RESUMO
The integration of CRISPR/Cas9 genome editing techniques with organoid technology has revolutionized the field of tumor modeling, enabling the creation of diverse tumor models with distinct mutational profiles. This protocol details the application of CRISPR knock-ins to engineer tumor organoids with reporter cassettes, which are regulated by endogenous promoters of specific genes of interest. This approach facilitates the precise fluorescent labeling, isolation, and subsequent manipulation of targeted tumor cell subpopulations. The utilization of these knock-in reporter cassettes not only allows the visualization and purification of specific tumor cell subsets but also enables conditional cell ablation and lineage tracing studies. In this chapter, we provide a comprehensive guide for the design, construction, delivery, and validation of CRISPR/Cas9 tools tailored for knock-in reporter cassette integration into specific marker genes of interest. By following this protocol, researchers can harness the potential of engineered tumor organoids to decipher intricate tumor heterogeneity, track metastatic trajectories, and unveil novel therapeutic vulnerabilities linked to specific tumor cell subpopulations.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Introdução de Genes , Organoides , Organoides/metabolismo , Organoides/patologia , Humanos , Técnicas de Introdução de Genes/métodos , Edição de Genes/métodos , Animais , Neoplasias/genética , Neoplasias/patologia , Genes ReporterRESUMO
Over the last decades, it has been established that the immune system is crucial for the impediment of cancer development by recognizing and destroying transformed cells. This process has been termed cancer immunosurveillance. Small animal models have significantly facilitated our understanding of it. Dissecting the contribution of any specific immune cell type participating in this process requires the ability to specifically target it while leaving the other immune components as well as the cancer model system unperturbed in vivo. Here, we provide a simple and rapid protocol for the generation of transgenic mice expressing Cre recombinase in a cell type-specific manner-in our example we chose cells expressing Ncr1, which encodes for the surface protein NKp46-and the use of those mice to ablate NKp46+ cells in order to study their role in a model of cancer immunosurveillance against experimental pulmonary metastases. This protocol can easily be adapted to target other cell types and other cancer models.
Assuntos
Antígenos Ly/genética , Vigilância Imunológica , Integrases/genética , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Animais , Antígenos Ly/imunologia , Antígenos Ly/metabolismo , Linhagem Celular Tumoral , Feminino , Genes Reporter/genética , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Células Matadoras Naturais/metabolismo , Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/genética , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologiaRESUMO
Autoimmune and inflammatory conditions are frequent complications in patients with reduced numbers of T cells. Here, we describe a mouse model of thymic stromal dysplasia resulting in peripheral T-cell lymphopenia. In Foxn1:CFP-NTR transgenic mice, the bacterial nitroreductase enzyme is expressed in thymic epithelial cells and converts the prodrug CB1954 into a cytotoxic agent. This strategy enables titratable and durable destruction of thymopoietic tissue in early embryogenesis. Our results indicate that the resulting low levels of thymic capacity for T-cell production create a predisposition for the development of a complex autoimmune syndrome, chiefly characterized by inflammatory bowel disease and lymphocytic organ infiltrations. We conclude that the Foxn1:CFP-NTR transgenic mouse strain represents a suitable animal model to optimize established clinical protocols, such as thymus transplantation, to correct various forms of thymic dysplasia and to explore novel treatment options.