Table Of ContentMethods in
Molecular Biology 1400
Jose L. Garcia-Pérez Editor
Transposons and
Retrotransposons
Methods and Protocols
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
For further volumes:
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Transposons
and Retrotransposons
Methods and Protocols
Edited by
Jose L. Garcia-Pérez
GENYO (Center for Genomics and Oncological Research), Pfizer/Universidad de
Granada/Junta de Andalucia, PTS Granada, Spain;
Institute of Genetics and Molecular Medicine (IGMM),
University of Edinburgh, United Kingdom
Editor
Jose L . Garcia-Pérez
GENYO (Center for Genomics and Oncological Research)
Pfizer/Universidad de Granada/Junta de Andalucia
PTS Granada, Spain
Institute of Genetics and Molecular Medicine (IGMM)
University of Edinburgh
United Kingdom
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-3370-9 ISBN 978-1-4939-3372-3 (eBook)
DOI 10.1007/978-1-4939-3372-3
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Prefa ce
Upon completion of the human genome and the ENCODE projects, together with “the
genomics revolution” that has (and is providing) provided an enormous amount of DNA
sequencing information of virtually any living creature in the world, today it is undeniable
that transposable elements (TEs) have been main drivers during genome evolution. Indeed,
and although often classifi ed as “selfi sh DNA” or termed “junk DNA,” it is obvious that
TEs manifest a tremendous impact on genome biology and its regulation.
More than 25 years have passed since the seminal discovery that revealed that some TEs
are still active in humans, acting sporadically as insertional mutagens. In the past years, the
fi eld of TE biology has dramatically expanded the horizons of research associated with “junk
DNA,” and the impact of TEs in genomes has provided exciting and provocative hypothesis
like their putative role in brain biology and cancer biology. Coupled with new sequencing
approaches, it is clear that the fi eld of TE biology has entered a new and exciting era in which
more and more scientists are challenging the concept of “junk DNA” using very different
approaches and experimental settings. Thus, it is my hope that this collection of detailed
protocols will serve as a driver to inspire new research in this fascinating area of research.
In this book, I intent to cover common but often laborious protocols developed by
very talented scientists and used by laboratories located in all parts of the world. Because of
their repeated nature, the identifi cation of new TE insertions has proven an extremely com-
plicated approach. This Method book covers some of the latest protocols designed to iden-
tify and characterize TEs in genomes, ancient or recently inserted. Additionally, this book
includes a series of protocols designed to understand how host genomes act to regulate the
activity of TEs, from elegant genetic mobilization assays to key biochemical methods that
have proven very important to understand the complex interplay between TEs and genomes.
Because of their high prevalence in genomes, understanding TE genome wide regulation
and its impact is an area of intensive research and a number of chapters will cover these top-
ics. Finally, this book also includes chapters that describe how TEs can be used for useful
biotechnological applications.
I am really indebted to a group of friends and very talented scientifi c colleagues that
have put together an outstanding compilation of Methods that are very easy to follow.
Generous, fun, and hard working scientists that have been brave to, despite their “junk”
nature, dedicate their careers to understand the impact of TEs in genomes form this fi eld
of research. And while doing that, they have developed sophisticated tools that I hope
might be of interest to many other researchers. I would also like to dedicate this book to
previous talented researchers that, despite not having a chapter in this book, have acted
as main drivers in the fi eld. Thank you to all contributors and past researchers in this fi eld!
It is a great honor to work with you in the preparation of this book and I hope it becomes
a reference book in the future that will be used by contributors but also will help others to
expand their horizons of research in the near term.
