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Protocol
Generation of Monoclonal iPSC Lines with Stable Cas9 Expression and High Cas9 Activity
CRISPR/Cas9-mediated gene editing has been rapidly and widely applied in many organisms for delicate genetic manipulation, including human-induced pluripotent stem cells (iPSCs). Gene editing in human iPSCs is...
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Protocol
A Fluorescence-Microscopic System for Monitoring the Turnover of the Autophagic Substrate p62/SQSTM1
In conditions of cellular stress and nutrient shortage, macroautophagy (hereafter referred to as autophagy) assures the degradation of dysfunctional macromolecules and organelles as it liberates energy resourc...
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Protocol
Investigating Fungal Biosynthetic Pathways Using Pichia pastoris as a Heterologous Host
Fungal natural products have extensive biological activities, and thus have been largely commercialized in the pharmaceutical, agricultural, and food industries. Recently, heterologous expression has become an...
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Protocol
Designing Overlap Extension PCR Primers for Protein Mutagenesis: A Programmatic Approach
Overlap extension PCR is one of the routinely used methods to generate mutagenic genes for the functional and structural study of proteins. However, it is time-consuming to design the overlap** mutagenic pri...
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Protocol
In Vivo Migration and Tfh Cell Interactions
Follicular T-helper cells play a key role in orchestrating the germinal center response. Cellular dynamics revealed by intravital imaging have helped deepen our understanding of how follicular T-helper cells d...
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Protocol
Reactive Oxygen Species-Responsive and Self-Illuminating Nanoparticles for Inflammation and Tumor Imaging
Reactive oxygen species (ROS) play a key role in various physiological and pathological processes. Abnormally elevated ROS levels are generally related to the pathogenesis of inflammatory diseases and tumors. ...
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Protocol
Detection of 5-Formylcytosine and 5-Formyluracil Based on Photo-Assisted Domino Reaction
5-Formylcytosine (5fC) is a recently identified DNA modification in mammalian genome, derived from 5-methylcytosine (5mC) by TET-mediated oxidation. It has been demonstrated to be a critical intermediate durin...
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Protocol
Gene Co-expression Network Analysis and Linking Modules to Phenoty** Response in Plants
Environmental factors, including different stresses, can have an impact on the expression of genes and subsequently the phenotype and development of plants. Since a large number of genes are involved in respon...
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Protocol
Functional Analysis of Actin-Binding Proteins in the Central Nervous System of Drosophila
Using Drosophila actin-binding protein Dunc-115 as an example, this chapter describes a MARCM (mosaic analysis with a repressible cell marker)-based method for analyzing cytoskeletal components for their function...
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Protocol
Tetramer-Associated T Cell Receptor Sequencing
Linking antigen specificity to T cell receptor (TCR) sequences is critical, albeit challenging, to both understanding T cell biology and develo** T cell-based therapeutics. Here, we describe in detail tetram...
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Protocol
Ligand-Directed GPCR Antibody Discovery
Develo** affinity reagents recognizing and modulating G-protein coupled receptors (GPCR) function by traditional animal immunization or in vitro screening methods is challenging. Some anti-GPCR antibodies ex...
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Protocol
Integration of Omics and Phenotypic Data for Precision Medicine
Over the past two decades, biomedical research is moving toward a big-data-driven approach. The underlying causes of this transition include the ability to gather genetic or molecular profiles of humans faster...
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Protocol
Single Transcript Unit CRISPR 2.0 Systems for Genome Editing in Rice
CRISPR-Cas9 and Cas12a (formerly Cpf1), RNA-guided DNA endonucleases found from adaptive immune system in prokaryotes, have been engineered and widely adopted as two of the most powerful genome editing systems...
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Protocol
Structural Characterization of Silk Fibers by Small-Angle X-Ray Scattering
Nature is rich in all kinds of unbelievably designed microstructures, which endows the natural materials with various fantastic performances. Unveiling the mystery of the sophisticated configurations and the r...
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Protocol
Discovery of Covalent Drugs Targeting the Key Enzymes of SARS-CoV-2 Using SCARdock
Historically, covalent drugs were avoided in drug development process due to the possible toxicity linked to the covalent binding of such drugs and cellular proteins. However, recent years have witnessed the f...
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Protocol
A Hybrid Biological–Biomaterial Vector for Antigen Delivery
A hybrid biological–biomaterial vector composed of a biocompatible polymeric biomaterial coating and an Escherichia coli core was designed and developed for antigen delivery. It provides a unique and efficient me...
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Protocol
Assembly of TALEN and mTALE-Act for Plant Genome Engineering
Transcription activator-like effector (TALE) is a DNA-binding domain that can be paired with a nuclease to create DNA double-strand breaks, or with an effector protein to alter gene transcription. The ability ...
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Protocol
Structural Characterization of Silk Fibers by Wide-Angle X-Ray Scattering
As one of the most advanced techniques to gain insight into the structure of the materials, wide-angle X-ray scattering (WAXS) records the scattering information at wide angles which typically larger than 5° (2θ)...
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Protocol
Improving a Quantitative Trait in Rice by Multigene Editing with CRISPR-Cas9
CRISPR-Cas9 system is one sequence-specific nuclease (SSN) that has several advantages over zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN), such as multiplex genome editi...
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Protocol
Efficient Multiplexed CRISPR-Cas12a Genome Editing in Plants
(formerly Cpf1) is a Class 2 Type V-A clustered regularly interspaced short palindrome repeats (CRISPR ) system that has been widely used in plant to target AT-rich regions. only requires short CRISPR RN...
