Transgenesis: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Transgenesis and Genome Engineering: A Historical Review
1 Origin and Meaning of Fundamental Concepts in Transgenesis and Genome Engineering
1.1 Transgene, Transgenic, and Transgenesis
1.2 Knockout, Knock-in, and Knockdown
1.3 Genetically Modified Organisms and Related Terms
1.4 Genome Engineering
1.5 Genome Editing or Gene Editing
2 Timeline of Transgenesis and Genome Engineering
2.1 1974
2.2 1975
2.3 1976
2.4 1980
2.5 1981
2.6 1982
2.7 1983
2.8 1984
2.9 1985
2.10 1986
2.11 1987
2.12 1988
2.13 1989
2.14 1990
2.15 1991
2.16 1992
2.17 1993
2.18 1994
2.19 1995
2.20 1996
2.21 1997
2.22 1998
2.23 1999
2.24 2000
2.25 2001
2.26 2002
2.27 2005
2.28 2006
2.29 2007
2.30 2008
2.31 2009
2.32 2011
2.33 2012
2.34 2013
2.35 2014
2.36 2015
2.37 2016
2.38 2017
2.39 2018
2.40 2019
2.41 2020
2.42 2021
3 Concluding Remarks
References
Chapter 2: Practical Application of the 3Rs in Rodent Transgenesis
1 Introduction
2 Materials
3 Methods
3.1 General
3.2 Choosing the Best Experimental Method
3.2.1 Non-sentient or Nonanimal Model
3.2.2 Existing Models
3.2.3 Type of Mutation
3.2.4 Harm-Benefit Analysis (HBA)/Weighing of Interests
3.3 Transgenic Techniques
3.3.1 Classical Transgenesis
3.3.2 ES Cell Mutagenesis
3.3.3 Endonucleases
3.4 Donor Females
3.5 Surgical Procedures
3.6 Embryo Transfer
3.6.1 Sterile Males
3.6.2 Surrogate Dams and Embryo Implantation
3.7 Identification of Harmful Phenotypes
3.8 Beyond Rodents
4 Notes
References
Chapter 3: Genetic and Molecular Quality Control of Genetically Engineered Mice
1 Introduction
2 Allele Design Considerations
2.1 Deletion Alleles
2.2 Insertion Alleles
2.3 Base Change Alleles
2.4 Transgenic Alleles
3 Methods
3.1 Generation of Chimeras and Founders
3.2 GLT Test Breeding
3.3 Genomic DNA Isolation for Screening and QC
3.4 Assay Design
3.5 Amplicon Sequencing
3.6 Endonuclease-Mediated Deletion Alleles
3.7 Endonuclease-Mediated Insertion Alleles
3.8 Endonuclease-Mediated Conditional Alleles
3.9 Endonuclease-Mediated Sequence Change Alleles
3.10 ES Cell-Derived Alleles
3.11 Transgenes
3.12 Colony Maintenance and Cohort Production
3.13 Molecular QC
3.14 Conclusion
4 Notes
References
Chapter 4: Genotyping Genome-Edited Founders and Subsequent Generation
1 Introduction
2 Materials
2.1 DNA Extraction
2.2 PCR and Sequencing
2.3 Droplet Digital PCR (ddPCR)
2.4 Equipment
2.5 Software and Online Tools
3 Methods
3.1 DNA Extraction
3.2 PCR and Sequencing
3.2.1 Primer Design
3.2.2 Primer Stocks
3.2.3 PCR
3.2.4 Electrophoresis
3.2.5 PCR Optimization
Template DNA
Assessing efficiency and specificity
Further Optimization
Purification of PCR Products for Sequencing
3.3 Sequence Analysis
3.3.1 General Method of Sequence Analysis
3.3.2 Non-specified Indels
3.4 Non-templated Deletions
3.5 Knock-ins, Floxed, and Specific Deletions
3.6 Droplet Digital PCR Copy-Counting