Granada, Spain J ose L. Garcia-Pérez Ph.D,
v
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Study of Transposable Elements and Their Genomic Impact . . . . . . . . . . . . . . 1
Martin Muñoz-Lopez, Raquel Vilar-Astasio, Pablo Tristan-Ramos,
Cesar Lopez-Ruiz, and Jose L. Garcia-Pérez
2 B acterial Group II Introns: Identification and Mobility Assay . . . . . . . . . . . . . 2 1
Nicolás Toro , María D olores M olina-Sánchez , Rafael Nisa-Martínez ,
Francisco Martínez-A barca , and Fernando M anuel García-Rodríguez
3 In Silico Methods to Identify Exapted Transposable Element Families. . . . . . . 3 3
LeeAnn Ramsay and Guillaume B ourque
4 R etrotransposon Capture Sequencing (RC-Seq): A Targeted, High-Throughput
Approach to Resolve Somatic L1 Retrotransposition in Humans. . . . . . . . . . . 47
Francisco J. Sanchez-Luque , Sandra R. Richardson ,
and Geoffrey J . F aulkner
5 L ong Interspersed Element Sequencing (L1-Seq): A Method
to Identify Somatic LINE-1 Insertions in the Human Genome. . . . . . . . . . . . 79
T ara T . Doucet and Haig H . K azazian Jr.
6 Combining Amplification Typing of L1 Active Subfamilies (ATLAS)
with High-Throughput Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 5
Raheleh Rahbari and R ichard M. B adge
7 RNA-Seq Analysis to Measure the Expression of SINE Retroelements. . . . . . . 1 07
Ángel C arlos R omán , A ntonio M orales-Hernández ,
and Pedro M. Fernández-Salguero
8 Q ualitative and Quantitative Assays of Transposition and Homologous
Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe . . . . 117
Maya Sangesland , Angela A twood-Moore , S udhir K. Rai ,
and Henry L. L evin
9 LINE Retrotransposition Assays in Saccharomyces cerevisiae. . . . . . . . . . . . . . . 131
Axel V . Horn and J effrey S . H an
10 LINE-1 Cultured Cell Retrotransposition Assay . . . . . . . . . . . . . . . . . . . . . . . 1 39
Huira C. K opera , Peter A. L arson , John B . M oldovan ,
Sandra R. Richardson , Ying L iu , and John V. Moran
11 L1 Retrotransposition in Neural Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . 1 57
Alysson R. Muotri
12 C haracterization of Engineered L1 Retrotransposition Events:
The Recovery Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 65
David Cano , Santiago M orell , Andres J . P ulgarin , Suyapa A mador ,
and Jose L. G arcia-Pérez
vii
viii Contents
13 SINE Retrotransposition: Evaluation of Alu Activity
and Recovery of De Novo Inserts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 83
Catherine A de and A strid M . R oy-Engel
14 The Engineered SVA Trans -mobilization Assay. . . . . . . . . . . . . . . . . . . . . . . . 203
Anja Bock and G erald G . S chumann
15 D etection of LINE-1 RNAs by Northern Blot . . . . . . . . . . . . . . . . . . . . . . . . 2 23
Prescott D eininger and Victoria P . Belancio
16 M onitoring Long Interspersed Nuclear Element 1 Expression
During Mouse Embryonic Stem Cell Differentiation. . . . . . . . . . . . . . . . . . . . 237
Maxime B odak and Constance Ciaudo
17 I mmunodetection of Human LINE-1 Expression
in Cultured Cells and Human Tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 61
Reema Sharma , Nemanja Rodić , K athleen H. Burns ,
and Martin S. Taylor
18 C ellular Localization of Engineered Human LINE-1 RNA and Proteins . . . . . 281
Aurélien J. D oucet , E ugénia B asyuk , and N icolas Gilbert
19 P urification of L1-Ribonucleoprotein Particles (L1-RNPs)
from Cultured Human Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Prabhat K . M andal and H aig H . K azazian Jr.
