Methods in Molecular Biology
1 Modeling Cancer Using Genetically Engineered Mice
1.1.1 Knockout and Knockin Models
1.1.2 Conditional Mutagenesis
1.1.3 Reversible Inducible Systems
2 Lung Adenocarcinomas: Comparison Between Mice and Men
1.1 The Human Lung Carcinoma Spectrum
2.2 Immunohistochemistry (IHC)
3.1 Histopathology of Adenocarcinomas
3.2 Immunohistochemistry of Adenocarcinomas
3.3 Progression of Adenocarcinomas
3.4 Specific Changes Induced by Genetic Modifications
3.4.1 Signet Ring Cell Formation
3.4.2 Oxyphilic/Oncocytic Changes
3.5 Do Mouse Adenocarcinomas Resemble Human Adenocarcinomas?
3.6 Differences in Mouse and Human Lung Morphology as Explanation for Different Adenocarcinoma Appearance
3.7 Genetic Differences Between Mouse and Human Adenocarcinomas
3.8 Cellular Origin of Adenocarcinomas
Part II: Individual Cancers
3 Mouse Models of Breast Cancer
2 Genetically Engineered Mouse Models (GEMs) for Human Breast Cancer
2.1 Transgenic Mice with a Mammary-Specific Expression of Oncogenes
2.2 LigandưControlled Oncogene Expression in the Mammary Gland
2.3 Conventional Knockout Models and Mammary Gland Transplantation
2.4 MammaryưSpecific Conditional Knockout Mouse Models
3 Morphological and Molecular Characteristics of GEM-Derived Mammary Tumors That Define Human Breast Cancer Subtypes
4 Modeling Breast Cancer Metastasis
4 Genetically Engineered Mouse Models to Study Prostate Cancer
1.1 Dissection and Sample Collection
1.2 Isolating Blood/Serum
1.3 Formalin Fixation and Processing of Tissue
1.4 Hematoxylin and Eosin Staining
1.5 Ki67 Immuno-histochemistry
2.2 Processing, Embedding, and Microtomy
2.3 Hematoxylin and Eosin Staining
3.1 Dissection and Sample Collection
3.2 Isolating Blood/Serum
3.3 Formalin Fixation and Processing of Tissue
3.4 Hematoxylin and Eosin Staining
3.5 Ki67 Immunohistochemistry
5 Practical Use of Advanced Mouse Models for Lung Cancer
2.1 Orthotopic Transplantation of Lung Tumor Tissue or Cell Lines
2.2 Orthotopic Mouse Model by Injection into Parenchyma
2.3 Cryopreservation of Lung Tumor Tissue Fragments
2.4 Thawing of Cryopreserved Tumor Tissue and Cell Lines
2.5 Conditional Transgenic Model; Cre-Lox or FLP-Frt System
2.6 Doxycycline Application for Tet-Inducible Mice Models
2.7 Administration of Tamoxifen for HormoneưInducible Transgenic Mice
3.1 Orthotopic Transplantation of Lung Tumor Cells or Cell Lines
3.1.1 Intratracheal Tumor Cell Injection
3.1.2 Injection into Lung Parenchyma
3.2 Cryopreservation of Lung Tumor Fragments
3.3 Thawing of Cryopreserved Tumor Pieces
3.4 Conditional Transgenic Model; Cre-Lox or FLP-Frt System
3.4.1 Intratracheal Infection
3.4.2 Intranasal Infection
3.5 Doxycycline Application for Tet-Inducible Mice Models
3.6 Administration of Tamoxifen for HormoneưInducible Transgenic Mice
3.6.1 Intraperitoneal Injection of Tamoxifen
3.6.2 Tamoxifen Administration via Drinking Water
6 Generation and Analysis of Mouse Intestinal Tumors and Organoids Harboring APC and K-Ras Mutations
2.1 Breeding, Genotyping, and Induction of Intestinal Tumors in the Lgr5GFP-ires-CreErt2_APC-LoxP/LoxP_ K-RasLoxPstopLoxP-G12D Compound Mice
2.1.3 Primers Sequences Used to Identify Mice via PCR Analysis
2.1.4 PCR Reaction Mixture Used for Genotyping
2.1.5 Analysis of Amplified PCR Products
2.1.6 Tumor Induction Upon Cre Activation via Tamoxifen Administration
2.1.7 Resection of the Mouse Intestine
2.2 Histological Analysis of the Intestinal Tumors Induced in the Lgr5GFP-iresưCreErt2_APC-LoxP/LoxP_ K-rasLoxPstopLoxP-ưG12D Compound Mice
