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M ETHODS IN M OLECULAR B 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

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Page 1: M ETHODS IN M OLECULAR B IOLOGY978-1-4939-1215-5/1.pdf · Shree Ram Singh Stem Cell Regulation and Animal Aging Section, Basic Research Laboratory National Cancer Institute, National

M E T H O D S I N M O L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life SciencesUniversity of Hertfordshire

Hat fi eld, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

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Mouse Genetics

Methods and Protocols

Edited by

Shree Ram SinghStem Cell Regulation and Animal Aging Section, Basic Research Laboratory, National Cancer Institute, National Institutes of Health, Frederick, MD, USA

Vincenzo CoppolaGenetically Engineered Mouse Modeling Core/Department of Molecular Virology,

Immunology and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA

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ISSN 1064-3745 ISSN 1940-6029 (electronic)ISBN 978-1-4939-1214-8 ISBN 978-1-4939-1215-5 (eBook) DOI 10.1007/978-1-4939-1215-5 Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2014942721

© Springer Science+Business Media New York 2014 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 SpringerSpringer is part of Springer Science+Business Media (www.springer.com)

Editors Shree Ram Singh Stem Cell Regulation and Animal Aging Section,

Basic Research LaboratoryNational Cancer Institute, National Institutes

of Health Frederick , MD , USA

Vincenzo Coppola Genetically Engineered Mouse Modeling

Core/Department of Molecular Virology, Immunology and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center

The Ohio State University Columbus , OH , USA

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v

Although evolution has separated mice and humans about 75 million years ago, they still share an incredible level of anatomical, physiological, and especially genomic resemblance. Modeling human disease in mice offers numerous advantages over other mammalian ani-mal models because they are small, easy to breed, available in large number of inbred strains, and their genome is fully sequenced. Most of all, early stage mouse embryos are conducive to in vitro manipulation, and many embryonic stem (ES) cell lines have shown robust homologous recombination and germline transmission in existence. All these ele-ments have allowed researchers to develop innovative technologies that effi ciently edit the mouse genome in vivo. It is now possible to engineer targeted alterations such as gene knockout and knockin or elegant conditional gene modifi cation, which allows temporal- spatial regulation and cell lineage tracing. Other genetic technologies such as Recombinase- Mediated Cassette Exchange (RMCE), which can generate allelic series of mutants and mutagenesis, can also be obtained by use of transposons. Over the years, almost all human diseases including cancer, diabetes, obesity, cystic fi brosis, arthritis, and heart and neurode-generative diseases have been modeled in mice.

Mouse Genetics : Methods and Protocols provides selected mouse genetic techniques and their application in modeling varieties of human diseases. The chapters are mainly focused on the generation of different transgenic mice to accomplish the manipulation of genes of interest, tracing cell lineages, and modeling human diseases. Composed in the highly suc-cessful Methods in Molecular Biology series format, each chapter contains a brief introduc-tion, a list of necessary materials, systematic methods, and a notes section, which shares tips on troubleshooting to avoid known pitfalls.

We hope that Mouse Genetics : Methods and Protocols would provide fundamental tech-niques and protocols to geneticists, molecular biologists, cell and developmental biologists, students, and postdoctoral fellows working in the various disciplines of mouse biology and modeling human disease.

We would like to thank Prof. John M. Walker and the staff at Springer for their invita-tion, editorial guidance, and assistance throughout the preparation of the book for publica-tion. We also would like to express our sincere appreciation and gratitude to the contributors for sharing their precious laboratory expertise with the mouse community. Finally yet importantly, we would like to thank our family members for their continued support.

Frederick, MD, USA Shree Ram Singh, Ph.D. Columbus, OH, USA Vincenzo Coppola, M.D.