20 Characterization of L1-Ribonucleoprotein Particles. . . . . . . . . . . . . . . . . . . . . 3 11
Martin S. Taylor , J ohn LaCava , L ixin Dai , Paolo Mita ,
Kathleen H . Burns , Michael P . Rout , and Jef D . Boeke
21 L EAP: L1 Element Amplification Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 39
Huira C . Kopera , Diane A. F lasch , Mitsuhiro N akamura ,
Tomoichiro Miyoshi , A urélien J . D oucet , and John V. Moran
22 Biochemical Approaches to Study LINE-1 Reverse Transcriptase
Activity In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 57
Sébastien V iollet , A urélien J . D oucet , and G aël Cristofari
23 Methylated DNA Immunoprecipitation Analysis of Mammalian
Endogenous Retroviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 77
Rita Rebollo and D ixie L . M ager
24 Profiling DNA Methylation and Hydroxymethylation
at Retrotransposable Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 87
Lorenzo de la R ica , Jatinder S . Stanley , and M iguel R. Branco
25 A Large-Scale Functional Screen to Identify Epigenetic Repressors
of Retrotransposon Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403
Gabriela E cco , Helen M . Rowe , and Didier Trono
26 Reprogramming of Human Fibroblasts to Induced Pluripotent Stem
Cells with Sleeping Beauty Transposon-B ased Stable Gene Delivery . . . . . . . . . 4 19
Attila Sebe and Z oltán I vics
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Contributors
CATHERINE ADE • Department of Epidemiology, Tulane Cancer Center,
Tulane University Health Sciences Center , New Orleans, L A, USA
SUYAPA A MADOR • Department of Human DNA Variability ,
Pfi zer/University of Granada and Andalusian Regional Government Center
for Genomics and Oncology (GENYO) , Granada, S pain
ANGELA ATWOOD-MOORE • Section on Eukaryotic Transposable Elements, Program in
Cellular Regulation and Metabolism, The Eunice Kennedy Shriver National Institute
of Child Health and Human Development, N ational Institutes of Health ,
Bethesda, M D, U SA
RICHARD M. BADGE • Department of Genetics, U niversity of Leicester , L eicester, U K
EUGÉNIA B ASYUK • Institut de Génétique Moléculaire de Montpellier , C NRS (UMR5535),
Montpellier cedex 5, F rance
VICTORIA P . BELANCIO • Department of Structural and Cellular biology,
Tulane School of Public Health , N ew Orleans, LA, U SA; Tulane Cancer Center
and Center for Aging, New Orleans, LA, USA
ANJA B OCK • Division of Medical Biotechnology , P aul-Ehrlich-Institut , L angen, G ermany
MAXIME B ODAK • Department of Biology, Swiss Federal Institute of Technology ,
Zurich, S witzerland
JEF D . BOEKE • Department of Biochemistry and Molecular Pharmacology,
Institute for Systems Genetics, N ew York University Langone School of Medicine , N ew
York , N Y , U SA
GUILLAUME BOURQUE • Department of Human Genetics, M cGill University ,
Montreal , Q C, Canada; M cGill University and Génome Québec Innovation Center,
Montréal, O C , Canada
MIGUEL R . BRANCO • Blizard Institute, School of Medicine and Dentistry , Q MUL, London,
UK
KATHLEEN H . BURNS • Department of Pathology, J ohns Hopkins University School
of Medicine , B altimore , M D , USA
DAVID CANO • Department of Human DNA Variability , Pfi zer/University of Granada
and Andalusian Regional Government Center for Genomics and Oncology (GENYO) ,
Granada, S pain
CONSTANCE CIAUDO • Department of Biology, Swiss Federal Institute of Technology , Z urich,
Switzerland
GAEL C RISTOFARI • INSERM/CNRS (U1081 and UMR 7284) Institute for Research
on Cancer and Aging of Nice (IRCAN) , N ice , F rance ; Faculty of Medicine,
University of Nice-Sophia-Antipolis, Nice, France
LIXIN DAI • Department of Molecular Biology and Genetics, High Throughput Biology
Center , Johns Hopkins University School of Medicine , B altimore , MD, USA ;
Modern Meadow Inc., Brooklyn, USA
ix
