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`Cellular Programming
`and Reprogramming
`
`Methods and Protocols
`
`Edited by
`Sheng Ding Ph.D.
`
`Department of Chemistry, The Scripps Research Institute, La Jolla, CA, USA
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`Editor
`Sheng Ding, Ph.D.
`Department of Chemistry
`The Scripps Research Institute
`La Jolla, CA
`USA
`sding@scripps.edu
`
`ISSN 1064-3745 e
`ISBN 978-1-60761-690-0
`DOI 10.1007/978-1-60761-691-7
`Springer New York Dordrecht Heidelberg London
`
`-ISSN 1940-6029
`e-ISBN 978-1-60761-691-7
`
`Library of Congress Control Number: 2010922991
`
`© Springer Science+Business Media, LLC 2010
`All rights reserved. This work may not be translated or copied in whole or in part without the written permission of
`the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013,
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`information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
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`The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
`as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.
`While the advice and information in this book are believed to be true and accurate at the date of going to press, 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.
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`Preface
`
`Advances in stem cell biology are making possible new approaches to treat devastating
`human diseases, including cardiovascular disease, neurodegenerative disease, musculosk-
`eletal disease, diabetes, and cancer. Such approaches may involve cell replacement therapy
`as well as the development of therapeutic drugs for stimulating the body’s own regenera-
`tive ability to repair cells damaged by disease and injury. However, obstacles such as con-
`trol of stem cell fate, immunorejection, and limited cell sources must be overcome before
`their therapeutic potentials can be realized. Recent studies have suggested that tissue-
`specific cells may overcome their intrinsic lineage-restriction to dedifferentiate or transdif-
`ferentiate upon exposure to a specific set of signals in vitro and in vivo. The ability to
`dedifferentiate or reverse lineage-committed cells to pluripotent/multipotent cells might
`overcome many of the obstacles (e.g., cell sources, immunocompatibility, and bioethical
`concerns) associated with using ES and adult stem cells in clinical applications. With an
`efficient dedifferentiation process, it is conceivable that healthy, abundant, and easily
`accessible somatic cells could be reprogrammed to become multipotent or pluripotent
`stem/progenitor cells, which can then be programmed to generate different types of func-
`tional cells for the repair of damaged tissues and organs. This series will cover the most
`recent technologies and their mechanistic understanding in cellular reprogramming and
`programming.
`
`La Jolla, CA
`
`Sheng Ding
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`Contents
`
`Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`v
`ix
`
`25
`
`45
`
`55
`
` 1 Human Embryonic Stem Cell Derivation, Maintenance,
`and Differentiation to Trophoblast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
`Ge Lin, Kristen Martins-Taylor, and Ren-He Xu
` 2 Isolation and Maintenance of Mouse Epiblast Stem Cells . . . . . . . . . . . . . . . . . . .
`Josh G. Chenoweth and Paul J. Tesar
` 3 Functional Assays for Hematopoietic Stem Cell Self-Renewal . . . . . . . . . . . . . . . .
