Mouse Genetics: Methods and Protocols (Methods in Molecular Biology, 2224)

✍ Scribed by Shree Ram Singh (editor), Robert M. Hoffman (editor), Amit Singh (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 222
• Category: Library

✦ Synopsis

This fully updated edition 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. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and up-to-date, Mouse Genetics: Methods and Protocols, Second Edition delivers fundamental techniques and protocols to geneticists, molecular biologists, cell and developmental biologists, students, and postdoctoral fellows working in the various disciplines of genetics, developmental biology, mouse genetics, and modeling human diseases.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Generation of Mouse Model (KI and CKO) via Easi-CRISPR
1 Introduction
2 Materials
2.1 Software for CRISPR Design and Genome Editing
2.2 CRISPR Reagents
2.3 DNA Reagents and Purification kits
2.4 LssDNA Generation
2.5 Zygote Injection and Electroporation
3 Methods
3.1 Optimal CRISPR Site (gRNA) Selection
3.1.1 CRISPR gRNA Design for Novel DNA KI Model (Insert GFP+GOI at GENE-X)
3.1.2 CRISPR gRNA Design for Exon-Floxed CKO Model (GENE-Y)
3.1.3 Different Forms of SpCas9 and gRNA for Genome Editing in Zygotes
3.2 Validation of CRISPR gRNA Cleavage Efficiency
3.2.1 PCR Amplification of Target Region from the Mouse Genome
3.2.2 In vitro Validation of Cleavage: via ICA (In vitro Cleavage Assay)
3.2.3 In vivo Validation of Cleavage: via Sequencing
3.3 Design and Generation of LssDNA Donor Template
3.3.1 Design and Generation of LssDNA for Novel DNA Insertion (GENE-X)
3.3.2 Design and Generation of LssDNA for Exon-Floxed CKO Model (GENE-Y)
3.4 Validation of LssDNA Donor
3.4.1 Check Length Intactness
3.4.2 Check Single-stranded Feature
3.4.3 Check Sequence Fidelity
3.5 Delivery of CRISPR Reagents and LssDNA into Mouse Zygote
3.5.1 Preparation of CRISPR Reagents and LssDNA Mixture
3.5.2 Pronuclear Injection
3.6 Screening and Genotyping of Founder (F0) Mice
3.6.1 PCR Screening Primers and Genotyping: Novel DNA Insertion (GENE-X)
3.6.2 PCR Screening Primers and Genotyping: Exon-Floxed CKO (GENE-Y)
3.6.3 Results and Breeding Scheme
4 Notes
References
Chapter 2: Analysis of Gene Expression Using lacZ Reporter Mouse Lines
1 Introduction
1.1 The lacZ Reporter System
1.2 lacZ Reporter Mice
2 Materials
2.1 Dissection and Fixation of Specimen
2.2 Histochemical Detection of β-Galactosidase Activity
2.2.1 X-Gal Staining of Whole Embryos or Isolated Organs
2.2.2 Clearing of X-Gal Stained Embryos/Organs
2.2.3 X-Gal Staining of Frozen Sections
2.3 Immunofluorescent Detection of β-Galactosidase
2.3.1 Immunostaining of Whole Embryos or Isolated Tissues
2.3.2 Clearing of Immunofluorescently Labeled Whole Embryos/Organs
2.3.3 Immunostaining of Frozen Sections
2.4 Identification of β-Galactosidase Expressing Live Cells Using a Fluorogenic Substrate
3 Methods
3.1 Breeding of Mice
3.2 Dissection and Fixation of Specimen
3.2.1 Immersion-Fixation
3.2.2 Fixation of Whole Animals by Perfusion
3.2.3 Fixation for Immunostaining of Whole-Mount Embryos or Isolated Tissue
3.3 Histochemical Detection of β-Galactosidase Activity