Pref ace

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Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I MOUSE GENETICS TECHNIQUES

1 Development of Pronuclear Injection-Based Targeted Transgenesis in Mice Through Cre–loxP Site-Specific Recombination . . . . . . . . . . . . . . . . . 3 Masato Ohtsuka

2 Generation of Conditional Knockout Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Kazuhito Sakamoto, Channabasavaiah B. Gurumurthy, and Kay-Uwe Wagner

3 Generating Mouse Lines for Lineage Tracing and Knockout Studies . . . . . . . . 37 Petra Kraus, V. Sivakamasundari, Xing Xing, and Thomas Lufkin

4 Generation of an Allelic Series of Knock-In Mice Using Recombinase-Mediated Cassette Exchange (RMCE). . . . . . . . . . . . . . . . . . . . 63 Anton J.M. Roebroek and Bart Van Gool

5 Generating Chimeric Mice from Embryonic Stem Cells via Vial Coculturing or Hypertonic Microinjection . . . . . . . . . . . . . . . . . . . . . 77 Kun-Hsiung Lee

6 Genetic Inducible Fate Mapping in Adult Mice Using Tamoxifen-Dependent Cre Recombinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Susanne Feil, Jana Krauss, Martin Thunemann, and Robert Feil

7 Intracytoplasmic Sperm Injection (ICSI)-Mediated Transgenesis in Mice. . . . . 141 Pedro N. Moreira and Lluís Montoliu

8 Generation of Bacterial Artificial Chromosome (BAC) Transgenic Mice . . . . . 157 Jane Beil and Thorsten Buch

9 Generation of Genetically Engineered Mice by the piggyBac Transposon System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Sheng Ding, Tian Xu, and Xiaohui Wu

10 Generation and Applications of MADM-Based Mouse Genetic Mosaic System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 Hui Zong

11 Generation of Mouse Lines Conditionally Over-expressing MicroRNA Using the Rosa26-Lox-Stop-Lox System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Claudia Piovan, Foued Amari, Francesca Lovat, Qun Chen, and Vincenzo Coppola

12 In Situ Hybridization (Both Radioactive and Nonradioactive) and Spatiotemporal Gene Expression Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 225 Olga Simmons, Esther M. Bolanis, Jian Wang, and Simon J. Conway

Contents

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PART II MOUSE GENETICS IN STEM CELLS

13 Isolation and Handling of Mouse Embryonic Fibroblasts . . . . . . . . . . . . . . . . 247 Kanika Jain, Paul J. Verma, and Jun Liu

14 Generation of Induced Pluripotent Stem Cells from Mouse Adipose Tissue . . . 253 Pollyanna Agnes Goh and Paul J. Verma

15 Pdx1 (GFP/w) Mice for Isolation, Characterization, and Differentiation of Pancreatic Progenitor Cells . . . . . . . . . . . . . . . . . . . . . . 271 Michael D. Williams, Wilson Wong, Amanda Rixon, Sarang N. Satoor, Anandwardhan A. Hardikar, and Mugdha V. Joglekar

16 Generation of Transgenic Mouse Fluorescent Reporter Lines for Studying Hematopoietic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 Andrei M. Vacaru, Joseph Vitale, Johnathan Nieves, and Margaret H. Baron

17 Analyzing Gene Function in Adult Long-Term Hematopoietic Stem Cells Using the Interferon Inducible Mx1-Cre Mouse System . . . . . . . . 313 Kristbjorn Orri Gudmundsson, Kevin Oakley, Yufen Han, and Yang Du

18 Generation of Transgenic Mice by Exploiting Spermatogonial Stem Cells In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Lalit Sehgal, Abul Usmani, Sorab N. Dalal, and Subeer S. Majumdar

PART III MOUSE GENETICS IN MODELING HUMAN DISEASE

19 Methods for the Detection of Genome Instability Derived from Replication Stress in Primary Mouse Embryonic Fibroblasts . . . . . . . . . . 341 Spencer W. Luebben, Naoko Shima, and Tsuyoshi Kawabata

20 Transgenic Nude Mice Ubiquitously Expressing Fluorescent Proteins for Color-Coded Imaging of the Tumor Microenvironment. . . . . . . . 353 Robert M. Hoffman

21 Genetically Engineered Insertional Mutagenesis in Mice to Model Cancer: Sleeping Beauty. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367 Viive M. Howell and Emily K. Colvin

22 Generating Double Knockout Mice to Model Genetic Intervention for Diabetic Cardiomyopathy in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 Vishalakshi Chavali, Shyam Sundar Nandi, Shree Ram Singh, and Paras Kumar Mishra

23 Experimental Osteoarthritis Models in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . 401 Julia Lorenz and Susanne Grässel

24 Mouse Models and Methods for Studying Human Disease, Acute Kidney Injury (AKI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421 Ganesan Ramesh and Punithavathi Ranganathan

