Imaging Gene Expression: Methods and Protocols

367 Pages·2013·6.24 MB·English

by  Tina B. Miranda, Ty C. Voss, Gordon L. Hager (auth.), Yaron Shav-Tal (eds.)

Methods in
Molecular Biology 1042
Yaron Shav-Tal Editor
Imaging Gene
Expression
Methods and Protocols
M M B ™
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

Imaging Gene Expression
Methods and Protocols
Edited by
Yaron Shav-Tal
The Mina and Everard Goodman Faculty of Life Sciences and Institute of Nanotechnology,
Bar-Ilan University, Ramat Gan, Israel
Editor
Yaron Shav-Tal
The Mina and Everard Goodman Faculty
of Life Sciences and Institute of Nanotechnology
Bar-Ilan University
Ramat Gan, Israel
Additional material to this book can be downloaded
from h ttp://extras.springer.com
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-62703-525-5 ISBN 978-1-62703-526-2 (eBook)
DOI 10.1007/978-1-62703-526-2
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2013942490
© Springer Science+Business Media, LLC 2 013
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this
legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifi cally for
the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work.
Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the
Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions
for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution
under the respective Copyright Law.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
While the advice and information in this book are believed to be true and accurate at the date of publication, neither
the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be
made. The publisher makes no warranty, express or implied, with respect to the material contained herein.
Printed on acid-free paper
Humana Press is a brand of Springer
Springer is part of Springer Science+Business Media (www.springer.com)
Dedication
To my mentors, Dov Zipori and Robert Singer.

Pref ace
Gene expression can be a term of many meanings, depending on the type of research fi eld
one belongs to. The activity of genes has been measured since the days when radioactive
labeling of nucleic acids became possible, and ever since the fi eld has been ploughed through
by scores of biochemical and molecular analyses, and recently, bioinformatics and genomics
approaches. Imaging of gene expression has also made an impact on our understanding of
the gene expression pathway, particularly since the fi eld uniquely deals with gene activity
within single cells rather than taking a population view like the above-mentioned tech-
niques. As imaging technologies and approaches have evolved, the scope of certain imaging
techniques has moved far beyond the production of purely illustrative images or appealing
time-lapse movies to providing the scientist with a rich range of ways to measure and quan-
tify the biological process and outcome of gene expression. Close interactions between
biologists and physicists trying to better understand the mechanistics of gene activity have
allowed the import of techniques and terminology once solely used in physics, thereby
bringing to light the emerging fi eld of biophysics.
This book aims to offer up-to-date approaches and protocols that scientists in the fi eld
have developed, which would benefi t the broader scientifi c community. The methods not
only describe the technical design of the experiments but also focus on the expected out-
come and provide tips and personal insights for the benefi t of the user, which we all know
can sometimes tip the outcome between the failure and success of a technique. This volume
is divided into three parts. The fi rst section deals with the output of a gene, namely the
RNA molecules that are transcribed from the gene and the way by which these molecules
can be tracked or quantifi ed in fi xed or living cells. The second section provides protocols
that focus on the gene, DNA, or chromatin. The third portrays a variety of ways by which
nuclear processes intertwined with gene expression can be followed and quantifi ed in living
cells as well as approaches for studying several subnuclear structures found in eukaryotic
cells. Finally, since cells make up tissues, it is imperative to be able to follow these processes
in whole tissues, or even better, in the actual living organism. Therefore imaging in the
context of a complete organism is given special notice in the book. The chapters have been
contributed by both well-established and young scientists, and I am ever grateful to all the
authors who have participated and invested time, thought, and energy in the bringing this
collection to light.
Ramat Gan, Israel Yaron Shav-Tal
vii

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
PART I IMAGING GENE EXPRESSION AND RNA DYNAMICS
IN CELLS AND ORGANISMS
1 High-Throughput Fluorescence-Based Screen to Identify Factors Involved
in Nuclear Receptor Recruitment to Response Elements. . . . . . . . . . . . . . . . . 3
Tina B. Miranda, Ty C. Voss, and Gordon L. Hager
2 Live-Cell Imaging Combined with Immunofluorescence, RNA,
or DNA FISH to Study the Nuclear Dynamics and Expression
of the X-Inactivation Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Tim Pollex, Tristan Piolot, and Edith Heard
3 Single-Molecule Resolution Fluorescent In Situ Hybridization (smFISH)
in the Yeast S. cerevisiae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Samir Rahman and Daniel Zenklusen
4 Measuring Transcription Dynamics in Living Cells
Using Fluctuation Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Matthew L. Ferguson and Daniel R. Larson
5 Tracking Nuclear Poly(A) RNA Movement Within and Among Speckle
Nuclear Bodies and the Surrounding Nucleoplasm . . . . . . . . . . . . . . . . . . . . . 61
Joan C. Ritland Politz and Thoru Pederson
6 Nuclear Trafficking and Export of Single, Native mRNPs
in Chironomus tentans Salivary Gland Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Tim P. Kaminski, Jan-Hendrik Spille, Claudio Nietzel,
Jan Peter Siebrasse, and Ulrich Kubitscheck
7 Single mRNP Tracking in Living Mammalian Cells. . . . . . . . . . . . . . . . . . . . . 87
Alon Kalo, Pinhas Kafri, and Yaron Shav-Tal
8 Imaging Nascent RNA Dynamics in Dictyostelium . . . . . . . . . . . . . . . . . . . . . 101
Jonathan R. Chubb, Michelle Stevense, Danielle Cannon,
Tetsuya Muramoto, and Adam M. Corrigan
PART II IMAGING THE GENOME AND CHROMATIN DYNAMICS
9 Monitoring Dynamic Binding of Chromatin Proteins In Vivo
by Single-Molecule Tracking. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Davide Mazza, Sourav Ganguly, and James G. McNally
ix