3.3.1 X-Gal Staining of Whole-Mount Mouse Embryos or Isolated Organs
3.3.2 Clearing of X-Gal Stained Whole-Mount Embryos/Organs for Documentation
3.3.3 X-Gal Staining of Frozen Sections
3.4 Immunofluorescent Detection of β-Galactosidase
3.4.1 Immunostaining of Whole-Mount Mouse Embryos or Isolated Tissue
3.4.2 Clearing of Immunofluorescent Stained Whole Embryos/Tissues for Documentation
3.4.3 Immunostaining of Cryosections
3.5 Identification of β-Galactosidase Expression in Living Cells
4 Notes
References
Chapter 3: Linear Density Sucrose Gradients to Study Mitoribosomal Biogenesis in Tissue-Specific Knockout Mice
1 Introduction
2 Materials
2.1 Specialized Equipment
2.2 Isolation of Mitochondria (See Note 2)
2.3 Sucrose Gradients
2.4 TCA Precipitation, SDS-PAGE, and Immunoblot Analysis of the Gradient Fractions
3 Methods
3.1 Isolation of Mitochondria from Mouse Heart
3.2 Ultracentrifugation through a Linear Density Sucrose Gradient
3.3 Gradient Fractionation and TCA Precipitation (See Note 9)
3.4 Analysis of the Fractions by SDS-PAGE and Immunoblotting
4 Notes
References
Chapter 4: Mouse Models for Studying Hippocampal Adult Neural Stem Cell Biology
1 Introduction
1.1 The Most Common Mouse Models Used to Study Hippocampal Neurogenesis
2 Materials
2.1 Transgenic Reporter Mice for Selective Labeling of Neural Stem Cells
2.1.1 Lfng-eGFP Mouse
2.1.2 Lfng-CreERT2 Mouse
3 Methods
3.1 Brain Perfusion and Post-Fixation
3.2 Sectioning
3.3 Immunofluorescence
3.4 Confocal Microscopy and Stereology
3.4.1 Identification of a Cell Type
3.4.2 Cell Counting
4 Notes
References
Chapter 5: Retina as a Model to Study In Vivo Transmission of α-Synuclein in the A53T Mouse Model of Parkinson´s Disease
1 Introduction
2 Methods
2.1 In Vivo Animal Experiments
2.2 Animals
3 Methods
3.1 Preparation and Inoculation of Brain Homogenates
3.2 Clinical Monitoring of Mice
3.3 In Vitro Animal Experiments
3.4 Representative Results
4 Notes
References
Chapter 6: Promoting Pro-Endocrine Differentiation and Graft Maturation Following Surgical Resection of the Mouse Pancreas
1 Introduction
2 Materials
2.1 Cell Transplantation
2.2 Pancreatectomy
2.3 Insulin ELISA
3 Methods
3.1 Cell Transplantation
3.2 Partial Pancreatectomy
3.3 Insulin ELISA
4 Notes
References
Chapter 7: Color-Coded Imaging of Cancer and Stromal-Cell Interaction in the Pancreatic-Cancer Tumor Microenvironment (TME)
1 Introduction
2 Materials
2.1 Animals
2.2 Animal Care and Treatment
2.3 Cells and Culture
2.4 Imaging System
3 Methods
3.1 Animal Care
3.2 Cell Culture
3.3 Establishment of a PDOX Model of Patient Tumors
3.4 Orthotopic Tumor Transplantations in Transgenic Fluorescent Protein-Expressing Nude Mice
3.5 Fluorescence Imaging
3.6 Confocal Microscopy
3.7 Histological Analysis
3.8 Results
3.8.1 GFP Host Stromal cells Infiltrate a Pancreatic Cancer PDOX
3.8.2 GFP Host Stromal Cells Infiltrate Peritoneal Disseminated Metastases of Pancreatic Cancer PDOX
3.8.3 RFP Host Stromal cells Infiltrate Pancreatic Cancer PDOX
3.8.4 GFP Host Stromal Cells Infiltrate Pancreatic PDOX Labeled with RFP Stroma to Form a Two-Color Stroma Model
3.8.5 CFP Host Stromal Cells Infiltrate Pancreatic Cancer PDOX Previously Grown in RFP and GFP Transgenic Mice to Form a Three...