2.2.1 Tissue Block Preparation
2.2.2 Tissue Section Preparation
2.2.3 Immunohistochemical Staining
2.3 Generation, Culturing, and Histological Analysis of the Intestinal Tumor Organoids Derived from the Lgr5GFPưires-CreErt2_APC-LoxP/LoxP_ K-rasLoxPstopLoxP-G12D Compound Mice
2.3.1 Small Intestinal Crypt Isolation
2.3.2 Small Intestinal Tumor/Crypts Culture
2.3.3 Passaging of Intestinal Organoids
2.3.4 Processing and Histological Analysis of Intestinal Organoids
3.1 Breeding, Genotyping, and Induction of Intestinal Tumors in the Lgr5GFP-iresưCreErt2_APC-LoxP/LoxP_ K-RasLoxPstopLoxP-ưG12D Compound Mice
3.1.1 Genomic DNA Extraction from Mouse Tails for Genotyping
3.1.2 PCR Reaction Mixture for Genotyping
3.1.3 PCR Cycle Conditions for Genotyping
3.1.4 Analysis PCR Products
3.1.5 Tumor Induction upon Cre Activation via Intra Peritoneal Tamoxifen Administration
3.1.6 Resection of the Intestine
3.2 Histological Analysis of the Intestinal Tumors Induced in the Lgr5GFP-iresưCreErt2_APC-LoxP/LoxP_ K-rasLoxPstopLoxP-ưG12D Compound Mice
3.2.1 Fixation of the Intestine for Tissue Sectioning
3.2.2 Preparation of Paraffin-Embedded Tissue Sections
3.2.3 Immunohistochemical Staining
3.3 Generation, Culturing, and Histological Analysis of the Intestinal Tumor Organoids Derived from the Lgr 5GFP-ưires-CreErt2_APC-LoxP/LoxP_ K-rasLoxPstopLoxP-G12D Compound Mice
3.3.1 Small Intestinal Crypt Isolation
3.3.2 Culturing Intestinal Organoids
3.3.3 Passaging of Intestinal Organoids
3.3.4 Processing and Histological Analysis of Organoids
7 Induction of Colorectal Cancer in Mice and Histomorphometric Evaluation of Tumors
2.1 Induction of Colorectal Tumors with AOM/DSS
2.2 Preparation of Swiss Roles
2.3 Hematoxylin and Eosin Staining
2.4 Quantification of CRC Tumor Area on H&EưStained Swiss Roles
2.5 PDGFR/FSP1 Immunohistochemistry of Stromal Cells
3.1 Induction of Colorectal Tumors with AOM/DSS
3.2 Preparation of Swiss Roles
3.3 Hematoxylin and Eosin Staining
3.4 Quantification of CRC Tumor Area on H&EưStained Swiss Roles
3.5 Characterization of the CRC Tumor Stroma by PDGFR/FSP1 Immunohistochemistry and Histomorphometry
3.5.1 PDGFR/Fsp1 Immunohistochemistry of Stromal Cells
3.6 Histomorphometric Evaluation of the Stromal Composition Using Definiens Tissue Studio Software
8 Mouse Models of Liver Cancer
1.1 Mechanisms of Hepatocarcinogenesis
1.1.1 Genetic Alterations
1.1.2 The Hepatic Microenvironment
1.1.3 Cellular Origin of HCC
1.2 Determinants for Choosing HCC Models
1.2.1 Best Representation of Genomic Alterations and Gene Expression Patterns
1.2.2 Presence of Chronic Inflammation, Injury, and Fibrosis
1.2.3 Mechanistic Versus Preclinical Studies
1.2.4 Limitations of Mouse Models
1.3 Key Features and Considerations of Select Models
1.3.4 TAK1 Liver Knockout Mice
1.3.5 PTEN Liver Knockout Mice
3.4 TAK1 Liver Knockout Mice
3.5 PTEN Liver Specific Deletion
3.6.1 Orthotopic Syngeneic Transplant Models
3.6.2 Orthotopic Xenotransplant Models
9 Current Methods in Mouse Models of Pancreatic Cancer
1.1 Orthotopic and Heterotopic Xenograft Implantation of PatientưDerived and Cultured PDAC
1.2 Genetically Engineered Mouse Models (GEMM) of Spontaneous, Endogenous PDAC
1.3 CaeruleinưInduced Acute Pancreatitis for Accelerated Tumor Development in GEMM
1.4 Isolation of Cancer and Stromal Cells from GEMMưBased PDAC
2.1 Orthotopic and Heterotopic Xenograft Implantation
2.2 Genetically Engineered Mice
2.3 Caerulein-Induced Acute Pancreatitis
2.4 Isolation of Single Cells from GEMMưBased PDAC
