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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Early detection of cancer and the monitoring of cancer recurrence in treated patients are significant challenges in esophageal squamous cell carcinoma (ESCC). Liquid biopsy is the identification of tumor biomarkers from minimally invasive samples of biological fluids, including urine, blood, stool, saliva, or cerebrospinal fluid. ⋯ These sources of information have the potential to significantly improve the management of patients with ESCC. In this chapter, we detail a method for the isolation of cell-free DNA from blood plasma and DNA associated with exosomes in blood from patients with esophageal squamous cell carcinomas.
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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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The discovery of induced pluripotent stem cell (iPSC) technology has provided a versatile platform for basic science research and regenerative medicine. With the rise of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems and the ease at which they can be utilized for gene editing, creating genetically modified iPSCs has never been more advantageous for studying both organism development and potential clinical applications. However, to better understand the behavior and true therapeutic potential of iPSCs and iPSC-derived cells, a tool for labeling and monitoring these cells in vitro and in vivo is needed. ⋯ The approach involves the integration of the EGFP transgene into the transcriptionally active adeno-associated virus integration site 1 (AAVS1) locus through homology directed repair. The knockin of this transgene results in the generation of iPSC lines with constitutive expression of the EGFP protein that also persists in differentiated iPSCs. These EGFP-labeled iPSC lines are ideal for assessing iPSC differentiation in vitro and evaluating the distribution of iPSC-derived cells in vivo after transplantation into model animals.
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Mass spectrometry-based proteomics analysis could categorize proteins and study their interactions in large scale in human cancers. By this method, many proteins are upregulated or downregulated in esophageal squamous cell carcinoma (ESCC) when compared to nonneoplastic esophageal mucosae. ⋯ Different biological matrices such as pathological tissue, body fluids, and cancer cell lines-based proteomics have widely been used. Herein, we described cell line-based label-free shotgun proteomics (in-solution tryptic digestion) to identify the protein biomarkers differently expressed in ESCC.