3.8.6 Noninvasive Imaging of Pancreatic Cancer PDOX with Labeled Stromal Cells
3.8.7 Color-Coded Imaging of Stromal-Cell and Cancer-Cell Response to Therapy
3.8.8 Fusion of Cancer and Stromal Cells
4 Notes
References
Chapter 8: Generating Ins2+/-/miR-133aTg Mice to Model miRNA-Driven Cardioprotection of Human Diabetic Heart
1 Introduction
2 Materials
3 Methods
3.1 Crossbreeding Ins2+/- and miR-133aTg Mice
3.2 Genotyping
3.2.1 DNA Extraction
3.2.2 Polymerase Chain Reaction (PCR)
3.2.3 Restriction Digest
3.2.4 Agarose Gel Electrophoresis
3.2.5 Analysis of Gel-Band
3.3 Blood Glucose Measurement
3.4 Real-Time PCR Measurement of miR-133a
4 Notes
References
Chapter 9: Generating a Podocyte-Specific Neonatal F Receptor (FcRn) Knockout Mouse
1 Introduction
2 Materials
2.1 Mice
2.2 Genotyping
2.3 Phenotype Characterization
2.4 Imaging
3 Methods
3.1 Creation of Podocyte-Specific FcRn Knockout Mice
3.2 Phenotypic Analysis of Control and Podocyte-Specific FcRn Knockout Mice
3.3 Immunolocalization of Intraglomerular Albumin and IgG in Control and Podocyte-Specific FcRn KO Mice
3.4 Imaging
4 Notes
References
Chapter 10: Mouse Models of Colitis-Associated Colon Cancer
1 Introduction
2 Materials
2.1 AOM-DSS-Induced Inflammation-Associated Colon Cancer
3 Methods
3.1 AOM-DSS-Induced Colon Cancer
3.1.1 Treating Mice with AOM
3.1.2 Treating Mice with DSS
3.2 Sacrificing Animals and Tissue Harvesting
3.3 Preparing the Colon for Histological Assessment
3.4 Assessment of Colitis Induction in Treated Mice
3.5 Assessment of Inflammation
3.6 Assessment of CAC Following AOM/DSS Treatment
3.7 Representative Results and Discussion
4 Notes
References
Chapter 11: Generation of Colon Cancer Model Based on Colonoscopy Injection
1 Introduction
2 Materials
2.1 Cell Line
2.2 Animals
2.3 Equipment
2.3.1 Cell Culture and Cell Preparation for Injection
2.3.2 Colonoscopy-Guided Mucosal Injection
3 Methods
3.1 Cell Preparation Procedure
3.2 Colonoscopy Preparation and Staff Member
3.3 Animal Preparation Procedure
3.4 Implantation Procedure
3.5 Tumor Follow-Up with Colonoscopy
4 Additional Tips
References
Chapter 12: Generation of Transgenic Fluorescent Reporter Lines for Studying Hematopoietic Development in the Mouse
1 Introduction
1.1 Ontogeny of the Mouse Hematopoietic System
1.2 General Considerations in Designing a Transgene
1.2.1 Regulatory Elements
1.2.2 Insertion of Exogenous DNA into the Genome
1.2.3 Alternative Approaches to Drive Hematopoietic Lineage-Specific Expression of Fluorescent Protein Reporters
1.3 Fluorescent Protein Reporters
1.3.1 Fluorescent Fusion Proteins
1.3.2 Photomodulatable FPs
1.3.3 General Considerations for Choosing Fluorescent Protein Reporters
1.4 Confirmation and Analysis of Transgene Expression
1.4.1 Mouse Background
1.4.2 Breeding
1.5 Analysis of Fluorescent Protein Expression Using Microscopy
1.6 Analysis of Fluorescent Protein Expression Using Flow Cytometry
