Data are presented as mean values ± SD ( n = 3 independent experiments, two-tailed t-test).Ī Colony formation of HCT116 cells expressing OsTIR1(WT), OsTIR1(F74G) or AtAFB2 after transfecting DHC1-tagging donors and a targeting CRISPR plasmid. The graph on the right shows the quantified reporter levels. Samples were taken at the indicated time points. HCT116 cells expressing OsTIR1(F74G) and the reporter were treated with 1 µM 5-Ph-IAA for 3 h before medium exchange. e Re-expression of the reporter after depletion by the AID2 system. The data were fitted with one phase decay. Data are presented as mean values ± SD ( n = 3 independent experiments). d A time-course of depletion of the reporter induced in cells expressing OsTIR1(WT) or OsTIR1(F74G) by treating with 100 µM IAA or 1 µM 5-Ph-IAA, respectively. The data were fitted with non-linear regression using four parameters. c Dose response of depletion of the reporter by IAA or 5-Ph-IAA in cells expressing OsTIR1(WT) or OsTIR1(F74G), respectively. Data are presented as mean values ± SD ( n = 3 independent experiments, two-tailed t-test). The graph on the right shows the quantified reporter levels relative to that of the cells expressing only the reporter. Indicated HCT116 cells were treated with the indicated ligand for 4 h. b Depletion of an mAID-EGFP-NLS reporter in isogenic cells using the AID and AID2 systems. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice.Ī Schematic illustration showing the AID and AID2 systems. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. 12 Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Yata 1111, Mishima, Shizuoka, 411-8540, Japan. 11 Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Yata 1111, Mishima, Shizuoka, 411-8540, Japan.10 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, 113-0033, Japan.9 Social Cooperation Program of Evolutional Chemical Safety Assessment System, LECSAS, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo, Tokyo, 113-0033, Japan.8 Department of Biochemistry, Okayama University of Science, Ridai-cho 1-1, Okayama, 700-0005, Japan.7 Department of Cancer Genome Research, Sasaki Institute, Sasaki Foundation, Kandasurugadai 2-2, Chiyoda-ku, Tokyo, 101-0062, Japan.6 Department of Genomics and Evolutionary Biology, National Institute of Genetics, ROIS, Yata 1111, Mishima, Shizuoka, 411-8540, Japan.5 Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Bunkyo, Tokyo, 113-0033, Japan.4 Department of Gene Function and Phenomics, National Institute of Genetics, ROIS, Yata 1111, Mishima, Shizuoka, 411-8540, Japan.2 Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Yata 1111, Mishima, Shizuoka, 411-8540, Japan.1 Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Yata 1111, Mishima, Shizuoka, 411-8540, Japan.
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