C. elegans: Methods and Applications

265 Pages·2015·7.869 MB·English

by  David Biron, Gal Haspel

Methods in
Molecular Biology 1327
David Biron
Gal Haspel Editors
C.elegans
Methods and Applications
Second Edition
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire , AL10 9AB, UK
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C. elegans
Methods and Applications
Second Edition
Edited by
David Biron
Department of Physics, The James Franck Institution, The Institute for Biophysical Dynamics,
The University of Chicago, Chicago, IL, USA
Gal Haspel
Federated Department of Biological Sciences, New Jersey Institute of Technology
and Rutgers University, Newark, NJ, USA
Editors
David Biron Gal H aspel
Department of Physics Federated Department of Biological Sciences
The James Franck Institution New Jersey Institute of Technology
The Institute for Biophysical Dynamics and Rutgers University
The University of Chicago Newark, NJ , U SA
Chicago, IL , USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-2841-5 ISBN 978-1-4939-2842-2 (eBook)
DOI 10.1007/978-1-4939-2842-2
Library of Congress Control Number: 2015945342
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Prefa ce
Tractable models such as the nematode C aenorhabditis elegans provide profound insight
into a wide range of biological phenomena. It is the objective of any scientifi c effort to
understand a natural phenomenon. Considerable effort and ingenuity are dedicated towards
developing novel methods and approaches that will enable breakthroughs and progress.
C. elegans has a short life cycle—it matures during three days at room temperature—
and is easy to cultivate. Under standard laboratory conditions, hermaphroditic nematodes
self-fertilize but mating with males can be induced for experimental purposes. Therefore,
genetic manipulations and the maintenance of mutant strains are comparatively simple.
C. elegans was the fi rst animal to have its genome fully sequenced and to this day, the
genome is being annotated with increasing detail and accuracy. Genetic, genomic, anatomi-
cal, physiological, and other data are curated in publically accessible databases including
Wormbase, Wormatlas, Wormweb, Wormwiring, and Wormbook.
The simple anatomy of C . elegans is a key advantage in and of itself. Determining the
cell lineage, which is mostly invariant between individuals, enabled fundamental discoveries
in developmental biology and the determination of cell fate. Likewise, the nervous system
of C . elegans is compact—a hermaphrodite has but 302 neurons. This enabled White and
colleagues to construct an anatomical map of neuronal connectivity, colloquially referred
to as the “Mind of a Worm.” The connectome of C. elegans —unique in its near
completeness—is being refi ned since, and updated information is periodically made avail-
able to researchers in the fi eld. It provides a powerful starting point for understanding how
neuronal and molecular pathways regulate behavior. At the same time, the importance of
nonsynaptic pathways is increasingly appreciated.
Novel experimental methods for genetic, cellular, and whole animal manipulations are
being developed at a staggering rate. In recent years, reading an entire genome has become
cost effective, writing or deleting genetic information in vivo has come to resemble editing
text, the properties of optical reporters have improved dramatically, and the diversity of
physiological parameters that can be monitored using unobtrusive measures has greatly
expanded. Concurrent advances in computation, imaging, microfl uidics, and prototyping
enable bench-top experiments that were not previously possible. The number of address-
able scientifi c questions scales rapidly with the number of available tools.
The aim of this volume is to provide a step-by-step guide for implementing a selection
of these novel techniques in the lab. Each protocol in this volume is presented as a stand-
alone chapter, specifi cally geared towards addressing practical needs without presuming
prior knowledge of the technique at hand. We hope this volume can assist in addressing the
subset of these questions that most intrigue you.
Chicago, IL, USA D avid Biron
Newark, NJ, USA Gal H aspel
v

Acknowledgment
This work was supported by the NSF IOS grant no. 1256989 (DB).
vii

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
1 Library Construction for Mutation Identification
by Whole-G enome Sequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Harold E. Smith
2 Fundamentals of Comparative Genome Analysis
in Caenorhabditis Nematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1
Eric S. Haag and Cristel G . T homas
3 G enetic Methods for Cellular Manipulations in C. elegans. . . . . . . . . . . . . . . . 23
Menachem Katz
4 A Fusion PCR Method for Expressing Genetic Tools in C. elegans. . . . . . . . . . 39
Yifat Eliezer and Alon Z aslaver
5 T ransposon-Assisted Genetic Engineering with Mos1-M ediated
Single-Copy Insertion (MosSCI). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Christian Frøkjær-Jensen
6 Creating Genome Modifications in C. elegans using
the CRISPR/Cas9 System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
John A. Calarco and Ari E. Friedland
7 Observing and Quantifying Fluorescent Reporters . . . . . . . . . . . . . . . . . . . . . 7 5
Michael Hendricks
8 M icrobial Rhodopsin Optogenetic Tools: Application for Analyses
of Synaptic Transmission and of Neuronal Network Activity in Behavior . . . . . 87
Caspar Glock , J atin N agpal , and Alexander Gottschalk
9 Simultaneous Optogenetic Stimulation of Individual Pharyngeal
Neurons and Monitoring of Feeding Behavior in Intact C. elegans. . . . . . . . . . 105
Nicholas F. Trojanowski and C hristopher Fang-Yen
10 High-Pressure Freeze and Freeze Substitution Electron Microscopy
in C. elegans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 21
Laura M anning and Janet R ichmond
11 Electron Tomography Methods for C. elegans. . . . . . . . . . . . . . . . . . . . . . . . . 141
David H. H all and W illiam J . R ice
12 Microfluidic Devices for Behavioral Analysis, Microscopy,
and Neuronal Imaging in Caenorhabditis elegans. . . . . . . . . . . . . . . . . . . . . . . 159
Ross C. L agoy and D irk R . A lbrecht
13 T racking Single C. elegans Using a USB Microscope
on a Motorized Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 81
Eviatar I. Y emini and André E . X. B rown
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