1.7 Analysis of Hematopoietic Progenitor Potential
2 Materials
2.1 DNA Purification for Microinjection
2.2 Isolation of Genomic DNA
2.3 Polymerase Chain Reaction (PCR)
2.4 Dissecting Tools
2.5 Glassware and Plasticware
2.6 Embryo dissection and Cell Preparation for Flow Cytometry
2.7 Flow Cytometry
2.8 Immunostaining and Microscopy
2.9 Primitive Erythroid (EryP) Progenitor Assay
2.10 Definitive Erythroid and Myeloid Progenitor Assay
2.11 B Lymphocyte Colony Assay
2.12 Megakaryocyte Colony Assay
3 Methods
3.1 DNA Preparation for Microinjection
3.2 Genotyping
3.2.1 Preparation of Tissue Samples for Genotyping
3.2.2 Genomic PCR
3.3 Dissection
3.3.1 Embryo Dissection
3.3.2 Isolation of Peripheral Blood from Embryos
3.3.3 Dissection of Yolk Sac and Placenta
3.3.4 Dissection of Fetal Liver
3.3.5 Isolation of the AGM Region
3.3.6 Isolation of Adult Bone Marrow
3.4 General Immunofluorescence Protocol
3.5 Flow Cytometry
3.5.1 Labeling of Cells for Flow Cytometry
3.5.2 Preparation of Cells for Sorting by FACS
3.6 Hematopoietic Progenitor Assays
3.6.1 Primitive Erythroid (EryP) progenitors
3.6.2 Erythroid and Myeloid Progenitors
3.6.3 Lymphocyte (B-Cell) Progenitors
3.6.4 Megakaryocyte Progenitors
4 Notes
References
Chapter 13: Gene Inactivation in Adult Long-Term Hematopoietic Stem Cells Using Inducible Mouse Models
1 Introduction
2 Materials
2.1 Mouse Strains
2.2 Isolation of Genomic DNA from Ear Punch or Tail Clippings
2.3 Primers for Genotyping Cre-ERT and Cre-ERT2 Mice
2.4 Preparation of Tamoxifen or 4-Hydroxytamoxifen for Injection
2.5 Preparation of Bone Marrow for Transplantation into Lethally Irradiated Pepcb/BoyJ (CD45.1) Recipient Mice
3 Methods
3.1 Generation of Conditionally Targeted Cre-ERT or Cre-ERT2 Mice
3.2 Identification of Conditionally Targeted Cre-ERT or Cre-ERT2 Mice by Genotyping
3.3 Preparation and Intraperitoneal Injection of Tamoxifen or 4-OHT
3.4 Bone Marrow Transplantation into Lethally Irradiated Pepcb/BoyJ (CD45.1) Recipient Mice
4 Notes
References
Chapter 14: Hematological Humanization of Immune-Deficient Mice
1 Introduction
2 Materials
2.1 Mobilized Peripheral Blood (MPB)
2.2 Reagents for Human CD34+ Isolation
2.3 NSG Mice (See Notes 2 and 3)
2.4 Irradiator
2.5 Flow Cytometry for Chimerism
3 Methods
3.1 Isolating Human CD34+ Cells (See Note 5)
3.2 Transplanting Human CD34+ Cells in NSG Mice
3.3 Testing Chimerism
4 Notes
References
Chapter 15: Antisense Oligonucleotide Treatment in a Humanized Mouse Model of Duchenne Muscular Dystrophy and Highly Sensitive...
1 Introduction
2 Materials
2.1 Retroorbital Injection of PMOs into Humanized DMD Mice
2.2 RNA Extraction
2.3 RT-PCR to Evaluate Exon Skipping
2.4 Dystrophin Western Blotting Analysis of Mouse Muscles
3 Methods
3.1 Retroorbital Injection of PMOs into Humanized DMD Mice
3.2 RNA Extraction
3.3 RT-PCR to Evaluate Exon Skipping
3.4 Western Blotting Analysis of Dystrophin
4 Notes
References
Index