Contents

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25 Mouse Models of Acute and Chronic Colitis . . . . . . . . . . . . . . . . . . . . . . . . . . 437 Santhakumar Manicassamy and Indumathi Manoharan

26 Manipulation and Assessment of Gut Microbiome for Metabolic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449 Sarang N. Satoor, Deepak P. Patil, Holly D. Kristensen, Mugdha V. Joglekar, Yogesh Shouche, and Anandwardhan A. Hardikar

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471

Contents

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FOUED AMARI • Genetically Engineered Mouse Modeling Core/Department of Molecular Virology, Immunology and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center , The Ohio State University , Columbus , OH , USA

MARGARET H. BARON, M.D., PH.D. • Department of Medicine , Icahn School of Medicine at Mount Sinai , New York , NY , USA ; Department of Developmental and Regenerative Biology , Icahn School of Medicine at Mount Sinai , New York , NY , USA ; Department of Oncological Sciences , Icahn School of Medicine at Mount Sinai , New York , NY , USA ; The Tisch Cancer Institute , Icahn School of Medicine at Mount Sinai , New York , NY , USA ; The Black Family Stem Cell Institute , Icahn School of Medicine at Mount Sinai , New York , NY , USA ; Graduate School of Biomedical Sciences , Icahn School of Medicine at Mount Sinai , New York , NY , USA

JANE BEIL • Institute for Medical Microbiology, Immunology, and Hygiene , Technische Universität München , Munich , Germany

ESTHER M. BOLANIS • HB Wells Center for Pediatric Research , Indiana University School of Medicine , Indianapolis , IN , USA

THORSTEN BUCH • Institute for Medical Microbiology, Immunology and Hygiene , Technische Universität München , Munich , Germany

VISHALAKSHI CHAVALI • Department of Cellular and Integrative Physiology , University of Nebraska Medical Center , Omaha , NE , USA

QUN CHEN • Genetically Engineered Mouse Modeling Core/Department of Molecular Virology, Immunology and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center , The Ohio State University , Columbus , OH , USA

EMILY K. COLVIN • Bill Walsh Translational Cancer Research Laboratory, Kolling Institute of Medical Research, Royal North Shore Hospital , University of Sydney , St. Leonards , NSW , Australia

SIMON J. CONWAY • HB Wells Center for Pediatric Research , Indiana University School of Medicine , Indianapolis , IN , USA

VINCENZO COPPOLA • Genetically Engineered Mouse Modeling Core/Department of Molecular Virology, Immunology and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center , The Ohio State University , Columbus , OH , USA

SORAB N. DALAL • Advanced Centre for Treatment Research Education and Cancer , Tata Memorial Centre , Navi Mumbai , India

SHENG DING • State Key Laboratory of Genetic Engineering and Institute of Developmental Biology and Molecular Medicine, Fudan-Yale Biomedical Research Center, School of Life Sciences , Fudan University , Shanghai , China ; Department of Genetics, Howard Hughes Medical Institute , Yale University School of Medicine , New Haven , CT , USA

YANG DU • Department of Pediatrics , Uniformed Services University of the Health Sciences , Bethesda , MD , USA

ROBERT FEIL • Interfakultäres Institut für Biochemie , Universität Tübingen , Tübingen , Germany

SUSANNE FEIL • Interfakultäres Institut für Biochemie , Universität Tübingen , Tübingen , Germany

Contributors

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POLLYANNA AGNES GOH • Research Department of Haematology, UCL Cancer Institute , University College London , London , UK

BART VAN GOOL • Laboratory for Experimental Mouse Genetics, Center for Human Genetics , KU Leuven , Leuven , Belgium

SUSANNE GRÄSSEL, PH.D. • Orthopaedic Surgery, Experimental Orthopaedics, Centre for Medical Biotechnology , University of Regensburg , Regensburg , Germany

KRISTBJORN ORRI GUDMUNDSSON • Department of Pediatrics , Uniformed Services University of the Health Sciences , Bethesda , MD , USA

CHANNABASAVAIAH B. GURUMURTHY • Department of Genetics, Cell Biology, & Anatomy , University of Nebraska Medical Center , Omaha , NE , USA

YUFEN HAN • Department of Pediatrics , Uniformed Services University of the Health Sciences , Bethesda , MD , USA

