Degron tagging for rapid protein degradation in mice

Disease Models & Mechanisms, Год журнала: 2024, Номер 17(4)

Опубликована: Апрель 1, 2024

Degron tagging allows proteins of interest to be rapidly degraded, in a reversible and tuneable manner, response chemical stimulus. This provides numerous opportunities for understanding disease mechanisms, modelling therapeutic interventions constructing synthetic gene networks. In recent years, many laboratories have applied degron successfully cultured mammalian cells, spurred by rapid advances the fields genome editing targeted protein degradation. this At Glance article, we focus on efforts apply mouse models, discussing distinct set challenges posed vivo environment.

Язык: Английский

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Distinct patterns of RNA polymerase II and transcriptional elongation characterize mammalian genome activation

Cell Reports, Год журнала: 2022, Номер 41(13), С. 111865 - 111865

Опубликована: Дек. 1, 2022

How transcription is regulated as development commences fundamental to understand how the transcriptionally silent mature gametes are reprogrammed. The embryonic genome activated for first time during zygotic activation (ZGA). RNA polymerase II (Pol II) and productive elongation this process remains elusive. Here, we generate genome-wide maps of Serine 5 2-phosphorylated Pol after ZGA in mouse embryos. We find that both phosphorylated forms display similar distributions across genes ZGA, with typical enrichment emerging ZGA. occurs at prior their activation, suggesting 2 phosphorylation may prime gene expression. Functional perturbations demonstrate CDK9 SPT5 major regulators prevents precocious some genes. Overall, our work sheds molecular insights into transcriptional regulation beginning mammalian development.

Язык: Английский

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RNA Pol II pausing facilitates phased pluripotency transitions by buffering transcription

Genes & Development, Год журнала: 2022, Номер 36(13-14), С. 770 - 789

Опубликована: Июль 1, 2022

Promoter-proximal RNA Pol II pausing is a critical step in transcriptional control. has been predominantly studied tissue culture systems. While shown to be required for mammalian development, the phenotypic and mechanistic details of this requirement are unknown. Here, we found that loss stalls pluripotent state transitions within epiblast early mouse embryo. Using Nelfb −/− mice NELFB degron stem cell model, show embryonic cells (ESCs) representing naïve pluripotency successfully initiate transition program but fail balance levels induced repressed genes enhancers absence NELF. We an increase chromatin-associated NELF during from later states. Overall, our work defines acute long-term molecular consequences reveals role continuum as modulator transitions.

Язык: Английский

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Differential Protein Degradation with a Super-Degron Tag and Thalidomide Analogs: EGFP is Degraded in Wild-Type Mouse Cell, While PD-1 Requires CRBN Humanization, Providing New Insights into T Cell Regulation

Опубликована: Янв. 1, 2025

It was previously considered that protein knockdown using a combination of zinc finger degron tag and thalidomide analog is impossible in mouse cells, however, EGFP expression as an indicator, we found possible super-degron (SD) with iberdomide or mezigdomide cells. Despite the efficient degradation SD-tagged wild-type PD-1 could not be degraded but human Jurkat cells human-type CEREBLON (CRBNI391V). In mice CRBNI391V, endogenous tagged SD efficiently suppressed T both cultured whole body. addition, comparison treatment anti-PD-1 antibody suggested transient downregulation might prevent cell exhaustion compared to treatment. brain also reduced by pomalidomide crosses brain-blood barrier. These results suggest analogs are effective for vitro vivo knockdown, although some conditional settings required.

Язык: Английский

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Rapid and sustained degradation of the essential centrosome protein CEP192 in live mice using the AID2 system

Science Advances, Год журнала: 2025, Номер 11(9)

Опубликована: Фев. 28, 2025

Studying essential genes required for dynamic processes in live mice is challenging as genetic perturbations are irreversible and limited by slow protein depletion kinetics. The auxin-inducible degron (AID) system a powerful tool analyzing inducible loss vitro, but it toxic to mice. Here, we use an optimized second-generation AID achieve the conditional reversible of centrosomal CEP192 We show that auxin derivative 5-phenyl-indole-3-acetic acid well tolerated over 2 weeks drives near-complete degradation less than 1 hour vivo. did not affect centriole duplication decreased γ-tubulin recruitment centrosomes impairing mitotic spindle assembly. Sustained vivo led cell division failure death proliferative tissues. Thus, suited rapid and/or sustained study functions

Язык: Английский

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Rapid protein degradation systems to determine gene function in vivo

