An auxin-based degron system for the rapid depletion of proteins in nonplant cells

Nature Methods, Journal Year: 2009, Volume and Issue: 6(12), P. 917 - 922

Published: Nov. 15, 2009

Language: Английский

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Phthalimide conjugation as a strategy for in vivo target protein degradation

Science, Journal Year: 2015, Volume and Issue: 348(6241), P. 1376 - 1381

Published: May 22, 2015

A degrading game plan for cancer therapy Certain classes of proteins that contribute to development are challenging target therapeutically. Winter et al. devised a chemical strategy that, in principle, permits the selective degradation any protein interest. The involves chemically attaching ligand known bind desired another molecule hijacks an enzyme whose function is direct cell's machinery. In proof-of-concept study, they demonstrated transcriptional coactivator called bromodomain-containing 4 and delayed progression leukemia mice. Science , this issue p. 1376

Language: Английский

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Oncolytic virotherapy

Nature Biotechnology, Journal Year: 2012, Volume and Issue: 30(7), P. 658 - 670

Published: July 1, 2012

Language: Английский

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A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells

PLoS ONE, Journal Year: 2009, Volume and Issue: 4(8), P. e6529 - e6529

Published: Aug. 5, 2009

The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful deliver DNA constructs that difficult transfect by other methods. Lentiviruses have additional advantage being able integrate into genomes non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones cDNA, small hairpin RNA (shRNA) microRNA (miRNA) and provide limited drug selection markers. Furthermore, recombinogenic bacteria, complicating generation maintenance desired clones. Here, we describe a collection 59 comprise an integrated system constitutive inducible cDNAs, shRNAs miRNAs, use wide variety These based on Gateway technology (Invitrogen) whereby shRNA miRNA interest cloned Entry then recombined Destination carries chosen backbone marker. This recombination reaction generates product with >95% efficiency greatly reduces frequency unwanted bacteria. We generated production both retroviruses lentiviruses. Further, characterized each its titer as well expressing depleting interest. also multiple types fusion confirmed each. demonstrated utility these functional studies. First, show FKBP12 Destabilization Domain can be used either protein mitotically-arrested Also, generate primary fibroblasts induced senesce presence absence damage. Finally, determined isoforms AT-Rich Interacting 4B (ARID4B) could induce G1 arrest when overexpressed. As new technologies emerge, this easily modified adapted without need extensive recloning.

Language: Английский

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p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells

Nature Medicine, Journal Year: 2018, Volume and Issue: 24(7), P. 939 - 946

Published: June 8, 2018

Language: Английский

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Target identification using drug affinity responsive target stability (DARTS)

Proceedings of the National Academy of Sciences, Journal Year: 2009, Volume and Issue: 106(51), P. 21984 - 21989

Published: Dec. 8, 2009

Identifying the molecular targets for beneficial or detrimental effects of small-molecule drugs is an important and currently unmet challenge. We have developed a method, drug affinity responsive target stability (DARTS), which takes advantage reduction in protease susceptibility protein upon binding. DARTS universally applicable because it requires no modification independent mechanism action. demonstrate use to identify known small-molecule-protein interactions reveal eukaryotic translation initiation machinery as longevity-enhancing plant natural product resveratrol. envisage that will also be useful global mapping protein-metabolite interaction networks label-free screening unlimited varieties compounds development imaging agents.

Language: Английский

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The dTAG system for immediate and target-specific protein degradation

Nature Chemical Biology, Journal Year: 2018, Volume and Issue: 14(5), P. 431 - 441

Published: March 26, 2018

Language: Английский

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Single-Cell Dynamics of Genome-Nuclear Lamina Interactions

Cell, Journal Year: 2013, Volume and Issue: 153(1), P. 178 - 192

Published: March 1, 2013

Language: Английский

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Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors

Cell Reports, Journal Year: 2016, Volume and Issue: 15(1), P. 210 - 218

Published: March 24, 2016

Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows depletion animal cells and fungi, but its application human has been limited by difficulties tagging endogenous proteins. We have developed simple scalable CRISPR/Cas-based tag HCT116 mouse embryonic stem (ES) using donor constructs that harbor synthetic short homology arms. Using combination AID with CRISPR/Cas, we generated conditional alleles nuclear cytoplasmic cells, which can then be depleted very rapidly after addition auxin culture medium. This approach should greatly facilitate functional analysis proteins, particularly those previously unknown function.

Language: Английский

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The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans

Development, Journal Year: 2015, Volume and Issue: unknown

Published: Jan. 1, 2015

Experimental manipulation of protein abundance in living cells or organisms is an essential strategy for investigation biological regulatory mechanisms. While powerful techniques expression have been developed C. elegans, existing tools conditional disruption function are far more limited. To address this, we adapted the auxin-inducible degradation (AID) system discovered plants to enable depletion elegans. We report that a modified Arabidopsis TIR1 F-box mediates robust auxin-dependent degron-tagged targets. document effectiveness this nuclear and cytoplasmic proteins diverse somatic germline tissues throughout development. Target were depleted as little 20-30 minutes, their could be reestablished upon auxin removal. engineered strains expressing under control various promoter 3' UTR sequences drive tissue-specific temporally regulated expression. The degron tag can efficiently introduced by CRISPR/Cas9-based genome editing. harnessed explore roles dynamically expressed hormone receptors molting, analyze meiosis-specific required proliferation. Together, our results demonstrate AID provides new tool spatiotemporal regulation analysis metazoan model organism.

Language: Английский

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