A super sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair

Nishimura, Kohei; Yamada, Ryotaro; Hagihara, Shinya; Iwasaki, Rie; Uchida, Naoyuki; Kamura, Takumi; Takahashi, Koji; Torii, Keiko U.; Fukagawa, Tatsuo

doi:10.1101/2020.01.20.912113

Cited by 9 publications

(17 citation statements)

References 14 publications

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“…BAF, emerin, and Lem2 were selected as target proteins for depletion because they are all known to be involved in NE reformation during telophase 27,[46][47][48] . We first employed the auxin-inducible degron (AID)-induced degradation system to specifically deplete a target protein at the time of interest, for example, only during mitosis 49,50 . Therefore, we integrated a sequence of mClover3-mAID into each of the three target genes (BAFN1, EMD, and LEMD2) using CRISPR/Cas9 editing 51 .…”

Section: Resultsmentioning

confidence: 99%

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Transfected plasmid DNA is incorporated into the nucleus via nuclear envelope reformation at telophase

et al. 2022

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DNA transfection is an important technology in life sciences, wherein nuclear entry of DNA is necessary to express exogenous DNA. Non-viral vectors and their transfection reagents are useful as safe transfection tools. However, they have no effect on the transfection of non-proliferating cells, the reason for which is not well understood. This study elucidates the mechanism through which transfected DNA enters the nucleus for gene expression. To monitor the behavior of transfected DNA, we introduce plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observe plasmid behavior and RFP expression in living cells. RFP expression appears only after mitosis. Electron microscopy reveals that plasmids are wrapped with nuclear envelope (NE)‒like membranes or associated with chromosomes at telophase. The depletion of BAF, which is involved in NE reformation, delays plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus during NE reformation at telophase.

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Section: Resultsmentioning

confidence: 99%

“…The AID-induced protein degradation system was used to deplete Lem2 50 . The plasmid pAID1.2-EF1a-NmClover3-mAID was developed from pAID1.2-EF1a-NGFP-mAID (Addgene plasmid #140607).…”

Section: Methodsmentioning

confidence: 99%

Transfected plasmid DNA is incorporated into the nucleus via nuclear envelope reformation at telophase

et al. 2022

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“…Both the AID and dTAG systems offer comparable advantages in terms of acute degradation of a genetically edited tagged target protein (Nabet et al, 2018; Nishimura et al, 2009). The second iteration of the AID system, AID2, overcame significant challenges of basal degradation and has been shown to be effective in degrading a tagged transgene reporter in vivo in mice (Nishimura et al, 2020; Yesbolatova et al, 2020). However, auxin derivatives have been shown to be bioactive and can potentially affect embryonic development (Nishimura et al, 2020).…”

Section: Designmentioning

confidence: 99%

“…The second iteration of the AID system, AID2, overcame significant challenges of basal degradation and has been shown to be effective in degrading a tagged transgene reporter in vivo in mice (Nishimura et al, 2020; Yesbolatova et al, 2020). However, auxin derivatives have been shown to be bioactive and can potentially affect embryonic development (Nishimura et al, 2020). Furthermore, all auxin systems require constitutive exogenous expression of a plant-based accessory protein (e.g.…”

Section: Designmentioning

confidence: 99%

Rapid and efficient adaptation of the dTAG system in mammalian development reveals stage specific requirements of NELF

Abuhashem

Hadjantonakis

2021

Preprint

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SUMMARYTargeted protein degradation methods offer a unique avenue to assess a protein’s function in a variety of model systems. Recently, these approaches have been applied to mammalian cell culture models, enabling unprecedented temporal control of protein function. However, the efficacy of these systems at the tissue and organismal levels in vivo is not well established. Here, we tested the functionality of the degradation tag (dTAG) degron system in mammalian development. We generated a homozygous knock-in mouse with a FKBPF36V tag fused to Negative elongation factor b (Nelfb) locus, a ubiquitously expressed protein regulator of transcription. In the first validation of targeted endogenous protein degradation across mammalian development, we demonstrate that irrespective of the route of administration the dTAG system is safe, rapid, and efficient in embryos from the zygote to midgestation stages. Additionally, acute early depletion of NELFB revealed a specific role in zygote-to-2-cell development and Zygotic Genome Activation (ZGA).HIGHLIGHTSgenetically engineered mouse model harboring a FKBPF36V knock-in to evaluate kinetics and efficacy of the dTAG degron system in vivosystem is non-toxic, and allows acute and efficient degradation of a FKBPF36V- tagged endogenous protein during in utero embryo developmentsystem nables fine temporal degradation and reversibility of depletion across embryonic stagesstage-specific depletion reveals a role for NELFB during mouse ZGA

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“…7−9 Furthermore, recent synthetic and protein engineering efforts have resulted in the generation of a more potent analogue mutant pair, enabling the effective depletion of target proteins in mammalian cells. 10,11 This was followed by Prof. Maja Koḧn (University of Freiburg, Germany) discussing mining the druggability of protein phosphatases. She first emphasized the importance of understanding and drugging both kinases and phosphatases to target disease.…”

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confidence: 98%

Empowering Global Chemical Biology at the Dawn of the New Decade

Hoogendoorn

Aye

2020

ACS Chem. Biol.

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On January 22−24, 2020, scientific luminaries across the far-flung corners of chemical biology gathered in Geneva, Switzerland, to deliver their latest and greatest discoveries in the field. Generously supported by the Swiss National Science Foundation (SNSF), our academic partners, and industrial and journal sponsors, this chemical biology symposium in our opinion will remain memorable for several years to come, not only because of the diversity in scientific topics delivered by our invited eminent speakers as detailed herein, but it is also one-of-a-kind conference which reflected multidimensional balancebalance in age and gender, across these speakers. Such a remarkable speaker line-up doubtless attracted >200 attendees from academia and industry in and around Switzerland and beyond, representing a huge swathe of subfields of science interfacing chemistry and biology. Poster presentations from students and postdocs further spotlighted the exciting diversity in the field: spanning biosynthesis, optochemical genetics, genetic code expansion, lipid chemical biology, redox perturbation, microfluidics screening, membrane signaling, immune modulation, DNA circuits, and synthetic and computational biology. This notable heterogeneity in scientific topics also went hand-in-hand with the diverse representations of student/postdoc trainees from 56 institutions covering 14 countries worldwide, allowing us to witness science as a truly global enterprise.

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A super sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair

Cited by 9 publications

References 14 publications

Transfected plasmid DNA is incorporated into the nucleus via nuclear envelope reformation at telophase

Transfected plasmid DNA is incorporated into the nucleus via nuclear envelope reformation at telophase

Rapid and efficient adaptation of the dTAG system in mammalian development reveals stage specific requirements of NELF

Empowering Global Chemical Biology at the Dawn of the New Decade

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