3.1 Orthotopic and Heterotopic Xenograft Implantation
3.2 Genetically Engineered Mice
3.3 Caerulein-Induced Acute Pancreatitis
3.4 Isolation of Single Cells from GEMMưBased PDAC
10 Mouse Models of Nonmelanoma Skin Cancer
1.1 Squamous Cell Carcinoma
Tumor Development/Morphology
1.1.2 Other GEMM Modeling SCC
1.1.3 Chemically/Inflammation-Induced SCC (DMBA/TPA Treatment)
1.2.1 Mouse Models of BCC
Tumor Development and Morphology
Tumor Development/Morphology
1.2.4 Other GEMM Modeling BCC
1.4 Keratoacanthoma/ Actinic Keratosis (Precancerous Lesions)
2.1 Isolation of Genomic DNA
Genotyping K5-hSOS-F Mice
2.2.3 Keratinocyte Culture
2.2.4 DMBA/TPA Preparation
2.3.2 Genotyping Ptch1 Mice
2.3.3 Genotyping SmoM2 Mice
2.3.4 Preparation of Tamoxifen
2.5.1 Hematoxylin and Eosin Staining
2.5.2 Immunohistochemistry Stainings on Paraffin Sections
3.1 Isolation of Mouse Genomic DNA
3.2 Tumor Induction in K5-hSOS-F (K5-SOS) Mice
3.3 Histology of the Tail Skin from K5-Sos Mice
3.4 Keratinocyte Cultures
3.5 DMBA-/TPA-Induced SCC
3.7 Tissue Preparation and Histology
3.7.2 Histology of Back Skin
3.7.3 Histology of Tail Skin
3.7.5 Whole Mounts of Ears
3.7.6 Whole Mounts of the Back Skin
3.7.7 Whole Mounts of the Tail Skin
3.8.1 Hematoxylin and Eosin Stainings on Paraffin Sections
3.8.2 Immunohistochemistry Staining on Paraffin Sections
3.8.3 Immunofluorescent Stainings on Cryo-sections
3.8.4 Staining of Epidermal Ear Whole Mounts
3.8.5 Staining of Epidermal Back Skin and Tail Skin Whole Mounts
3.8.6 Frequently used Antibodies
11 Clinicopathological Characterization of Mouse Models of Melanoma
2.2 Clinical Follow-Up and Dermoscopy
2.3 Tumor Excision, Fixation, and Paraffin Embedding Protocol
2.4 Hematoxylin and Eosin (H&E) Staining
2.5 TRP-1 Immunohistochemistry
2.6 SOX-10 Immunohistochemistry
3.2 Clinical Follow-Up and Dermoscopy
3.3 Tumor Excision, Fixation, and Paraffin Embedding Protocol
3.4 Hematoxylin and Eosin (H&E) Staining
3.6 Confirmation of Melanocyte Origin or Lesions
3.7 TRP1 Immunohistochemistry
3.8 SOX10 Immunohistochemistry Protocol
12 Modeling BCR/ABL-Driven Malignancies in the Mouse
2.1 Cell Culture and In Vitro Assays
2.2 Mice and In Vivo Assays
3.1 In Vitro Generation of Stable v-ABLp160+ or BCR/ABLp185+ Lymphoid Leukemic Cell Lines (B-ALL)
3.1.1 Generation of Stable Retroviral Producer Cell Lines
3.1.2 Production of Retroviral Supernatants Using Stable Producer Cells
3.1.3 Production of Retroviral Supernatants Using Phoenix Cells
3.1.4 Infection of Primary Murine Hematopoietic Cells via Viral Supernatant
3.1.5 Infection of Primary Murine Hematopoietic Cells via Coculture with Stable Retroviral Producer Cells
3.1.6 Applications for Stable Murine B-ALL Cell Lines
3.2 In Vivo Mouse Experiments
3.2.1 Newborn Infections with v-ABLp160: A Model for Systemic B-ALL
3.2.2 Intravenous Injection of BCR/ABLp185+ or v-ABLp160+ Cell Lines: A Model for Systemic B-ALL
3.2.3 Bone Marrow Transplants of BCR/ABLp210-Infected Cells: A Model for CML
3.2.4 Inducible Gene Deletion During Established Leukemia via the Mx1-Cre/loxP System
3.2.5 Models for Solid Lymphoid Tumors
3.2.6 Mouse Strains Suitable for Leukemia Models
13 Methods to Generate Genetically Engineered Mouse Models of Soft Tissue Sarcoma
2.1 Ad-Cre Tumor Generation
2.2 TamoxifenưGenerated Tumors
2.3 Satellite CellưGenerated Tumors
2.4 Antibodies for FACS Sorting of Satellite Cells (See Table 1)
3.1 Adenoviral-CreưInitiated Tumors in LSL-KrasG12D/+; p53flox/flox (KP) Mice (See Note 2)
