Methods in molecular biology
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Macroscopic examination of the surgical specimen of esophageal squamous cell carcinoma by pathologist is important for quality clinical management, research, as well as education purposes. The process includes dissection of the specimen, identification of the lesion, measurements, and taking appropriate samples for histopathological examination. ⋯ In addition, examination of proximal resection margin and circumferential resection margin are important in the cancer. A standardized approach for macroscopic examination by professionals is needed for accurate diagnosis and to optimize the use of the surgical specimen with esophageal squamous cell carcinoma.
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Pathological staging is the most important factor that determines the prognosis and management of patients with esophageal squamous cell carcinoma. The method for the pathological staging in esophageal squamous cell carcinoma involves assessment of standard parameters-extent of tumor (T), lymph node status (N), presence of distant metastasis (M), as well as grade (G) and anatomical location of the carcinoma. In addition, other relevant factors, such as use of neoadjuvant therapy, could affect the pathological staging of esophageal squamous cell carcinoma.
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Orthotopic xenograft model recapitulates the faithful organ-specific microenvironment and facilitates analyses involving tumor-stromal interactions that are crucial for developing new-generation cancer therapy. Herein, we describe the detailed rationales and protocols of a versatile orthotopic xenograft model for esophageal squamous cell carcinoma.
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Mouse models are important in the study of pathogenesis, testing new treatment, and monitoring the progress of treatment in patients with esophageal squamous cell carcinoma (ESCC). The mice commonly used are immunosuppressed. The first category of models is for basic research and includes genetically engineered mouse models and carcinogen- or diet-induced mouse models. ⋯ This model appears promising for personalized medicine and of high resemblance to the nature of human ESCC, but there are many limitations for the use. It is likely to be used more in research in ESCC in the future. In this chapter, we detailed the preparation and experiments of PDX model from a patient with ESCC.
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Rapid evaluation of the CRISPR gRNA activity is an essential step of employing the technology in editing genes. Through machine learning strategy, the rule sets for in silico designing gRNAs with high activity has greatly improved. However, there are still discrepancies between different prediction rule sets, and between the predicted and actual gRNA activities. ⋯ We had previously developed a dual-fluorescent surrogate system, called C-Check, which based on single-strand annealing repair of the DNA double-strand breaks introduced by CRISPR-Cas9 to generate a functional EGFP. The system offers a tool for rapid functional evaluation of CRISPR gRNA activity, as well as for enrichment of gene edited cells. In this chapter, we will give a step-by-step instruction on the design, generation, and application of the C-Check system for quantifying gRNA activities.