3.6.1 Primer/Probe Design
3.6.2 DNA controls
3.6.3 Reaction Mix and Droplet Generation
3.6.4 Thermocycling and Droplet Reading
3.6.5 ddPCR Analysis
3.7 Genotyping for Indel and Point Mutations
3.7.1 G0 Screening
3.7.2 G1 Screening and Validation
3.8 Genotyping for Deletion Alleles
3.8.1 G0 Screening for Deletion (DEL) Alleles
3.8.2 G1 screening and Validation of Deletion (DEL) Alleles
3.9 Genotyping for Large KIs
3.9.1 G0 Screening for Large KIs
3.9.2 G1 Screening and Validation for Large KIs
3.10 Genotyping for Floxed Alleles
3.10.1 G0 Screening for a Floxed Allele
3.10.2 G1 Screening and Validation for a Floxed Allele
3.11 Off-Target Analysis: All Allele Types
4 Notes
References
Chapter 5: ASIS-Seq: Transgene Insertion Site Mapping by Nanopore Adaptive Sampling
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents Flow Cell R9.4.1 (ONT FLO-MIN106D)
2.3 Buffers and Other Solutions
2.4 Software and Internet Resources
3 Methods
3.1 Generating a Mouse Model by Random Transgenesis
3.2 Genomic DNA Isolation from Mouse Tail Biopsies
3.3 Shearing Genomic DNA
3.4 Visualize Sheared Genomic DNA by Agarose Gel Electrophoresis
3.5 Sequencing Library Preparation
3.6 MinKNOW Control Software Settings for Adaptive Sampling
3.7 Flow Cell Wash and Reloading Libraries
3.8 Data Analysis
3.9 Confirmation of Transgene Insertion Sites by Cas9-Targeted Sequencing
4 Notes
References
Chapter 6: High-Throughput Analysis of CRISPR-Cas9 Editing Outcomes in Cell and Animal Models Using CRIS.py
1 Introduction
2 Materials
2.1 Wet Lab
2.2 Programming Resources
3 Methods
3.1 Overview of NGS Library Setup for Amplicon Sequencing
3.2 Gene-Specific Primer Design
3.3 Primer Design for KO and Small Modification Projects
3.4 Primer Design for Large Deletion Projects
3.5 Primer Design for Large KI Projects
3.5.1 Primer Design for Detecting KI Modifications Using ssDNA Donor Templates
3.5.2 Primer Design for KI Modifications Using Plasmid Donors
3.5.3 PCR #1 to Amplify the Target Region
3.5.4 PCR #2 to Add Unique Indexes to Each Sample
3.6 Using CRIS.py to Analyze NGS Data
3.6.1 Installing Python and CRIS.py
3.6.2 Setting Up a Folder with the CRIS.py Script and NGS Data
3.6.3 CRIS.py Program Layout
3.6.4 Variables of the CRIS.py Program
3.6.5 How Does CRIS.py Work?
3.7 Setup and Analysis of CRIS.py-Generated Data for Various Editing Scenarios
3.7.1 KO Projects
3.7.2 Analyzing and Interpreting Data for a KO Project
3.7.3 Interpreting the Output .txt File
3.8 Small Modification Projects
3.9 Deletion Projects
3.10 Large KI Projects
3.11 Interpreting the CRIS.py .csv Output File for Clonal Data
3.12 Summary
4 Notes
References
Chapter 7: Advanced Technologies and Automation in mES Cell Workflow
1 Introduction
2 Materials
2.1 MultiMACS Materials
2.2 Biomek i5 Materials
2.3 Materials for Two-Channel Droplet Digital PCR
2.4 Materials for Five-Channel Digital PCR