3.2 TamoxifenưInitiated Tumors with CreER Technology in Pax7CreER/+; p53flox/flox; LSL-KRasG12D/+ (P7KP) Mice (See Note 4)
3.3 Satellite Cell Isolation, Transformation, and Transplantation (See Note 5)
14 Characterization of Mouse Model-Derived Osteosarcoma (OS) Cells In Vitro and In Vivo
3.1 Isolation and Generation of OS Cell Lines from Bone Tumors
3.2 Generation of OS Cell Lines from Stromal Cultures
3.3 Characterization of Cell Lines
3.3.1 Alkaline Phosphatase Staining
3.3.2 Alizarin Red Staining
3.4 Assessment of Tumorigenic Capacity In Vivo
3.4.1 Subcutaneous Tumor Cell Implantation
3.4.2 Intra-tibial Tumor Cell Implantation
3.5 Imaging Techniques for Ex Vivo and In Vivo Mouse Models
15 Genetically Engineered Mouse and Orthotopic Human Tumor Xenograft Models of Retinoblastoma
1.1 Genetic Mouse Models of Retinoblastoma
1.1.1 Retina-Specific Cre-Expressing Transgenics
Pax6 α-Enhancer Cre (α-Cre)
1.1.2 Rb Mouse Models of Retinoblastoma
1.2 Orthotopic Human Xenograft of Retinoblastoma
2.1 Genetic Engineered Mouse Models
2.2 Orthotopic Human Xenografts
2.3 Retina Camera Measurement
2.4 Intraocular Pressure (IOP) Measurement
3.1 Genetic Engineered Mouse Models
3.2 Orthotopic Human Xenograft Model
3.3 Retina Camera Measurement
3.4 Intraocular Pressure Measurement (IOP)
Part III: Specific Aspects
16 Tumor Imaging Technologies in Mouse Models
3.1 GFP Retrovirus Production
3.2 RFP Retrovirus Production
3.3 Production of Histone H2B-GFP Vector
3.4 RFP or GFP Gene Transduction of Cancer Cell Lines
3.5 Double RFP and Histone H2B-GFP Gene Transduction of Cancer Cells
3.6 Establishment of Imageable Tumor Models
3.6.1 Cell Injection to Establish Experimental Metastasis Model
3.6.2 Subcutaneous Injection of Cancer Cells
3.6.3 Surgical Orthotopic Implantation (SOI) to Establish Spontaneous Metastasis Model (See Note 3)
3.7 Whole-Body Imaging of Mice
3.7.1 Whole-Body Imaging with a Microscope
3.7.2 Whole-Body Imaging with a Flashlight
3.7.3 Whole-Body Imaging in a Light box
3.7.4 Whole-Body Imaging in a Chamber
3.9 Comparison of Fluorescence Imaging to MRI
3.10 Comparison of Fluorescence Imaging to Ultrasound
17 Tumor Angiogenesis: Methods to Analyze Tumor Vasculature and Vessel Normalization in Mouse Models of Cancer
3.1 Immunohistochemistry and Microvessel Density Determination
3.2 Immunofluorescence from Fresh Frozen Tumor Tissues
3.3 Blood Vessel Lumen Diameter and Vascular Branching
3.5 Vessel Perfusion Assay with Fluorescent Lectins
3.6 In Vivo Perfusion with Fluorescent Microspheres (See refs. 24, 25)
3.7 Vessel Permeability Assay with Fluorescent Compounds
3.8 Vessel Wall Permeability Assay with Evans Blue
3.9 In Vivo Measurement of Tumor Bioavailability of Doxorubicin
18 Transplantable Mouse Tumor Models of Breast Cancer Metastasis
2.2 Mouse Tumor Cells from Primary Tumor or Cell Line
2.4 Primary Mammary Tumor Cell Isolation Reagents
3.1 Preparation of Single Cells from Mouse Mammary Tumor for Syngenic Transplantable Injection
3.2 Preparation of Single Cells from Mouse or Human Breast Tumor Cell Lines
3.3 Mammary Fat Pad (MFP) Injection for Spontaneous Metastasis Assay
3.4 Intravenous (Tail Vein) Injection for Experimental Lung Metastasis Assay
3.5 Intracardiac Injection for Experimental Bone and Brain Metastasis Assay
3.6 Bio-Luminescent Imaging (BLI)
19 Methods to Study Primary Tumor Cells and Residual Tumor Cells in Mouse Models of Oncogene Dependence
1.1 Initiation of Mammary Tumors in Transgenic Mice by Intraductal Delivery of a Tetracycline Transactivator in a Standard Retroviral Vector
