A transgenic mouse for in vivo detection of endogenous labeled mRNA.
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 β-actin gene. As β-actin-MBS was ubiquitously expressed, we could uniquely address endogenous mRNA regulation in any tissue or cell type. We simultaneously followed transcription from the β-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 β-actin mRNA molecules in various mouse tissues.
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USA.
Timothée
T
Lionnet
Kevin
K
Czaplinski
Xavier
X
Darzacq
Yaron
Y
Shav-Tal
Amber L
AL
Wells
Jeffrey A
JA
Chao
Hye Yoon
HY
Park
Valeria
V
de Turris
Melissa
M
Lopez-Jones
Robert H
RH
Singer
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D014158:Transcription, Genetic
D012333:RNA, Messenger
Science. 2009 Jul 24;325(5939):471-3
19628867
Curr Opin Cell Biol. 2005 Jun;17(3):332-9
15901505
Biophys J. 2002 May;82(5):2775-83
11964263
Curr Biol. 2003 Jan 21;13(2):161-167
12546792
Cancer Res. 1999 Mar 15;59(6):1202-5
10096548
Annu Rev Biophys. 2009;38:173-96
19416065
Nat Rev Mol Cell Biol. 2007 Feb;8(2):113-26
17245413
Cell. 2004 Mar 5;116(5):683-98
15006351
DNA Res. 2009 Feb;16(1):45-58
19001483
Cell. 1986 May 9;45(3):407-15
3698103
J Cell Biol. 1994 Oct;127(2):441-51
7929587
Nat Struct Mol Biol. 2007 Sep;14(9):796-806
17676063
J Neurosci. 2003 Apr 15;23(8):3251-61
12716932
Cell. 2005 Dec 16;123(6):1025-36
16360033
Cell. 2002 Feb 22;108(4):439-51
11909516
J Cell Sci. 2010 May 15;123(Pt 10):1761-74
20427315
Science. 2004 Jun 18;304(5678):1797-800
15205532
J Cell Biochem. 1990 Jan;42(1):13-31
2153691
Nat Cell Biol. 2010 Jun;12(6):543-52
20453848
Curr Biol. 2006 May 23;16(10):1018-25
16713960
Nature. 2010 Sep 30;467(7315):604-7
20844488
Science. 1998 Apr 24;280(5363):585-90
9554849
J Cell Sci. 2008 Mar 15;121(Pt 6):887-94
18303053
Nat Methods. 2008 Oct;5(10):877-9
18806792
Nat Methods. 2010 Aug;7(8):631-3
20639867
Nat Methods. 2005 Sep;2(9):663-5
16118636
J Cell Sci. 2009 Aug 15;122(Pt 16):2980-8
19638415
Proc Natl Acad Sci U S A. 2006 Oct 31;103(44):16406-11
17062750
Mol Cell. 1998 Oct;2(4):437-45
9809065
Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7045-50
11416185
Trends Biochem Sci. 2006 Dec;31(12):687-93
17084632
Cell. 2009 Aug 21;138(4):673-84
19703394
Nat Struct Mol Biol. 2007 Nov;14(11):1049-55
17984967
Science. 2007 Nov 30;318(5855):1412-6
18048681