Lab Animal, Год журнала: 2025, Номер unknown

Опубликована: Фев. 28, 2025

Язык: Английский

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Spatio-Temporal Control of GFP-tagged Protein Degradation in the Mouse

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Апрель 18, 2025

Abstract Loss of function studies are a central approach to understanding gene/protein function. In mice, this often relies upon heritable recombination at the DNA level. This is slow and non-reversible, which limits both spatial temporal resolution analysis. Recently, degron techniques that directly target proteins for degradation have been successfully used quickly reversibly knockdown proteins. Currently, these systems limited by lack tissue/cell type specificity. Here, we generated mice allow control GFP-tagged protein degradation. Degron GFP line leads in different cellular compartments distinct cell types. Further, it rapid reversible. We probe glucocorticoid receptor epidermis demonstrate has functions proliferative differentiated cells – an analysis would not possible with traditional approaches. propose ability use knock-in lines loss will provide additional motivation generation useful tools.

Язык: Английский

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Use of the dTAG system in vivo to degrade CDK2 and CDK5 in adult mice and explore potential safety liabilities

Toxicological Sciences, Год журнала: 2023, Номер 194(1), С. 53 - 69

Опубликована: Май 25, 2023

Abstract The degradation tag (dTAG) system for target protein can remove proteins from biological systems without the drawbacks of some genetic methods, such as slow kinetics, lack reversibility, low specificity, and inability to titrate dosage. These make it difficult compare toxicity resulting pharmacological interventions, especially in vivo. Because dTAG has not been studied extensively vivo, we explored use this study physiological sequalae CDK2 or CDK5 adult mice. Mice with homozygous knock-in sequence onto were born at Mendelian ratios despite decreased levels comparison wild-type In bone marrow cells duodenum organoids derived these mice, treatment degrader dTAG-13 resulted rapid robust but caused no appreciable change viability transcriptome. Repeated delivery vivo studies proved challenging; multiple formulations an effort maximize while minimizing formulation-related toxicity. Degradation all organs except brain, where likely did cross blood brain barrier, only microscopic changes testis CDK2dTAG findings corroborated conditional knockout Our results suggest that provide loss mice causes previously unknown phenotypes.

Язык: Английский

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Molecular mechanisms underlying totipotency

Life Science Alliance, Год журнала: 2023, Номер 6(11), С. e202302225 - e202302225

Опубликована: Сен. 4, 2023

Numerous efforts to understand pluripotency in mammals, using pluripotent stem cells culture, have enabled the generation of artificially induced cells, which serve as a valuable source for regenerative medicine and creation disease models. In contrast these tremendous successes field past few decades, our understanding totipotency, is highlighted by its broader plasticity than pluripotency, still limited. This largely attributable scarcity available materials lack vitro However, recent technological advances unveiled molecular features that characterize totipotent cells. Single-cell or low-input sequencing technologies allow dissection pre- post-fertilization developmental processes at level with high resolution. this review, we describe some key findings totipotency discuss how acquired beginning life.

Язык: Английский

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A degron-based approach to manipulate Eomes functions in the context of the developing mouse embryo

Proceedings of the National Academy of Sciences, Год журнала: 2023, Номер 120(44)

Опубликована: Окт. 23, 2023

The T-box transcription factor Eomesodermin (Eomes), also known as Tbr2, plays essential roles in the early mouse embryo. Loss-of-function mutant embryos arrest at implantation due to Eomes requirements trophectoderm cell lineage. Slightly later, expression visceral endoderm promotes anterior formation and anterior-posterior axis specification. Early induction epiblast beginning day 6 is necessary for nascent mesoderm undergo epithelial mesenchymal transition (EMT). acts a temporally spatially restricted manner sequentially specify yolk sac haemogenic endothelium, cardiac mesoderm, definitive endoderm, axial progenitors during gastrulation. Little about underlying molecular mechanisms governing actions of these distinct progenitor populations. Here, we introduced degron-tag mCherry reporter sequence into locus. Our experiments analyzing homozygously tagged embryonic stem cells demonstrate that degron-tagged protein fully functional. dTAG (degradation fusion tag) treatment vitro results rapid degradation recapitulates Eomes-null phenotype. However utero administration resulted variable lineage-specific degradation, likely reflecting diverse type-specific dynamics. Finally, rapidly recovers following wash-out vitro. ability manipulate combination with marking by mCherry-reporter offers powerful tool dissecting Eomes-dependent functional types

Язык: Английский

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