`John M. Perry and Linheng Li
` 4 Isolation Procedure and Characterization of Multipotent Adult
`Progenitor Cells from Rat Bone Marrow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Kartik Subramanian, Martine Geraerts, Karen A. Pauwelyn,
`Yonsil Park, D. Jason Owens, Manja Muijtjens, Fernando Ulloa-Montoya,
`Yeuhua Jiang, Catherine M. Verfaillie, and Wei-Shou Hu
` 5 Generation of Functional Insulin-Producing Cells from Human
`Embryonic Stem Cells In Vitro. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Yan Shi
` 6 Mesoderm Cell Development from ES Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Takumi Era
` 7 Directed Differentiation of Red Blood Cells from Human
`Embryonic Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
`Shi-Jiang Lu, Qiang Feng, Jennifer S. Park, and Robert Lanza
` 8 Directed Differentiation of Neural-stem cells and Subtype-Specific
`Neurons from hESCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
`Bao-Yang Hu and Su-Chun Zhang
` 9 Directing Human Embryonic Stem Cells to a Retinal Fate . . . . . . . . . . . . . . . . . . 139
`Thomas A. Reh, Deepak Lamba, and Juliane Gust
`10 Bovine Somatic Cell Nuclear Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
`Pablo J. Ross and Jose B. Cibelli
`11 Cell Fusion-Induced Reprogramming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
`Jeong Tae Do and Hans R. Schöler
`12 An Improved Method for Generating and Identifying Human
`Induced Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
`Prashant Mali, Zhaohui Ye, Bin-Kuan Chou, Jonathan Yen,
`and Linzhao Cheng
`13 Using Small Molecules to Improve Generation of Induced
`Pluripotent Stem Cells from Somatic Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
`Caroline Desponts and Sheng Ding
`
`79
`
`87
`
`vii
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`

`viii
`
`Contents
`
`14 Reprogramming of Committed Lymphoid Cells by Enforced
`Transcription Factor Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
`Huafeng Xie, Catherine V. Laiosa, and Thomas Graf
`15 Reprogramming of B Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
`César Cobaleda
`16 Adult Cell Fate Reprogramming: Converting Liver to Pancreas. . . . . . . . . . . . . . . 251
`Irit Meivar-Levy and Sarah Ferber
`17 In Vitro Reprogramming of Pancreatic Cells to Hepatocytes. . . . . . . . . . . . . . . . . 285
`Daniel Eberhard, Kathy O’Neill, Zoë D. Burke, and David Tosh
`18 Generation of Novel Rat and Human Pluripotent Stem Cells
`by Reprogramming and Chemical Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
`Wenlin Li and Sheng Ding
`19 Small Molecule Screen in Zebrafish and HSC Expansion. . . . . . . . . . . . . . . . . . . . 301
`Eirini Trompouki and Leonard I. Zon
`20 Zebrafish Small Molecule Screen in Reprogramming/Cell
`Fate Modulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
`Jing-Ruey J. Yeh and Kathleen M. Munson
`Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
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`Contributors
`
`Zoë D. Burke • Department of Biology & Biochemistry, Centre for Regenerative
`Medicine, University of Bath, Bath, UK
`LinZhao Cheng • Institute for Cell Engineering, Johns Hopkins
`University School of Medicine, Baltimore, MD, USA
`Josh g. Chenoweth • Laboratory of Molecular Biology, National Institute
`of Neurological Disorders and Stroke, National Institutes of Health,
`Bethesda, MD, USA
`Bin-kuan Chou • Institute for Cell Engineering, Johns Hopkins
`University School of Medicine, Baltimore, MD, USA
`Jose B. CiBeLLi • Department of Animal Science, Michigan State
`University, East Lansing, MI, USA
`César CoBaLeDa • Departamento de Fisiología y Farmacología,
`Universidad de Salamanca, Salamanca, Spain
`CaroLine Desponts • Department of Chemistry, The Scripps Research Institute,
`La Jolla, CA, USA
`sheng Ding • Department of Chemistry, The Scripps Research Institute,
`La Jolla, CA, USA
`Jeong tae Do • CHA Stem Cell Institute & CHA Biotech, Pochon CHA
