Somatic Cell Nuclear Transfer Technology

Dedication
Preface
Contents
Contributors
Chapter 1: Cloning by SCNT: Integrating Technical and Biology-Driven Advances
1 Introduction
2 Nuclear Equivalency and Animal Cloning
3 Mammalian Cloning from Embryonic Nuclei
4 The Development of SCNT in Mammals
5 Applications of Mammalian Cloning by SCNT
6 Technical Adaptations of SCNT Protocols in Mammals
7 Improving SCNT-Mediated Reprogramming
8 Genomics Revealed Roadblocks to Nuclear Reprogramming
9 Concluding Remarks and Perspectives
References
Chapter 2: Epigenetic Reprogramming and Somatic Cell Nuclear Transfer
1 Epigenetics
1.1 DNA Methylation
1.2 Histone Code
1.3 Non-Coding RNA
2 Epigenetic Reprogramming During Mammalian Preimplantation Development
2.1 Genomic Imprinting
2.2 X Chromosome Inactivation
3 Epigenetic Reprogramming After Somatic Cell Nuclear Transfer
3.1 Molecular Strategies to Improve SCNT Efficiency
References
Chapter 3: Early Cell Specification in Mammalian Fertilized and Somatic Cell Nuclear Transfer Embryos
1 Introduction
2 Early Cell Specification During Mammalian Preimplantation Development
2.1 Differentiation of the Trophectoderm and the Inner Cell Mass
2.2 Differentiation of the Epiblast and the Primitive Endoderm
2.3 Epigenetic Dynamics Related to Early Cell Specification
2.4 X Chromosome Inactivation and Cell Lineage Specification
3 Early Cell Lineage Specification in SCNT Embryos
3.1 Differences in ICM or TE Cell Numbers in SCNT Embryos
3.2 Expression of Differentiation-Associated Genes in SCNT Embryos
3.3 Derivation of ESC Lines from SCNT Embryos
3.4 Aberrant X Chromosome Inactivation in SCNT Embryos
4 Perspectives and Conclusions
References
Chapter 4: Mitochondrial Inheritance Following Nuclear Transfer: From Cloned Animals to Patients with Mitochondrial Disease
1 Introduction
2 Mitochondrial Function and Dynamics
3 Mitochondrial DNA Structure and Maintenance
4 Mitochondrial DNA Inheritance
5 Mitochondrial Inheritance in Cloned Animals
6 Manipulation of Mitochondrial Inheritance in Humans
7 Conclusions and Future Prospects
References
Chapter 5: Stem Cells as Nuclear Donors for Mammalian Cloning
1 Introduction
2 Use of Stem Cells for Mammalian Cloning
2.1 Cattle (Bos taurus)
2.2 Buffalo (Bubalus bubalis)
2.3 Swine (Sus scrofa)
2.4 Horses (Equus caballus)
2.5 Goats (Capra hircus) and Sheep (Ovis aries)
2.6 Dogs (Canis lupus familiaris)
2.7 Red Deer (Cervus elaphus)
2.8 Mouse (Mus musculus)
3 Perspectives on Using Stem Cells for Mammalian Cloning
References
Chapter 6: Animal Transgenesis and Cloning: Combined Development and Future Perspectives
1 Livestock Genomes as the Context for Transgenesis
2 The Dawn of Recombinant DNA Technology and Animal Transgenesis
3 GMO and Regulatory Aspects
4 Genome-Edited Organisms (GEO)
5 GEO and Regulatory Aspects
6 Genome Editing Applied to Livestock Animals
6.1 Rabbits
6.2 Sheep and Goats
6.3 Pigs
6.4 Cattle
7 Concluding Remarks
References
Chapter 7: Mouse Cloning Using Outbred Oocyte Donors and Nontoxic Reagents
1 Introduction
2 Materials
2.1 Equipment
2.2 Mouse Strains
2.3 Media and Solutions
2.4 Culture Media
2.5 Preparation of Holding, Enucleation, and Injection Micropipettes
2.6 Preparation of Media Dishes