3 Methods
3.1 Plate Preparation on Day of Sorting
3.2 Prepare MultiMACS Block While Cells Are Mixed with Microbeads
3.3 Biomek i5 Method for DNA Preparation in 96-Well Plates of mES Cells
3.4 Biomek i5 Method for DNA Preparation in 24-Well Plates of mES Cells
3.5 Droplet Digital PCR Method for a Two-Channel ddPCR Instrument
3.6 Droplet Digital PCR Method for a Five-Channel ddPCR Instrument
4 Notes
References
Chapter 8: Gene Editing in Mouse Zygotes Using the CRISPR/Cas9 System
1 Introduction
1.1 Gene Editing in Mouse Zygotes
1.2 Timing of CRISPR/Cas9 Gene Editing in Zygotes
1.3 Design of Gene Editing Experiments
1.3.1 Targeting Strategies
1.3.2 Design of Guide RNAs
1.3.3 Preparation of Donor Template Molecules
1.3.4 CRISPR/Cas9 Reagents
1.3.5 Genotyping Strategies
2 Materials
2.1 Preparation of Fertilized Embryos
2.2 Targeting Constructs
2.3 Pronuclear Injection of Fertilized Embryos
2.4 Electroporation of Fertilized Embryos
2.5 In Vitro Blastocyst Analysis
2.6 Embryo Transfer
2.7 Genotyping
3 Methods
3.1 Zygote Preparation
3.2 Zygote Treatment
3.3 Option A: Microinjection
3.4 Option B: Electroporation
3.4.1 For Electroporation in Cuvettes
3.4.2 For Electroporation in Slide Electrodes
3.4.3 Option C: Electroporation Combined with AAV Incubation
3.5 Optional: In Vitro Blastocyst Analysis
3.6 Embryo Transfer of Manipulated Zygotes
3.7 Genotyping
3.7.1 Isolation of Genomic DNA from Tissue Biopsies
3.7.2 PCR Amplification of Targeted Region
3.7.3 Analysis by Restriction Enzyme Digest or Sanger Sequencing
4 Notes
References
Chapter 9: Floxing by Electroporating Single-Cell Embryos with Two CRISPR RNPs and Two ssODNs
1 Introduction
2 Materials
2.1 Validation of gRNAs and ssODNs in Cultured Cells
2.2 In Vitro Validation of RNPs
2.3 Embryo Electroporation
2.3.1 Sample Prep for Electroporation
2.3.2 Embryo Electroporation With or Without Acid Treatment of Zona
2.4 Sperm Cryopreservation
2.5 In Vitro Fertilization (IVF) with Frozen Sperm
2.6 IVF with Fresh Sperm
2.7 NGS-Based Genotyping
2.8 In Vitro Cre Assay
3 Methods
3.1 Criteria for Designing and Choosing Insertion Sites
3.2 gRNA Design
3.3 ssODN Design
3.4 Choice of Founders for Retargeting
3.5 Validation in Cell Culture
3.6 Validation In Vitro
3.7 Sample Prep for Embryo Electroporation
3.8 Embryo Electroporation with Acid Treatment of Zona
3.9 Embryo Electroporation Without Acid Treatment of Zona
3.10 Mouse Sperm Cryopreservation: CPA Preparation
3.11 Mouse Sperm Cryopreservation: Sperm Freezing
3.12 In Vitro Fertilization with Frozen Sperm
3.13 In Vitro Fertilization with Fresh Sperm
3.14 NGS-Based Genotyping
3.14.1 NGS-Based Genotyping of Founder
3.14.2 Genotyping Blastocysts
3.15 In Vitro Cre Assay for Phase Determination
4 Notes
References
Chapter 10: CRISPR/Cas9 Endonuclease-Mediated Mouse Genome Editing of One-Cell and/or Two-Cell Embryos by Electroporation, and...