`University, Seoul, Korea
`DanieL eBerharD • Department of Biology & Biochemistry, Centre for Regenerative
`Medicine, University of Bath, Bath, UK
`takumi era • Division of Molecular Neurobiology, Institute of Molecular Embryology
`and Genetics, Kumamoto University, Kumamoto, Japan
`Qiang Feng • Advanced Cell Technology, Worcester, MA, USA
`sarah FerBer • Endocrine Institute, Sheba Medical Center, Tel-Hashomer, Israel;
`Department of Human Genetics and Molecular Medicine,
`Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel
`martine geraerts • Stem Cell Institute Leuven, Catholic University of Leuven,
`Leuven, Belgium
`thomas graF • Center for Genomic Regulation and ICREA, Barcelona, Spain
`JuLiane gust • Department of Biological Structure, University of Washington,
`Seattle, WA, USA
`Bao-Yang hu • Department of Anatomy and Department of Neurology,
`Waisman Center, School of Medicine and Public Health, the WiCell Institute,
`University of Wisconsin-Madison, Madison, WI, USA
`wei-shou hu • Stem Cell Institute and Department of Chemical Engineering
`and Materials Science, University of Minnesota, Minneapolis, MN, USA
`Yeuhua Jiang • Stem Cell Institute Leuven, Catholic University of Leuven,
`Leuven, Belgium
`
`ix
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`x
`
`Contributors
`
`Catherine V. Laiosa • Mount Sinai Hospital, New York, NY, USA
`Deepak LamBa • Department of Biological Structure, University of Washington,
`Seattle, WA, USA
`roBert LanZa • Advanced Cell Technology, Worcester, MA, USA
`Linheng Li • Stowers Institute for Medical Research, Kansas City, MO, USA;
`Department of Pathology and Laboratory Medicine, Kansas University Medical
`Center, Kansas City, KS, USA
`wenLin Li • Department of Chemistry, The Scripps Research Institute,
`La Jolla, CA, USA
`ge Lin • Stem Cell Institute and Department of Genetics and Developmental Biology,
`University of Connecticut Health Center, Farmington, CT, USA
`shi-Jiang Lu • Advanced Cell Technology, Worcester, MA, USA; Stem Cell and
`Regenerative Medicine International, Worcester, MA, USA
`prashant maLi • Institute for Cell Engineering, Johns Hopkins
`University School of Medicine, Baltimore, MD, USA
`kristen martins-taYLor • Stem Cell Institute and Department of Genetics
`and Developmental Biology, University of Connecticut Health Center,
`Farmington, CT, USA
`irit meiVar-LeVY • Sheba Medical Center, Endocrine Institute, Tel-Hashomer, Israel;
`Department of Human Genetics and Molecular Medicine,
`Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel
`manJa muiJtJens • Stem Cell Institute Leuven, Catholic University of Leuven,
`Leuven, Belgium
`kathLeen m. munson • Developmental Biology Laboratory, Cardiovascular
`Research Center, Massachusetts General Hospital, Charlestown, MA, USA;
`Department of Medicine, Harvard Medical School, Boston, MA, USA
`kathY o’neiLL • Department of Biology & Biochemistry, Centre for Regenerative
`Medicine, University of Bath, Bath, UK
`D. Jason owens • Stem Cell Institute and Department of Chemical Engineering
`and Materials Science, University of Minnesota, Minneapolis, MN, USA
`JenniFer s. park • Advanced Cell Technology, Worcester, MA, USA
`YonsiL park • Department of Chemical Engineering and Materials Science and
`Department of Biomedical Engineering, University of Minnesota, Minneapolis,
`MN, USA
`karen a. pauweLYn • Stem Cell Institute Leuven, Catholic University of Leuven,
`Leuven, Belgium
`John m. perrY • Stowers Institute for Medical Research, Kansas City, MO, USA
`thomas a. reh • Department of Biological Structure, University of Washington,
`Seattle, WA, USA
`paBLo J. ross • Department of Animal Science, Michigan State University,
`East Lansing, MI, USA
`hans r. sChöLer • Department of Cell and Developmental Biology,
`Max Planck Institute for Molecular Biomedicine, Münster, Germany
`Yan shi • Laboratory of Chemical Genomics, Shenzhen Graduate School of Peking
`University, Shenzhen, Guangdong, China