2.7 Micromanipulator Setup
3 Methods
3.1 Recipient Oocyte Preparation
3.2 Donor Cell Preparation
3.2.1 Preparation of Cumulus Cells (Optional)
3.2.2 Preparation of Tail Tip Fibroblasts (Optional)
3.2.3 Preparation of Embryonic Stem Cells (Optional)
3.3 Donor Cell Preparation Using PLC-PVP Medium (Alternative Method)
3.4 Enucleation of Oocytes
3.5 Donor Nucleus Injection
3.6 Oocyte Activation and Embryo Culture
3.7 Embryo Culture Without Oocyte Chemical Activation
3.8 Embryo Transfer
3.9 Cesarean Section
4 Notes
References
Chapter 8: Somatic Cell Nuclear Transfer in Rabbits
1 Introduction
2 Materials
2.1 Equipment
2.2 Tools and Consumables
2.3 Media and Solutions
2.4 Preparation of Micromanipulation Pipettes
2.4.1 Holding Pipette
2.4.2 Injection Pipette
2.5 Micromanipulation Setup
3 Methods
3.1 Preparation of Donor Cells
3.1.1 Isolation of Primary Fibroblast Cells
3.1.2 Culture of Fibroblast Cells
3.2 Oocyte Collection and Denuding
3.3 Somatic Cell Nuclear Transfer
3.3.1 Oocyte Enucleation
3.3.2 Oocyte Reconstruction
3.4 Cell Fusion
3.5 Oocyte Activation
3.6 Embryo Culture
3.7 Embryo Transfer
3.8 Caesarean Section
4 Notes
References
Chapter 9: Production of Cloned Pigs by Handmade Cloning
1 Introduction
2 Materials
2.1 Equipment
2.2 Tools and Consumables
2.3 Media and Solutions
2.4 HMC Setup
3 Methods
3.1 Donor Cell and Embryo Culture Dish Preparation
3.2 Oocyte Collection and In Vitro Maturation (IVM)
3.3 Oocyte Denudation and Zona Pellucida Digestion
3.4 Somatic Cell Nuclear Transfer
3.4.1 Oocyte Enucleation by Bisection
3.4.2 Cell Fusion
3.5 Oocyte Activation
3.6 Embryo Culture
3.7 Embryo Transfer
3.7.1 Recipient Preparation
3.7.2 Surgical Method for Embryo Transfer
4 Notes
References
Chapter 10: Somatic Cell Nuclear Transfer in Pigs
1 Introduction
2 Materials
2.1 Equipment
2.2 Media and Solutions
3 Methods
3.1 Tissue Collection and Culture of Somatic Cells
3.2 Cell Cycle Synchronization of Donor Cells
3.3 Oocyte Collection and IVM
3.4 Oocyte Denuding
3.5 Somatic Cell Nuclear Transfer
3.5.1 Oocyte Enucleation
3.5.2 Oocyte Reconstruction
3.5.3 Cell Fusion
3.6 Oocyte Activation
3.7 Embryo Culture
3.8 Embryo Transfer
4 Notes
References
Chapter 11: Somatic Cell Nuclear Transfer Using Freeze-Dried Protaminized Donor Nuclei
1 Introduction
2 Materials
2.1 Equipment
2.2 Tools and Consumables
2.3 Media and Solutions
2.4 Micromanipulation Setup
3 Methods
3.1 Adult Sheep Fibroblast Culture
3.2 Cell Transfection with phPrm1
3.3 Lyophilization of Protaminized Somatic Cells
3.4 Oocyte Collection and IVM
3.5 Somatic Cell Nuclear Transfer
3.5.1 Oocyte Enucleation
3.5.2 Oocyte Reconstruction
3.6 Oocyte Activation
3.7 Embryo Culture
4 Notes
References
Chapter 12: Cattle Cloning by Somatic Cell Nuclear Transfer
1 Introduction
2 Materials
2.1 Equipment
2.2 Media and Solutions
2.3 Preparation of Dishes for Oocyte Handling
2.4 Micromanipulator Setup
3 Methods
3.1 Preparation of Somatic Cells
3.2 Fibroblast Cell Cycle Synchronization
3.3 Oocyte Collection and In Vitro Maturation
3.4 Oocyte Denuding and Staining
3.5 Somatic Cell Nuclear Transfer
3.5.1 Oocyte Enucleation
3.6 Oocyte Reconstruction
3.7 Cell Fusion
3.8 Oocyte Chemical Activation
3.9 Embryo Culture
3.10 Embryo Transfer