1 Introduction
2 Materials
2.1 Embryo Preparation and Culture
2.2 RNP Components (sgRNA and Cas9)
2.3 Donor DNA (ssODN and lssDNA) and Rad51
2.4 Electroporation
3 Methods
3.1 Embryo Collection and Culture
3.2 Electroporation (at One-Cell or Two-Cell Embryonic Developmental Stage)
3.3 GEMM Individual Project Plan Details
3.4 Conclusion
4 Notes
References
Chapter 11: Electroporation-Mediated CRISPR/Cas9 Genome Editing in Rat Zygotes
1 Introduction
2 Materials
2.1 Superovulation and Zygote Production
2.2 Zygote Collection
2.3 sgRNA/Cas9 RNP Formation
2.4 Zygote Electroporation
2.5 Surgical Embryo Transfer
3 Methods
3.1 Zygote Superovulation
3.2 Zygote Collection
3.3 Zygote Electroporation of CRISPR Reagents
3.4 Surgical Embryo Transfer
4 Notes
References
Chapter 12: CRISMERE Chromosome Engineering in Mouse and Rat
1 Introduction
2 Materials
2.1 RNA Synthesis
2.2 RNA
2.3 Cas9 Protein
2.4 Agarose Gels
2.5 DNA Ladder
2.6 Sample Lysis and PCR
2.7 Droplet Digital PCR
3 Methods
3.1 Strategies for Generating the SV
3.2 CRISPR/Cas9 RNA Component
3.3 Preparation of the Mix for Microinjection or Electroporation
3.4 Preparation of Mouse One-Cell Embryos
3.5 Preparation of Rat One-Cell Embryos
3.6 Delivery of CRISPR/Cas9 in One-Cell Embryos
3.6.1 Microinjection
3.6.2 Electroporation
3.7 Characterization of Founders
3.7.1 DNA Extraction
3.7.2 Junction PCRs for the Identification of the New Junctions
3.7.3 Junction PCR Products Are Sent for Sanger Sequencing
3.7.4 Droplet Digital PCR for Copy Number Counting
3.8 Interpretations of the F0 Results
3.9 Establishment of a Validated F1 Line
3.10 Establishment of the SV Line
3.11 Conclusions and Final Recommendations
4 Notes
References
Chapter 13: Targeted Integration of Transgenes at the Mouse Gt(ROSA)26Sor Locus
1 Introduction
2 Materials
2.1 Cloning of the Transgene of Interest
2.2 Recombinase-Mediated Cassette Exchange in ES Cells
3 Methods
3.1 Cloning of the Transgene of Interest
3.2 Recombinase-Mediated Cassette Exchange in ES Cells
3.2.1 ES Cell Electroporation
3.2.2 Preparation of Genomic DNA and PCR Screening for Correct RMCE Integration
3.2.3 Thawing of Correctly Recombined Clones and Further Validation by Southern Blot
3.2.4 Downstream Steps
4 Notes
References
Chapter 14: Improved Genome Editing via Oviductal Nucleic Acids Delivery (i-GONAD): Protocol Steps and Additional Notes
1 Introduction
2 Materials
2.1 Mice
2.2 Equipment
2.3 Reagents
3 Methods
3.1 Preparing Pregnant Female Mice
3.2 Preparation of Genome Editing Solution
3.3 Surgical Procedure to Expose the Oviduct
3.4 Injection of Genome Editing Solution into the Oviduct
3.5 In Vivo EP
3.6 I-GONAD Procedure on the Right Oviduct
3.7 Postoperative Procedures
3.8 Genotyping of Founder (F0) Individuals
4 Notes
References
Chapter 15: Gene Targeting in Rat Embryonic Stem Cells
1 Introduction
2 Materials
2.1 Primary Mouse Embryonic Fibroblast (PMEF) Medium
2.2 Rat ES Cell Culture Medium Components
2.3 Rat ES Cell Targeting and Cryobanking
3 Methods
3.1 Prepare Rat ES Cell Medium Stock Solutions
3.2 Rat ES Cell Medium with Two Inhibitors (N2B27 + 2i)
3.3 Prepare PMEF Feeder Cell Media
3.4 ES Cell Targeting and Cryobanking Solutions
3.5 PMEF Feeder Cell Culture
3.6 Rat ES Cell Culture
3.7 ES Cell Passage
3.8 ES Cell Freezing
3.9 G418 Killing Curve
