id":"T11439","span":{"begin":753,"end":756},"obj":"PR_EXT:000003035"},{"id":"T11440","span":{"begin":756,"end":757},"obj":"SO_EXT:normal_or_wild_type_or_present"},{"id":"T11441","span":{"begin":762,"end":764},"obj":"CL:0002322"},{"id":"T11442","span":{"begin":765,"end":771},"obj":"SO_EXT:sequence_cloned_entity"},{"id":"T11443","span":{"begin":810,"end":818},"obj":"GO:0042571"},{"id":"T11444","span":{"begin":827,"end":830},"obj":"PR_EXT:000003035"},{"id":"T11445","span":{"begin":845,"end":852},"obj":"GO:0010467"},{"id":"T11446","span":{"begin":925,"end":931},"obj":"SO_EXT:sequence_fusion_entity_or_process"},{"id":"T11447","span":{"begin":942,"end":945},"obj":"PR_EXT:000003035"},{"id":"T11448","span":{"begin":965,"end":968},"obj":"PR_EXT:000003035"},{"id":"T11449","span":{"begin":1003,"end":1006},"obj":"PR_EXT:000003035"},{"id":"T11450","span":{"begin":1041,"end":1050},"obj":"SO_EXT:wild_type_entity_or_quality"},{"id":"T11451","span":{"begin":1051,"end":1054},"obj":"PR_EXT:000003035"},{"id":"T11452","span":{"begin":1056,"end":1059},"obj":"PR_EXT:000003035"},{"id":"T11453","span":{"begin":1059,"end":1061},"obj":"SO_EXT:wild_type_entity_or_quality"},{"id":"T11454","span":{"begin":1077,"end":1082},"obj":"CL_GO_EXT:cell"},{"id":"T11455","span":{"begin":1121,"end":1124},"obj":"CHEBI_SO_EXT:DNA"},{"id":"T11456","span":{"begin":1161,"end":1164},"obj":"PR_EXT:000003035"},{"id":"T11457","span":{"begin":1164,"end":1166},"obj":"SO_EXT:wild_type_entity_or_quality"},{"id":"T11458","span":{"begin":1171,"end":1174},"obj":"PR_EXT:000003035"},{"id":"T11459","span":{"begin":1197,"end":1207},"obj":"CHEBI:28748"}],"text":"We 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)."}

{"project":"craft-ca-core-dev","denotations":[{"id":"T11263","span":{"begin":41,"end":49},"obj":"CL:0002322"},{"id":"T11264","span":{"begin":90,"end":93},"obj":"PR:000003035"},{"id":"T11265","span":{"begin":108,"end":111},"obj":"PR:000003035"},{"id":"T11266","span":{"begin":135,"end":138},"obj":"PR:000003035"},{"id":"T11267","span":{"begin":393,"end":400},"obj":"CL:0002322"},{"id":"T11268","span":{"begin":753,"end":756},"obj":"PR:000003035"},{"id":"T11269","span":{"begin":762,"end":764},"obj":"CL:0002322"},{"id":"T11270","span":{"begin":810,"end":818},"obj":"GO:0042571"},{"id":"T11271","span":{"begin":827,"end":830},"obj":"PR:000003035"},{"id":"T11272","span":{"begin":845,"end":852},"obj":"GO:0010467"},{"id":"T11273","span":{"begin":942,"end":945},"obj":"PR:000003035"},{"id":"T11274","span":{"begin":965,"end":968},"obj":"PR:000003035"},{"id":"T11275","span":{"begin":1003,"end":1006},"obj":"PR:000003035"},{"id":"T11276","span":{"begin":1051,"end":1054},"obj":"PR:000003035"},{"id":"T11277","span":{"begin":1056,"end":1059},"obj":"PR:000003035"},{"id":"T11278","span":{"begin":1161,"end":1164},"obj":"PR:000003035"},{"id":"T11279","span":{"begin":1171,"end":1174},"obj":"PR:000003035"},{"id":"T11280","span":{"begin":1197,"end":1207},"obj":"CHEBI:28748"}],"text":"We 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)."}