3.11 Delivery of Cloned Offspring and Neonatal Care
4 Notes
References
Chapter 13: Production of Water Buffalo SCNT Embryos by Handmade Cloning
1 Introduction
2 Materials
2.1 Equipment
2.2 Tools and Consumables
2.3 Media and Solutions
3 Methods
3.1 Preparation of Donor Cells
3.2 Oocyte Collection and In Vitro Maturation
3.3 Oocyte Preparation for Enucleation
3.4 Somatic Cell Nuclear Transfer
3.4.1 Oocyte Enucleation
3.4.2 Oocyte Reconstruction
3.5 Cell Fusion
3.6 Oocyte Activation
3.7 Embryo Culture
3.8 Embryo Evaluation
3.8.1 Blastocyst Morphology
3.8.2 Blastocyst Total Cell Number
4 Notes
References
Chapter 14: Bovid Interspecies Somatic Cell Nuclear Transfer with Ooplasm Transfer
1 Introduction
2 Materials
2.1 Equipment
2.2 Media and Solutions
3 Methods
3.1 Oocyte Collection and IVM
3.2 Oocyte Denuding and Staining
3.3 Somatic Cell Nuclear Transfer with Ooplasm Transfer
3.3.1 Oocyte Enucleation
3.3.2 Oocyte Reconstruction and Ooplasm Transfer
3.4 Electrofusion of Reconstructed Embryos
3.5 Chemical Activation and In Vitro Culture (IVC)
4 Notes
References
Chapter 15: Horse Somatic Cell Nuclear Transfer Using Zona Pellucida-Enclosed and Zona-Free Oocytes
1 Introduction
2 Materials
2.1 Equipment
2.2 Tools and Consumables
2.3 Media and Solutions
2.4 Micromanipulation Setup
2.4.1 Dishes for Handling, Culture, and Micromanipulation
3 Methods
3.1 Preparation of Donor Somatic Cells
3.2 Cell Cycle Synchronization of Donor Cells
3.3 Oocyte Collection
3.4 Oocyte In Vitro Maturation and Denuding
3.5 Zona Pellucida Removal
3.6 Somatic Cell Nuclear Transfer (SCNT)
3.6.1 Enucleation and Reconstruction of ZP-Enclosed Oocytes
3.6.2 Enucleation and Reconstruction of ZP-Free Oocytes
3.7 Cell Fusion
3.8 Oocyte Activation
3.8.1 Chemical Activation for SCNT Using ZP-Free Oocytes
3.8.2 Chemical Activation for SCNT Using ZP-Enclosed Oocytes
3.9 Embryo Culture and Grading
4 Notes
References
Chapter 16: A Modified Handmade Cloning Method for Dromedary Camels
1 Introduction
2 Materials
2.1 Equipment
2.2 Tools and Consumables
2.3 Media and Solutions
3 The mHMC Setup
3.1 Preparation of Enucleation Micropipette
3.2 Preparation of Donor Cells
3.3 Cell Cycle Synchronization of Donor Cells
3.4 Media and Dish Preparation for Handling or Culture of Oocytes
3.5 Preparation of Recipient Oocytes
3.5.1 Preparation of In Vivo Matured Oocytes
3.5.2 Preparation of In Vitro Matured Oocytes
Collection and Storage of Abattoir-Derived Ovaries
Follicular Aspiration and IVM
3.5.3 Oocyte Denuding
3.5.4 Removal of the Zona Pellucida
3.6 Somatic Cell Nuclear Transfer
3.6.1 Oocyte Enucleation Assisted by Demecolcine Treatment
3.6.2 Oocyte Reconstruction
3.7 Cell Fusion
3.8 Oocyte Activation
3.9 Embryo Culture
3.10 Vitrification of Embryos
3.11 Embryo Transfer
4 Notes
References
Chapter 17: Derivation of Bovine Primed Embryonic Stem Cells from Somatic Cell Nuclear Transfer Embryos
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Handling of Blastocysts for ESC Derivation
2.3 Immunofluorescence Staining
3 Methods
3.1 Plating of MEF Feeders
3.2 Bovine ESC Derivation from SCNT Embryos
3.3 Passaging of Bovine bESCs
3.4 Cryopreservation of Bovine ESCs
3.5 Immunofluorescence Staining
4 Notes
References
Index