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`Contributors
`
`xi
`
`kartik suBramanian • Stem Cell Institute and Department of Chemical Engineering
`and Materials Science, University of Minnesota, Minneapolis, MN, USA
`pauL J. tesar • Department of Genetics, Case Western Reserve University,
`Cleveland, OH, USA
`DaViD tosh • Department of Biology & Biochemistry, Centre for Regenerative
`Medicine, University of Bath, Bath, UK
`eirini trompouki • Stem Cell Program and Hematology/Oncology, Children’s
`Hospital, Harvard Medical School, Howard Hughes Medical Institute,
`Harvard Stem Cell Institute, Boston, MA, USA
`FernanDo uLLoa-montoYa • Stem Cell Institute Leuven, Catholic
`University of Leuven, Leuven, Belgium; Stem Cell Institute and Department
`of Chemical Engineering and Materials Science, University of Minnesota,
`Minneapolis, MN, USA
`Catherine m. VerFaiLLie • Stem Cell Institute Leuven,
`Catholic University of Leuven, Leuven, Belgium
`huaFeng Xie • Dana Farber Cancer Institute, Boston, MA, USA
`ren-he Xu • Stem Cell Institute and Department of Genetics and Developmental
`Biology, University of Connecticut Health Center, Farmington, CT, USA
`Zhaohui Ye • Institute for Cell Engineering, Johns Hopkins University
`School of Medicine, Baltimore, MD, USA
`Jing-rueY J. Yeh • Developmental Biology Laboratory, Cardiovascular
`Research Center, Massachusetts General Hospital, Charlestown, MA, USA;
`Department of Medicine, Harvard Medical School, Boston, MA, USA
`Jonathan Yen • Institute for Cell Engineering, Johns Hopkins
`University School of Medicine, Baltimore, MD, USA
`su-Chun Zhang • Department of Anatomy and Department of Neurology,
`School of Medicine and Public Health, Waisman Center, the WiCell Institute,
`University of Wisconsin-Madison, Madison, WI, USA
`LeonarD i. Zon • Stem Cell Program and Hematology/Oncology,
`Children’s Hospital, Harvard Medical School, Howard Hughes Medical Institute,
`Harvard Stem Cell Institute, Boston, MA, USA
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`

`Chapter 10
`
`Bovine Somatic Cell Nuclear Transfer
`
`Pablo J. Ross and Jose B. Cibelli
`
`Abstract
`
`Somatic cell nuclear transfer (SCNT) is a technique by which the nucleus of a differentiated cell is introduced
`into an oocyte from which its genetic material has been removed by a process called enucleation. In mam-
`mals, the reconstructed embryo is artificially induced to initiate embryonic development (activation).
`The oocyte turns the somatic cell nucleus into an embryonic nucleus. This process is called nuclear repro-
`gramming and involves an important change of cell fate, by which the somatic cell nucleus becomes
`capable of generating all the cell types required for the formation of a new individual, including extraem-
`bryonic tissues. Therefore, after transfer of a cloned embryo to a surrogate mother, an offspring geneti-
`cally identical to the animal from which the somatic cells where isolated, is born. Cloning by nuclear
`transfer has potential applications in agriculture and biomedicine, but is limited by low efficiency. Cattle
`were the second mammalian species to be cloned after Dolly the sheep, and it is probably the most widely
`used species for SCNT experiments. This is, in part due to the high availability of bovine oocytes and the
`relatively higher efficiency levels usually obtained in cattle. Given the wide utilization of this species for
`cloning, several alternatives to this basic protocol can be found in the literature. Here we describe a basic
`protocol for bovine SCNT currently being used in our laboratory, which is amenable for the use of the
`nuclear transplantation technique for research or commercial purposes.
`
`Key words: Cloning, SCNT, Reprogramming, Oocyte enucleation, Nuclear transfer, Cell fusion,
`Oocyte activation, Micromanipulation, Bovine, Cattle
`
`1. Introduction
`
`Somatic cell nuclear transfer (SCNT) is a technique by which the
`nucleus of a somatic cell is introduced into an enucleated oocyte.
