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G protein-coupled receptors are a major class of membrane receptors that mediate physiological and pathophysiological cellular signaling. Many aspects of receptor activation and signaling can be investigated using genetically encoded luminescent fusion proteins. However, the use of these biosensors in live cell systems requires the exogenous expression of the tagged protein of interest. To maintain the normal cellular context here we use CRISPR/Cas9-mediated homology-directed repair to insert luminescent tags into the endogenous genome. Using NanoLuc and bioluminescence resonance energy transfer we demonstrate fluorescent ligand binding at genome-edited chemokine receptors. We also demonstrate that split-NanoLuc complementation can be used to investigate conformational changes and internalization of CXCR4 and that recruitment of β-arrestin2 to CXCR4 can be monitored when both proteins are natively expressed. These results show that genetically encoded luminescent biosensors can be used to investigate numerous aspects of receptor function at native expression levels.
This article was published in the following journal.
Name: Cell chemical biology
Recent advances in genome editing technologies have enabled the insertion of epitope tags at endogenous loci with relative efficiency. We describe an approach for investigation of protein interaction ...
Fluorescence imaging at single-cell resolution is a crucial approach to analyze the spatiotemporal regulation of proteins within individual cells of complex neural networks. Here we present a non-vira...
The association of reverse transcriptases (RTs) with CRISPR-Cas system has recently attracted interest because the RT activity appears to facilitate the RT-dependent acquisition of spacers from RNA mo...
This is a review describing advances in CRISPR/Cas-mediated therapies for neuromuscular disorders (NMDs). We explore both CRISPR-mediated editing and dead Cas approaches as potential therapeutic strat...
High-throughput CRISPR-Cas9 knockout screens using a tiling-sgRNA design permit in situ evaluation of protein domain function. Here, to facilitate de novo identification of essential protein domains f...
The purpose of this study is to determine whether Barium or BariumFerumoxsil is better for fecal tagging in MR colonography. Patients referred to colonoscopy are offered MR colonography be...
The aim of this study is to investigate the amino acid kinetics in blood after a bout of strength training and ingestion of different milk protein supplements (native whey, whey protein co...
Multiple solid tumors have positive targets of mesothelin expressed on the surfaces of the tumor cells, we use the technique of CRISPR-Cas9 to knocked out the PD-1 and TCR of chimeric anti...
This observational Mycobacterium tuberculosis (MTB) diagnostics evaluation study is a prospective study of pulmonary TB suspects who are undergoing sputum or bronchoalveolar lavage fluid (...
Computed tomography (CT) colonography has gained widespread multi-disciplinary interest as an evolving noninvasive colorectal screening examination, with the potential of improved patient ...
Protein components of the CRISPR-CAS SYSTEMS for anti-viral defense in ARCHAEA and BACTERIA. These are proteins that carry out a variety of functions during the creation and expansion of the CRISPR ARRAYS, the capture of new CRISPR SPACERS, biogenesis of SMALL INTERFERING RNA (CRISPR or crRNAs), and the targeting and silencing of invading viruses and plasmids. They include DNA HELICASES; RNA-BINDING PROTEINS; ENDONUCLEASES; and RNA and DNA POLYMERASES.
Adaptive antiviral defense mechanisms, in archaea and bacteria, based on DNA repeat arrays called CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR elements) that function in conjunction with CRISPR-ASSOCIATED PROTEINS (Cas proteins). Several types have been distinguished, including Type I, Type II, and Type III, based on signature motifs of CRISPR-ASSOCIATED PROTEINS.
The body's defense mechanism against foreign organisms or substances and deviant native cells. It includes the humoral immune response and the cell-mediated response and consists of a complex of interrelated cellular, molecular, and genetic components.
The exposure of protein to chemicals, or heat, which disrupt the non-covalent bonds and/or disulfide bonds responsible for maintaining the three-dimensional shape and activity of the native protein, while leaving the peptide bonds intact.
Immunologic method used for detecting or quantifying immunoreactive substances. The substance is identified by first immobilizing it by blotting onto a membrane and then tagging it with labeled antibodies.
Within medicine, nutrition (the study of food and the effect of its components on the body) has many different roles. Appropriate nutrition can help prevent certain diseases, or treat others. In critically ill patients, artificial feeding by tubes need t...
Cytokines include chemokines, lymphokines, and monokines. Cells of the immune system communicate with one another by releasing and responding to chemical messengers called cytokines. These proteins are secreted by immune cells and act on other cells to...