ANANDWARDHAN A. HARDIKAR • NHMRC Clinical Trials Centre , The University of Sydney , Camperdown , NSW , Australia

ROBERT M. HOFFMAN • AntiCancer, Inc. , San Diego , CA , USA ; Department of Surgery, UCSD Medical Center , San Diego , CA , USA

VIIVE M. HOWELL • Bill Walsh Translational Cancer Research Laboratory, Kolling Institute of Medical Research, Royal North Shore Hospital , University of Sydney , St. Leonards , NSW , Australia

KANIKA JAIN • Stem Cells and Reprogramming Group, Biological Engineering, Faculty of Engineering , Monash University , Clayton , VIC , Australia

MUGDHA V. JOGLEKAR, PH.D. • NHMRC Clinical Trials Centre , The University of Sydney , Camperdown , NSW , Australia

TSUYOSHI KAWABATA • Department of Genetics, Cell Biology and Development , University of Minnesota , Minneapolis , MN , USA

PETRA KRAUS • Department of Biology , Clarkson University , Potsdam , USA JANA KRAUSS • Interfakultäres Institut für Biochemie , Universität Tübingen , Tübingen ,

Germany HOLLY D. KRISTENSEN • NHMRC Clinical Trials Centre , The University of Sydney ,

Camperdown , NSW , Australia KUN-HSIUNG LEE • Division of Biotechnology , Animal Technology Institute Taiwan ,

Chunan , Miaoli , Taiwan JUN LIU • Stem Cells and Reprogramming Group, Biological Engineering Laboratories,

Faculty of Engineering , Monash University , Clayton , VIC , Australia JULIA LORENZ • Orthopaedic Surgery, Experimental Orthopaedics, Centre for Medical

Biotechnology , University of Regensburg , Regensburg , Germany FRANCESCA LOVAT • Department of Molecular Virology, Immunology and Medical Genetics,

Wexner Medical Center, Comprehensive Cancer Center , The Ohio State University , Columbus , OH , USA

SPENCER W. LUEBBEN • Department of Genetics, Cell Biology and Development , University of Minnesota , Minneapolis , MN , USA

THOMAS LUFKIN • Department of Biology , Clarkson University , Potsdam , USA SUBEER S. MAJUMDAR • National Institute of Immunology , New Delhi , India SANTHAKUMAR MANICASSAMY, PH.D. • Department of Medicine and Cancer Center , Georgia

Regents University , Augusta , GA , USA INDUMATHI MANOHARAN • Department of Medicine and Cancer Center , Georgia Regents

University , Augusta , GA , USA

Contributors

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PARAS KUMAR MISHRA, PH.D. • Department of Cellular and Integrative Physiology , University of Nebraska Medical Center , Omaha , NE , USA ; Department of Anesthesiology , University of Nebraska Medical Center , Omaha , NE , USA

LLUÍS MONTOLIU • National Centre for Biotechnology (CNB-CSIC) , Madrid , Spain ; CIBERER-ISCIII , Madrid , Spain

PEDRO N. MOREIRA • Mouse Biology Unit , EMBL Monterotondo, Monterotondo , Italy SHYAM SUNDAR NANDI • Department of Cellular and Integrative Physiology , University

of Nebraska Medical Center , Omaha , NE , USA JOHNATHAN NIEVES • Department of Medicine , Icahn School of Medicine at Mount Sinai ,

New York , NY , USA ; The Tisch Cancer Institute , Icahn School of Medicine at Mount Sinai , New York , NY , USA

KEVIN OAKLEY • Department of Pediatrics , Uniformed Services University of the Health Sciences , Bethesda , MD , USA

MASATO OHTSUKA • Division of Basic Medical Science and Molecular Medicine, Department of Molecular Life Science , Tokai University School of Medicine , Isehara , Kanagawa , Japan

DEEPAK P. PATIL • Department of Pharmacology, Weill Medical College , Cornell University , New York , NY , USA

CLAUDIA PIOVAN • Department of Molecular Virology, Immunology, and Medical Genetics, Wexner Medical Center, Comprehensive Cancer Center , The Ohio State University , Columbus , OH , USA

GANESAN RAMESH, PH.D. • Department of Medicine and Vascular Biology Center , Georgia Health Sciences University , Augusta , GA , USA