`As a result, the somatic nucleus is modified by the recipient
`oocyte’s cytoplasm, allowing the development of the recon-
`structed embryo into a whole individual. The result is a genetic
`clone of the animal from which the donor cell was derived. The
`original idea of generating an animal from a somatic cell was
`
`S. Ding (ed.), Cellular Programming and Reprogramming: Methods and Protocols, Methods in Molecular Biology, vol. 636,
`DOI 10.1007/978-1-60761-691-7_10, © Springer Science+Business Media, LLC 2010
`155
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`156
`
`Ross and Cibelli
`
`proposed by Spemann as a way to test the developmental potential
`of a cell nucleus (1). However, the required technology to per-
`form Spemann’s proposed experiment was not available until the
`1950s when Briggs and King developed nuclear transfer tech-
`niques in frogs, obtaining adult animals when injecting blastula
`cells into enucleated oocytes (2). Later, Gurdon produced
`feeding tadpoles from frog somatic cells (3). In mammals, nuclear
`transfer technology was developed several decades later. The
`transfer of a blastocyst cell (blastomere) nucleus into an enucle-
`ated mouse zygote was reported by Illmensee and Hoppe in
`1981, with the development of adult animals (4). However, con-
`troversy surrounded these results, as other groups were unable to
`repeat the experiment. McGrath and Solter developed a more
`efficient technique by which the donor cell was fused, instead of
`the nucleus injected, with an enucleated zygote; however, they
`were not able to produce offspring when two-cell embryos and
`older were used as cell donors. (5). In 1986, Willadsen obtained
`offspring after fusing sheep MII oocytes with 8- or 16-cell embry-
`onic blastomeres (6). Later, animal cloning from embryonic cells
`was successfully repeated in several species including cattle (7),
`rabbits (8), pigs (9), mice (10), and monkeys (11). Dolly the
`sheep was the first mammal to be cloned from a somatic cell
`nucleus (12) in 1997 and several other species followed, including
`cow (13), mouse (14), goat (15), pig (16), gaur (17), mouflon
`(18), rabbit (19), cat (20), rat (21), mule (22), horse (23), African
`Wildcat (24), dog (25), ferret (26), wolf (27), buffalo (28), and
`red deer (29). In each of these species, the efficiency remains very
`low, with less than 1% of nuclear transfers from adult cells devel-
`oping into normal offspring. Nevertheless, the success of SCNT-
`cloning in several species underscores the totipotent potential of
`the somatic cell nucleus and the reprogramming ability of the
`MII oocyte, and presents nuclear transplantation as a powerful
`methodology to study the molecular mechanisms that regulate
`cell fate commitment, differentiation, and pluripotency.
`In more applied areas, cloning by nuclear transfer has the
`potential to contribute substantially to animal agriculture, bio-
`technology, biomedicine, and preservation of endangered species.
`The success of adult SCNT with almost all agriculturally impor-
`tant species (12, 13, 15, 19, 30) confirms its usefulness for the
`clonal expansion of animals with superior genotypes. Moreover,
`SCNT makes possible germline genetic modifications in domestic
`species (13). Traits which have been considered for genetic modi-
`fication include feed utilization, resistance to disease (thus reduc-
`ing drug/antibiotic use), reduction of animal waste, and
`diversification of agricultural products, i.e., providing new eco-
`nomic opportunities in rural areas, and generation of new con-
`sumer products (31). SCNT can also be used for gene targeting,
`making additions or deletions in the genome feasible. Using this
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`Bovine Somatic Cell Nuclear Transfer
`
`157
`
`approach, cattle that lack the prion gene responsible for bovine
`spongiform encephalopathy were recently produced (32).
`Targeted modifications have also been successfully achieved in
`sheep (33) and pigs (34). Farm animals carrying genetic modifi-
`cations have great potential in biotechnology. Engineered animals
`are being used as bioreactors for the production of pharmaceuti-
`cals and as potential organ donors for the human population.
`Further, SCNT offers an alternative means to preserve endan-
`gered, and even to recover extinct species. Wells et al. reported
`the use of SCNT to clone the last surviving animal of the Enderby
`Island cattle breed (35), and Lanza et al., using interspecies
`nuclear transfer, were able to clone an endangered species (Bos
`gaurus) (36). The same approach was used to clone Mouflons (an
`endangered breed of sheep) with tissue collected from dead ani-
`mals (18). Although all of the above-described applications for
`SCNT are far-reaching, its broad implementation is hindered by
`low efficiency.
`Advances in micromanipulation techniques have allowed an
`improvement in preimplantation development of reconstructed
`embryos; however, the full-term developmental potential of
`embryos produced by SCNT remains low. High rates of early
`pregnancy loss are commonly observed and a higher incidence of
`late-term abortion is often reported for SCNT embryos compared
`to embryos produced by fertilization. Also, higher mortality rates
`of offspring born from NT embryos are often reported.