1.2 Analysis of Epithelial Cell Proliferation with CFSE
2.1 Materials for in vivo transduction
2.1.1 Materials for In Vivo Stimulation of Mammary Cell Division
2.1.2 Materials for Intraductal Injection
2.2 Materials for CFSE Labeling
3.1 Stimulation of Mammary Epithelial Cell Turnover for Efficient Retroviral Infection
3.1.1 Subcutaneous Hormone Injections. TIMING < 10 min.
3.1.2 Tissue Harvest for Documenting Proliferation. TIMING < 20 min.
3.2 Intraductal Injections of Retroviral Vectors Carrying the Tetracycline Transactivator
3.2.1 Intraductal Injections. TIMING ≥1 h.
3.2.2 Documenting the Success of the Injection. TIMING ~ 10 min (see Note 6).
3.2.3 Documenting the Success of the Transduction. TIMING ~10 min (see Note 7).
3.2.4 Documenting the Success of Tumor Induction. TIMING ~10 min.
3.3 Analysis of Epithelial Cell Proliferation With CFSE
3.3.1 Mammary Cell Labeling. TIMING~30 min
3.3.2 Analysis of CFSEưLabeled Cells by Flow Cytometry (see Note 8).
20 Generation of Transgenic Mouse Model Using PTTG as an Oncogene
2.1 Selection of Plasmid and Promoter
2.2 Collection of Zygotes and Microinjection
2.3 Implantation of Microinjected Zygotes into Recipient Mice
2.4 Preparation of Tissue Samples for Genotyping
2.5 Southern Hybridization
2.6 Examination of Expression of Transgene
2.7 Breeding of Positive Founders
3.1 Selection of a Plasmid and Promoter to Drive the Expression of the Candidate Gene
3.2 Construction of CMV-PTTGưEGFP Transgene
3.3 Collection of Zygotes and Microinjection of Transgene
3.4 Implantation of Microinjected Zygotes into Pseudopregnant Recipient Mice
3.5 Preparation of Tissue Samples for Genotyping Founder Mice
3.6 Southern Hybridization to Confirm Positive Founders
3.7 Examination of Expression of Transgene
3.7.1 Expression of Transgene Using RT/PCR
3.7.2 Examination of Expression of Transgene by Immunohistochemistry or Fluorescence Analysis
3.8 Breeding of Positive Founders
21 Modeling the Study of DNA Damage Responses in Mice
2 Modeling Genomic Instability in Mice
3 Technologies for the Generation of Mouse Models
3.1.1 cDNA-Based Transgenesis
3.2.1 Constitutive Knockout
3.2.2 Conditional Knockout
3.2.4 Conditional Knockin
3.2.5 Gene-Trapping Strategies
22 Methods to Study Tumor Surveillance Using Tumor Cell Transplantation into Genetically Engineered Mice
3.1 Transplantation of Tumor Cells
3.1.1 The B16F10 Melanoma Model
3.1.2 Subcutaneous RMA-S Tumor Formation
3.1.3 Subcutaneous EL4/EG7 Tumor Formation
3.1.4 Subcutaneous MC38 Tumor Formation
3.2 Analysis of Infiltrating Immune Cells in Tumor Tissue
3.3 Depletion of Specific Tumor Immune Surveillance Mediators (NK1.1+ Cell and CD8+ T Cell Depletion)
3.3.1 Purification of PK136 (NK1.1) and 53-6.72 (CD8a) Antibodies
3.3.2 NK1.1+ Cell and CD8+ T Cell Depletion
3.4 Adoptive Cell Transfer (NK Cells, CD8+ T Cells)
3.4.1 Adoptive Transfer of NK Cells
3.4.2 Adoptive CD8+ T Cell Transfer Using the OT-1 System
3.4.3 Adoptive Transfer of In Vivo Activated CD8+ T Cells
3.5 Useful Mouse Models for Tumor Cell Transplantations
3.5.1 Conditional Knockout Mice
3.5.2 Mice on Rag2ưDeficient Background
3.5.3 Bone Marrow Chimeric Mice
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