Methods in molecular biology
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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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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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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.
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Immunohistochemistry is the identification of a cell protein by a specific antibody targeting that protein. It is the most common ancillary test to study the pathology of cancer. Immunohistochemical protein markers are used to differentiate poorly differentiated squamous cell carcinoma from poorly differentiated adenocarcinoma or neuroendocrine carcinomas. ⋯ Successful application of the immunochemistry depends on understanding the mechanisms and principles as well as the limitations of the procedure. Automation of the procedure by different models of automatic stainers is widely used in diagnostic laboratories. The use of autostainers streamlines the workflows and certainly reduces the labor, time, and cost of using immunohistochemistry in clinical and research settings.
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Pathological assessment of frozen sections of tissues is important in the clinical management (intraoperative consultation) and research in patients with esophageal squamous cell carcinoma. Frozen sections may be used in the assessment of status of resection margins, extent of cancer metastasis (pathological staging), confirmation of the pathology, and increased volume of cancer cells for tissue banking. However, the applications of frozen sections have many technical limitations. Thus, interpretation of frozen sections needs expertise, collaborations, and attention to proper technical skills in the sectioning.