`Here we describe a basic protocol for bovine SCNT currently
`being used in our laboratory, which is amenable for the use of the
`nuclear transplantation technique for research or commercial
`purposes.
`
` 1. Inverted fluorescent microscope with 4× and 20× Hoffman
`modulation contrast optics. Thermoplate/sheet (38.5°C) is
`recommended. Fluorescence illumination is required to visu-
`alize the HOECHST 33342 stained DNA at the time of enu-
`cleation. A pedal-controlled shutter that blocks UV light
`from the path of light is important to minimize exposure of
`the oocytes to UV irradiation. Also, a condenser that limits
`UV light to the center of the field of view will help minimize
`oocyte exposure to UV light.
` 2. Micromanipulation equipment attached to microscope. For
`holding pipette (usually mounted on the left side), a coarse
`manipulator is sufficient, since after setting up the position of
`this pipette it is not necessary to make continuous adjustments.
`
`2. Materials
`
`2.1. Equipment
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`158
`
`Ross and Cibelli
`
`Fig. 1. Micromanipulation setup. (a) Microscope and micromanipulation equipment. (b) Layout of micromanipulation
`chamber
`
`For the enucleation/transfer manipulator (usually mounted
`on the right side) a hydraulic controlled manipulator is
`required (Fig. 1).
` 3. Microinjectors: An air microinjector can be used for holding
`the oocyte. For enucleation/cell transfer, an oil-filled injector
`is preferred to achieve greater flux control.
` 4. Electrofusion generator: A square DC pulse generator capa-
`ble of voltage and pulse duration adjustments.
` 5. Fusion chamber with 0.5 mm gap between electrodes.
` 6. CO2 incubator.
` 7. Microdispensers (Drummond Scientific Co., Broomall, PA):
`Used for handling of oocytes/embryos. Alternatively, mouth-
`pipettes or tomcat catheters can be used.
` 8. Pipette puller.
` 9. Micro forge: Used to cut and model glass pipettes.
` 10. Micro grinder: Used to produce a bevel in the pipette tip.
`
` 1. Saline solution: 8.5 mg/mL NaCl.
` 2. Hepes-Buffered Hamster Embryo Culture Medium (HH)
`(37): 114 mM NaCl, 3.2 mM KCl, 2 mM CaCl2, 0.5 mM
`MgCl2, 0.1 mM Na pyruvate, 2 mM NaHCO3, 10 mM
`HEPES, 17 mM Na lactate, 1× MEM nonessential amino
`acids, 100 IU/mL penicillin G, 100 mg/mL streptomycin,
`3 mg/mL BSA. pH: 7.3–7.4; Osmolarity: 275 ± 10 mOsm/
`kg; Filter sterilize and store at 4°C for up to 30 days.
` 3. Medium 199 (Sigma).
` 4. Pyruvate stock solution: 11 mg of sodium pyruvate in 5 mL
`of M199, store at 4°C for up to 30 days.
` 5. LH stock: 3 mg Luteinizing Hormone (Sioux Biochem),
`10 mg fatty acid free BSA, 1 mL saline solution. Aliquot and
`store at −20°C for up to 6 months.
`
`2.2. Oocyte Collection
`and Maturation
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`2.3. Micropipette
`Preparation
`
`2.4. Somatic Cell
`Nuclear Transfer
`
`Bovine Somatic Cell Nuclear Transfer
`
`159
`
` 6. FSH stock: 3 mg Follicular-Stimulating Hormone (Sioux
`Biochem), 10 mg fatty acid free BSA, 1 mL saline solution.
`Aliquot and store at −20°C for up to 6 months.
` 7. Estradiol stock: 1 mg 17 beta-estradiol in 1 mL absolute eth-
`anol. Store at −20°C.
` 8. Gentamicin: 10 mg/mL (Gibco).
` 9. Fetal bovine serum (FBS).
