PMID- 23424748
OWN - NLM
STAT- MEDLINE
DCOM- 20130619
LR  - 20220129
IS  - 1750-2799 (Electronic)
IS  - 1754-2189 (Print)
IS  - 1750-2799 (Linking)
VI  - 8
IP  - 2
DP  - 2013 Feb
TI  - Quantifying the transcriptional output of single alleles in single living 
      mammalian cells.
PG  - 393-408
AB  - Transcription kinetics of actively transcribing genes in vivo have generally been 
      measured using tandem gene arrays. However, tandem arrays do not reflect the 
      endogenous state of genome organization in which genes appear as single alleles. 
      Here we present a robust technique for the quantification of mRNA synthesis from 
      a single allele in real time in single living mammalian cells. The protocol 
      describes how to generate cell clones harboring an MS2-tagged allele and how to 
      detect in vivo transcription from this tagged allele at high spatial and temporal 
      resolution throughout the cell cycle. Quantification of nascent mRNAs produced 
      from the single tagged allele is performed using RNA fluorescence in situ 
      hybridization (FISH) and live-cell imaging. Subsequent analyses and data modeling 
      detailed in the protocol include measurements of transcription rates of RNA 
      polymerase II, determination of the number of polymerases recruited to the tagged 
      allele and measurement of the spacing between polymerases. Generation of the 
      cells containing the single tagged alleles should take up to 1 month; RNA FISH or 
      live-cell imaging will require an additional week.
FAU - Yunger, Sharon
AU  - Yunger S
AD  - The Mina and Everard Goodman Faculty of Life Sciences, Institute of 
      Nanotechnology, Bar-Ilan University, Ramat Gan, Israel.
FAU - Rosenfeld, Liat
AU  - Rosenfeld L
FAU - Garini, Yuval
AU  - Garini Y
FAU - Shav-Tal, Yaron
AU  - Shav-Tal Y
LA  - eng
GR  - 260441/ERC_/European Research Council/International
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Nat Protoc
JT  - Nature protocols
JID - 101284307
RN  - 0 (RNA, Messenger)
RN  - EC 2.7.7.- (RNA Polymerase II)
SB  - IM
MH  - *Alleles
MH  - Animals
MH  - Cloning, Molecular/*methods
MH  - Gene Expression Regulation/genetics/*physiology
MH  - In Situ Hybridization, Fluorescence
MH  - RNA Polymerase II/metabolism
MH  - RNA, Messenger/*biosynthesis
MH  - Transcription, Genetic/genetics/*physiology
PMC - PMC3597184
MID - EMS52245
OID - NLM: EMS52245
EDAT- 2013/02/22 06:00
MHDA- 2013/06/20 06:00
PMCR- 2013/08/01
CRDT- 2013/02/22 06:00
PHST- 2013/02/22 06:00 [entrez]
PHST- 2013/02/22 06:00 [pubmed]
PHST- 2013/06/20 06:00 [medline]
PHST- 2013/08/01 00:00 [pmc-release]
AID - 10.1038/nprot.2013.008 [doi]
PST - ppublish
SO  - Nat Protoc. 2013 Feb;8(2):393-408. doi: 10.1038/nprot.2013.008.