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CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging does not cause heterochromatinization of endogenous loci

ABSTRACT: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging does not cause heterochromatinization of endogenous loci

PROVIDER: PRJNA454207 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7081830_1.fastq.gz	Fastqsanger.gz
	SRR7081830_2.fastq.gz	Fastqsanger.gz
	SRR7081831_1.fastq.gz	Fastqsanger.gz
	SRR7081831_2.fastq.gz	Fastqsanger.gz
	SRR7081832_1.fastq.gz	Fastqsanger.gz

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CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging does not cause heterochromatinization of endogenous loci

Project description:We recently developed an easy, efficient and scalable method for tagging and live cell imaging of non-repetitive, endogenous chromosome regions via CRISPR/Cas9 mediated knock-in of a TetO repeat. For this purpose, we created optimized and irregular 48-mer and 96-mer TetO repeats. Since it is known that repetitive regions in the human genome can induce H3K9me3-mediated heterochromatin formation, we tested whether 48-mer and/or 96-mer TetO repeats induce H3K9me3 flanking their insertion sites. Using a newly developed method called as CUT&RUN, we showed that there was no significant difference in the H3K9me3 pattern flanking the insertion sites of TetO repeats when compared to wild-type cells.

2018-05-22 | GSE113832 | GEO

CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci.

Project description:Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into lamina-associated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by long-term live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

| S-EPMC6158506 | biostudies-literature

FiTAc-seq analysis of PDX 14-07 and MMTV-rtTA/tetO/Her2 tumors treated with abemaciclib

Project description:We measured changes in H3K27ac after treatment of PDX 14-07 or MMTV-rtTA/tetO-Her2 tumors implanted in nude mice with abemaciclib

2020-09-03 | GSE157384 | GEO

Live-animal imaging of native hematopoietic stem and progenitor cells

Project description:Live-animal imaging of native hematopoietic stem and progenitor cells

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MALDI-imaging-guided transcriptomics

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Multiplexed protein and drug imaging by super-resolution ion beam imaging

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Comparison of gene expression in M. tuberculosis TrxB2-TetON-tetO-WT mutant treated or not with atc

2016-05-12 | GSE72328 | GEO

Quantitative live-cell imaging of secretion activity reveals dynamic immune responses

Project description:Quantitative live-cell imaging of secretion activity reveals dynamic immune responses

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Quantitative live-cell imaging of secretion activity reveals dynamic immune responses

Project description:Quantification of cytokine secretion has facilitated advances in the field of immunology, yet the dynamic and varied secretion profiles of individual cells, particularly those obtained from limited human samples, remain obscure. Herein, we introduce a new technology for quantitative live-cell imaging of secretion activity (qLCI-S) that enables high-throughput and dual-color monitoring of secretion activity at the single-cell level over several days, followed by transcriptome analysis of individual cells based on their phenotype. The efficacy of qLCI-S was demonstrated by visualizing the characteristic temporal pattern of cytokine secretion of group 2 innate lymphoid cells, which constitute less than 0.01% of human peripheral blood mononuclear cells, and by revealing minor subpopulations with enhanced cytokine production. The underlying mechanism of this feature was linked to the gene expression of stimuli receptors. This new technology paves the way for exploring gene expression signatures linked to the spatiotemporal dynamic nature of various secretory functions.

2024-02-01 | GSE253100 | GEO

Comparison of gene expression in M. tuberculosis TrxB2-TetON-tetO-WT mutant treated or not with atc

Project description:M. tuberculosis thioredoxin reductase (TrxB2) is essential for Mtb physiology and pathogenesis. To gain insight into its biological functions, we generated the TrxB2-TetON-tetO mutants, in which TrxB2 is partially depleted in the absence of atc without impairing bacterial viability. We grew TrxB2-TetON-tetO-WT in 7H9 medium with 400 ng/ml atc, until the OD reached 0.5~0.6. Then Mtb was washed with 7H9 medium 3 times and suspended in 7H9 medium with or without atc. Then samples were taken 24, 48, 72 and 120 hrs later.

2016-05-12 | E-GEOD-72328 | biostudies-arrayexpress

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