`
` 1. Borosilicate glass capillaries 1 mm OD (outside diame-
`ter) × 0.75 mm ID (inside diameter).
` 2. Borosilicate glass capillaries 1 mm OD × 0.58 mm ID.
` 3. Chromerge glass cleaner.
` 4. 70% Ethanol.
`
` 1. Hyaluronidase solution: 1 mg/mL hyaluronidase in HH
`medium. Divide in 1 mL single use aliquots and store at −20°C.
` 2. CB stock: 1 mg of Cytochalasin B in 200 mL DMSO. Aliquot
`and store at −20°C.
` 3. HOECHST stock: 1 mg Bisbenzimide (Hoechst 33342) in
`1 mL of PBS. Store at −20°C protected from light.
` 4. Mineral oil.
` 5. Fluorinert FC-40 (Sigma).
` 6. Pronase: 10 IU/mL Pronase in HH medium, store at 4°C.
` 7. Fusion medium: 250 mM d-Sorbitol, 0.5 mM MgOAc,
`1 mg/mL BSA, pH: 7.2.
` 8. Osmolarity: 255 ± 10 mOsm/kg. Filter, sterilize, and store at
`4°C for 20 days.
` 9. Ionomycin stock (5 mM): Dissolve 1 mg Ionomycin in 267.6 mL
`DMSO. Aliquot and store at −20°C for up to 12 months.
` 10. DMAP stock (200 mM): Dissolve 163 mg of DMAP in 5 mL
`ddH2O in a 90°C water bath. Aliquot and store at −20°C.
` 11. KSOM embryo culture medium: Available commercially
`(Millipore) supplemented with 3 mg/mL BSA.
`
`3. Methods
`
`3.1. Oocyte Collection
`and Maturation
`
`Oocytes for bovine SCNT are typically harvested from slaughter-
`house-derived ovaries and matured in vitro. Alternatively, oocytes
`can be collected from animals by ultrasound-guided oocyte aspi-
`ration at mature or immature stages. We describe the protocol for
`collecting oocyte from slaughterhouse-derived ovaries only.
`
`Exhibit 1031
`Select Sires, et al. v. ABS Global
`
`

`

`160
`
`Ross and Cibelli
`
` 1. Prepare oocyte maturation medium by adding the following
`to 9 mL of Medium 199: 1 mL of FBS, 10 mL of FSH stock,
`10 mL of LH stock, 100 mL of pyruvate stock, and 25 mL of
`gentamicin. Filter sterilize and then add 10 mL of estradiol
`stock (do not take the estradiol out of the −20°C freezer to
`avoid ethanol evaporation and estradiol concentration).
`Equilibrate in the incubator for at least 4 h.
` 2. Ovaries are transported from slaughterhouse to lab in a ther-
`mal container at room temperature.
` 3. Place ovaries in a colander and rinse them thoroughly using
`warm tap water.
` 4. Transfer oocytes to a beaker and add warm saline solution.
` 5. Keep the beaker with oocytes in a 30°C waterbath.
` 6. Aspirate follicles using an 18-G hypodermic needle. The needle
`is connected to a vacuum source that can be a 10-mL syringe
`or a vacuum pump. The use of a vacuum pump allows for faster
`oocyte collection. A 50-mL tube acting as a vacuum trap is
`used to collect the follicular fluid containing the oocytes (Fig. 2).
`
`Fig. 2. Oocyte collection. (a) Aspiration assembly. (b) Dissembled aspiration assembly. (c) Immature oocytes. (d) Matured oocytes
`
`Exhibit 1031
`Select Sires, et al. v. ABS Global
`
`

`

`Bovine Somatic Cell Nuclear Transfer
`
`161
`
`An aspiration assembly is constructed with a rubber stopper
`and a 1-mL glass pipette. Bend the glass pipette at a 90° angle
`and cut the narrow end. Drill a hole in the center of the rubber
`stopper. Insert the pipette in the stopper through the hole. Cut
`a 1-mL plastic syringe in the middle and assemble the end that
`connects to the needle to the glass pipette using a small piece
`of tygon tubing. Ensure that the connection is air tight. Cut
`another 1-mL syringe and connect it to an 18-G needle, then
`insert the needle through the rubber stopper. Connect the
`vacuum pump to this piece of syringe with tygon tubing. For
`oocyte aspiration attach an 18-G needle to the aspiration
`assembly with the opening of the needle facing down and
`mount the assembly on a 50-mL tube. After using, rinse the
`assembly thoroughly with distilled water and spray with 70%
`ethanol. Let dry in a clean container.
