A transgenic mouse for in vivo detection of endogenous labeled mRNA

被引：224

作者：

Lionnet, Timothee ^{[1
,2
]}

Czaplinski, Kevin ^{[1
]}

Darzacq, Xavier ^{[3
]}

Shav-Tal, Yaron ^{[4
,5
]}

Wells, Amber L. ^{[1
]}

Chao, Jeffrey A. ^{[1
]}

Park, Hye Yoon ^{[1
,2
]}

de Turris, Valeria ^{[1
]}

Lopez-Jones, Melissa ^{[1
]}

Singer, Robert H. ^{[1
,2
]}

机构：

[1] Albert Einstein Coll Med, Dept Anat & Struct Biol, Bronx, NY 10467 USA

[2] Albert Einstein Coll Med, Gruss Lipper Biophoton Ctr, Bronx, NY 10467 USA

[3] Ecole Normale Super, CNRS, Inst Biol, Unite Mixte Rech 8197, Paris, France

[4] Bar Ilan Univ, Mina & Everard Goodman Fac Life Sci, Ramat Gan, Israel

[5] Bar Ilan Univ, Inst Nanotechnol & Adv Mat, Ramat Gan, Israel

来源：

NATURE METHODS | 2011年 / 8卷 / 02期

基金：

美国国家卫生研究院;

关键词：

LIVING MAMMALIAN-CELLS; NUCLEOCYTOPLASMIC TRANSPORT; GENE-EXPRESSION; INTRACELLULAR-LOCALIZATION; SINGLE CELLS; DYNAMICS; TRANSCRIPTION; DROSOPHILA; KINETICS; PROBES;

D O I：

10.1038/NMETH.1551

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Live-cell single mRNA imaging is a powerful tool but has been restricted in higher eukaryotes to artificial cell lines and reporter genes. We describe an approach that enables live-cell imaging of single endogenous labeled mRNA molecules transcribed in primary mammalian cells and tissue. We generated a knock-in mouse line with an MS2 binding site (MBS) cassette targeted to the 3' untranslated region of the essential beta-actin gene. As beta-actin-MBS was ubiquitously expressed, we could uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the beta-actin alleles in real time and observed transcriptional bursting in response to serum stimulation with precise temporal resolution. We tracked single endogenous labeled mRNA particles being transported in primary hippocampal neurons. The MBS cassette also enabled high-sensitivity fluorescence in situ hybridization (FISH), allowing detection and localization of single beta-actin mRNA molecules in various mouse tissues.

引用

页码：165 / U96

页数：9

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