PUNITHAVATHI RANGANATHAN • Department of Medicine and Vascular Biology Center , Georgia Health Sciences University , Augusta , GA , USA

AMANDA RIXON • Department of Surgery, O’Brien Institute, St. Vincent’s Hospital , University of Melbourne , Melbourne , VIC , Australia ; Experimental Medical and Surgical Unit (EMSU) , St Vincent’s Hospital , Melbourne , VIC , Australia

ANTON J.M. ROEBROEK • Laboratory for Experimental Mouse Genetics, Center for Human Genetics , KU Leuven , Leuven , Belgium

KAZUHITO SAKAMOTO • Eppley Institute for Research in Cancer and Allied Diseases , University of Nebraska Medical Center , Omaha , NE , USA

SARANG N. SATOOR • NHMRC Clinical Trials Centre , The University of Sydney , Camperdown , NSW , Australia

LALIT SEHGAL • Advanced Centre for Treatment Research Education and Cancer, Tata Memorial Centre , Kharghar Node , Navi Mumbai , India ; Department of Lymphoma/Myeloma , UT MD Anderson Cancer Center , Houston , TX , USA

NAOKO SHIMA, PH.D. • Department of Genetics, Cell Biology and Development , University of Minnesota , Minneapolis , MN , USA

YOGESH SHOUCHE • Microbial Collection Centre, National Centre for Cell Science , Pune , India

OLGA SIMMONS • HB Wells Center for Pediatric Research , Indiana University School of Medicine , Indianapolis , IN , USA

SHREE RAM SINGH • Stem Cell Regulation and Animal Aging Section, Basic Research Laboratory , National Cancer Institute, National Institutes of Health , Frederick , MD , USA

V. SIVAKAMASUNDARI • Stem Cell and Developmental Biology , Genome Institute , Singapore , Singapore

Contributors

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MARTIN THUNEMANN • Interfakultäres Institut für Biochemie , Universität Tübingen , Tübingen , Germany

ABUL USMANI • National Institute of Immunology , New Delhi , India ANDREI M. VACARU • Department of Medicine , Icahn School of Medicine at Mount Sinai ,

New York , NY , USA ; The Tisch Cancer Institute , Icahn School of Medicine at Mount Sinai , New York , NY , USA

PAUL J. VERMA • Stem Cells and Reprogramming Group, Biological Engineering Laboratories, Faculty of Engineering , Monash University , Clayton , VIC , Australia ; Turretfi eld Research Centre , South Australian Research & Development Institute , Rosedale , SA , Australia

JOSEPH VITALE • Department of Medicine , Icahn School of Medicine at Mount Sinai , New York , NY , USA ; The Tisch Cancer Institute , Icahn School of Medicine at Mount Sinai , New York , NY , USA

KAY-UWE WAGNER • Eppley Institute for Research in Cancer and Allied Diseases , University of Nebraska Medical Center , Omaha , NE , USA ; Department of Genetics, Cell Biology, & Anatomy , University of Nebraska Medical Center , Omaha , NE , USA

JIAN WANG • HB Wells Center for Pediatric Research , Indiana University School of Medicine , Indianapolis , IN , USA

MICHAEL D. WILLIAMS • NHMRC Clinical Trials Centre , The University of Sydney , Camperdown , NSW , Australia ; Department of Surgery, O’Brien Institute, St. Vincent’s Hospital , University of Melbourne , Melbourne , VIC , Australia

WILSON WONG • NHMRC Clinical Trials Centre , The University of Sydney , Camperdown , NSW , Australia

XIAOHUI WU • State Key Laboratory of Genetic Engineering and Institute of Developmental Biology and Molecular Medicine, Fudan-Yale Biomedical Research Center, School of Life Sciences , Fudan University , Shanghai , China

XING XING • Stem Cell and Developmental Biology, Genome Institute , Singapore , Singapore TIAN XU • State Key Laboratory of Genetic Engineering and Institute of Developmental

Biology and Molecular Medicine, Fudan-Yale Biomedical Research Center, School of Life Sciences , Fudan University , Shanghai , China ; Department of Genetics, Howard Hughes Medical Institute , Yale University School of Medicine , New Haven , CT , USA

HUI ZONG • Department of Microbiology, Immunology, and Cancer Biology, Center for Cell Signaling , University of Virginia , Charlottesville , VA , USA

Contributors