` 7. Remove groups of 10–20 ovaries from the beaker and dry
`their surfaces with paper towels.
` 8. Aspirate follicles 2–8 mm in diameter. To aspirate the follicle
`content first penetrate the ovarian parenchyma and then the
`follicle. This will prevent a potential follicle rupture and loss
`of oocyte. Also, several follicles can be aspirated through the
`same hole by advancing the needle through the oocyte
`cortex.
` 9. Let the oocytes sediment in the follicular fluid and collect the
`sediment using a disposable plastic Pasteur pipette.
` 10. Add 2–3 mL of HH medium to a gridded 100-mm petri dish
`(the grid can be drawn with a marker on the external surface
`of the dish).
` 11. Disperse the liquid in the dish but without touching the
`edges.
` 12. Add the oocytes to the dish and allow 1 min for them to sedi-
`ment (see Note 1).
` 13. Collect and transfer the oocytes to a 1-mL drop of HH
`medium.
` 14. Select good quality oocytes (homogeneous oocyte cytoplasm
`and at least three layers of cumulus cells; Fig. 10.2) and trans-
`fer them in groups of 50 into 100 mL drops of HH medium.
`Immediately after releasing the oocytes in clean HH drops,
`aspirate loose cells; this will help clean the oocytes in fewer
`washes therefore reduce handling of the oocytes.
` 15. Wash through another drop of HH and then transfer to a
`four-well dish containing 500 mL of preequilibrated matura-
`tion medium.
` 16. Incubate at 38.5°C, humidity to saturation, and 5% CO2
`in air.
`
`Exhibit 1031
`Select Sires, et al. v. ABS Global
`
`

`

`162
`
`Ross and Cibelli
`
`3.2. Micropipette
`Preparation
`
`3.2.1. Holding Pipette
`
`Preparing good manipulation tools accounts for a great part of
`success in nuclear transfer technique. The micropipettes required
`to do nuclear transfer consist of a holding pipette, enucleation
`pipette, and cell transfer pipette. These pipettes differ in size and
`shape and are fashioned from glass capillaries. Making these
`pipettes will require some practice and trial and error at first, but
`proficiency in making micromanipulation tools is generally gained
`in a short period of time (few weeks).
`
`The holding pipette is used to position the oocyte for enucleation
`and cell transfer. The external diameter of this pipette can range
`from 50 to 90% of that of the oocyte. To manipulate bovine
`oocytes, we typically prepare holding pipettes with an external
`diameter of 150 mM. The opening of the pipette is set at 20–30%
`the oocyte diameter, in our case approximately 30 mM. To pro-
`duce holding pipettes we use 1 mm OD × 0.58 mm ID glass capil-
`laries. The pipette is pulled using a pipette puller to achieve a
`lightly tapered and long tip. Then, the tip of the pipette is cut at
`the desired width using a diamond-tip pen. This can be easily
`performed by placing the pipette in the microforge where the
`desired diameter can be measured with the micrometer scale in
`the eyepiece (Fig. 3).
` 1. Pull the glass.
` 2. Place the pipette on the microforge in horizontal position
`and locate the desired width to be broken.
` 3. Pass a diamond-tip pen across the top surface of the pipette
`two or three times.
` 4. Apply pressure to the tip to break it. This should result in an
`even cut. If the cut is not even, discard the pipette and start
`over. Alternatively, the pipette can be cut using the technique
`described below for enucleation and transfer pipettes,
`although because of the larger size of holding pipette, this
`could be cumbersome.
` 5. Place the pipette in vertical position on top of the glass bead
`present in the microforge.
` 6. Set the h