Methods in molecular biology
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Histological assessment of esophageal squamous malignancies is crucial for management of patients with the cancer as well as working in research on the cancer. The squamous malignancies in the esophagus comprise squamous dysplasia and squamous cell carcinoma. Current classification of squamous dysplasia in the esophagus is to divide it into low grade and high grade. ⋯ Preoperative chemoradiation is used commonly in the treatment of esophageal squamous cell carcinoma and induces changes in morphology. Tumor regression grading systems based on the percentage of the remaining carcinoma cells are used to assess the response to the neoadjuvant therapy in the cancer. Additional histological parameters including lymphovascular invasion, perineural invasion, clearance of resection margins, and carcinoma in the nodal and distant metastatic sites provide essential information for the management of the patient with the cancer.
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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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Genome editing in eukaryotes has greatly improved through the application of targeted editing tools. The development of the CRISPR/Cas9 technology has facilitated genome editing in mammalian cells. However, efficient delivery of CRISPR components into cells growing in suspension remains a challenge. ⋯ Stable Cas9 expression is obtained by retroviral transduction, before sgRNA is transiently delivered into the Cas9+ cells. This method improves the on-target efficiency of genome editing and, through the transient presence of sgRNA, reduces the potential off-target sites. The current method can be easily applied to other cell types that are difficult to edit with CRISPR/Cas9.
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Coronaviruses (CoVs), enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. CoVs cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs, and upper respiratory tract and kidney disease in chickens to lethal human respiratory infections. Most recently, the novel coronavirus, SARS-CoV-2, which was first identified in Wuhan, China in December 2019, is the cause of a catastrophic pandemic, COVID-19, with more than 8 million infections diagnosed worldwide by mid-June 2020. Here we provide a brief introduction to CoVs discussing their replication, pathogenicity, and current prevention and treatment strategies. We will also discuss the outbreaks of the highly pathogenic Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV), which are relevant for understanding COVID-19.
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A critical stage in performing gene editing experiments using the CRISPR/Cas9 system is the design of guide RNA (gRNA). In this chapter, we conduct a review of the current gRNA design rules for maximizing on-target Cas9 activity while minimizing off-target activity. In addition, we present some of the currently available computational tools for gRNA activity prediction and assay design.