3.10 Nucleofection
3.11 ES Cell Selection
3.12 Colony Pick-Up
3.13 Preparation for Freezing and Genotyping
4 Notes
References
Chapter 16: Rat Embryonic Stem Cell Transgenesis
1 Introduction
2 Materials
2.1 Embryonic Stem Cell (ESC) Culture
2.2 Blastocyst Collection
2.3 Microinjection of ESCs into Blastocysts
2.4 Embryo Transfer
2.5 Surgical Embryo Transfer
2.6 Non-surgical Embryo Transfer
3 Methods
3.1 Embryonic Stem Cell (ESC) Culture
3.2 Blastocyst Collection
3.3 Microinjection of ESCs into Blastocysts
3.4 Embryo Transfer
3.5 Surgical Embryo Transfer
3.6 Non-surgical Embryo Transfer
4 Notes
References
Chapter 17: CRISPR/Cas9-Mediated Genome Editing in Zebrafish
1 Introduction
2 Materials
2.1 Equipment
2.2 Kits
2.3 Buffers and Chemicals
3 Methods
3.1 CRISPR/Cas9 sgRNA Design for Genomic Edits
3.2 sgRNA Production by In Vitro Transcription (IVT)
3.3 Generation of Knockout Lines
3.4 Crispant Genotyping
4 Notes
References
Chapter 18: Generation of Rabbit Chimeras by Eight-Cell Stage Embryo Injection
1 Introduction
2 Materials
2.1 Equipment
2.2 Consumables
2.3 Media and Solutions
2.4 Animals
3 Methods
3.1 Superovulation
3.2 Zygote Collection
3.3 PSCs Injection
3.4 Embryo Transfer
3.5 Evaluation of the Chimera
4 Notes
References
Chapter 19: Genome Editing in Pigs
1 Introduction
2 Materials
2.1 Generation of Genetically Engineered Pigs Via SCNT
2.2 Generation of Genetically Engineered Pigs Via Microinjection
2.2.1 Collection of Ovaries and In Vitro Maturation of Oocytes
2.2.2 In Vitro Fertilization
2.2.3 Microinjection
2.2.4 Embryo Transfer
3 Methods
3.1 Generation of Genetically Engineered Pigs Via SCNT
3.1.1 Isolation of Primary Porcine Kidney Fibroblasts (pKF)
3.1.2 Isolation of Adipose-Derived Mesenchymal Stem Cells (pADMSC)
3.1.3 Transfection of Primary Cells Via Lipofection
3.1.4 Determination of Adequate Antibiotic Concentrations for Selection
3.1.5 Selection, Isolation, and Screening of CRISPR/Cas9-Mediated knockout Cells
3.1.6 Selection, Isolation, and Screening of CRISPR/Cas9-Assisted Knock-in Clones
3.1.7 SCNT
3.2 Generation of Genetically Engineered Pigs Via Microinjection
3.2.1 Collection of Ovaries and In Vitro Maturation of Oocytes
3.2.2 In Vitro Fertilization
3.2.3 Microinjection
3.2.4 Preparations for Embryo Transfer
3.2.5 Embryo Transfer
4 Notes
References
Chapter 20: Generation of Genome-Edited Chicken Through Targeting of Primordial Germ Cells
1 Introduction
2 Materials
2.1 Avian Knockout DMEM (KO-DMEM): A No-Calcium, Low Osmolarity DMEM for Culturing Embryonic Cells
2.2 PGC Basal Medium
2.3 5000x Vitamin B12 (Sigma-Aldrich: V6629) Prepared as Follows (See Note 2)
2.4 PGC Growth Factors
2.5 Complete PGC Culture Medium
2.6 PGC Derivation, Culture, and Cryopreservation
2.7 PGC Transfection, Selection, and Clonal Expansion
2.8 PGC Injection into Surrogate Embryos
2.9 Equipment and General Reagents
3 Methods
3.1 Collection of Blood from Embryos (See Note 8)
3.2 PCR-Aided Determination of the Sex of Chicken Embryos
3.3 In Vitro Propagation of PGCs
3.4 Cryopreservation of PGCs
3.5 Thawing of PGCs
3.6 Transfection of PGCs
3.7 Selection for CRISPR-Transfected Cells (See Note 12)
3.8 Genome Analysis of Transfected PGCs
3.9 Single-Cell Clonal Culture
3.10 Injection of PGCs into Surrogate Host Embryos
4 Notes
References
Index