A Highly Sensitive Trap Vector System for Isolating Reporter Cells and Identification of Responsive Genes

Overview

Journal Biol Methods Protoc

Publisher Oxford University Press

Specialty Biology

Date 2020 Mar 13

PMID 32161797

Citations 5

Authors

Kosuke Ishikawa

Yuta Kobayashi

Yutaro Wakabayashi

Shinya Watanabe

Kentaro Semba

Affiliations

Soon will be listed here.

Abstract

We devised a versatile vector system for efficient isolation of reporter cells responding to a certain condition of interest. This system combines nontoxic GAL4-UAS and transposon systems, allowing application to mammalian cells and improved expression of a fluorescent reporter protein for cell sorting. Case studies under conditions of gene induction or endoplasmic reticulum (ER) stress with thapsigargin on mouse or human cell lines confirmed easy and efficient isolation of responsive reporter cells. Sequence analyses of the integrated loci of the thapsigargin-responsive clones identified responsive genes including and . is a novel ER stress-responsive gene and we confirmed that endogenous mRNA expression of is upregulated by thapsigargin, and is repressed by IRE1α inhibitors, 4μ8C and toyocamycin, but not significantly by a PERK inhibitor, GSK2656157. These results demonstrate that this approach can be used to discover novel genes regulated by any stimuli without the need for microarray analysis, and that it can concomitantly produce reporter cells without identification of stimuli-responsive promoter/enhancer elements. Therefore, this system has a variety of benefits for basic and clinical research.

Citing Articles

Tyrosine Kinase Inhibitor Profiling Using Multiple Forskolin-Responsive Reporter Cells.

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Identification of microbial metabolites that accelerate the ubiquitin-dependent degradation of c-Myc.

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Isolation of Reporter Cells That Respond to Vitamin A and/or D Using a Transposon Promoter-Trapping Vector System.

Ishikawa K, Tamamura S, Takahashi N, Takagi M, Semba K, Watanabe S Int J Mol Sci. 2022; 23(16).

PMID: 36012634 PMC: 9409033. DOI: 10.3390/ijms23169366.

CRISPR/Cas9-Directed Gene Trap Constitutes a Selection System for Corrected Leukemic Cells in CML.

Vuelta E, Ordonez J, Sanz D, Ballesteros S, Hernandez-Rivas J, Mendez-Sanchez L Int J Mol Sci. 2022; 23(12).

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Genetic manipulation of the mammary gland and potential applications.

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