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Methods: a Companion to Methods in Enzymology

Jan-Hendrik Spille, Micca Hecht, Valentin Grube, Won-Ki Cho, Choongman Lee, Ibrahim I Cissé
The MS2 system is a powerful tool for investigating transcription dynamics at the single molecule directly in live cells. In the past, insertion of the RNA-labelling cassette at specific gene loci has been a major hurdle. Here, we present a CRISPR/Cas9-based approach to insert an MS2 cassette with selectable marker at the start of the 3' untranslated region of any coding gene. We demonstrate applicability of our approach by tagging RNA of the stem cell transcription factor Esrrb in mouse embryonic stem cells...
September 11, 2018: Methods: a Companion to Methods in Enzymology
Katy L Everett, Matthew Kraman, Francisca P G Wollerton, Carlo Zimarino, Katarzyna Kmiecik, Miguel Gaspar, Sarka Pechouckova, Natalie L Allen, Jacqueline F Doody, Mihriban Tuna
The immunoglobulin superfamily protein lymphocyte-activation gene 3 (LAG-3) participates in immune suppression and has been identified as a suitable target for cancer therapies. In order to generate bispecific antibodies targeting LAG-3, Fcabs (Fc-region with antigen binding) targeting human and murine LAG-3 were generated from phage libraries. These Fcabs bind to LAG-3, inhibiting its interaction with MHC class II, and induce IL-2 production in a T cell assay. Bispecific antibodies, known as mAb2 , were produced by replacing the Fc region of a monoclonal antibody with Fcab sequences in the CH3 domain...
September 9, 2018: Methods: a Companion to Methods in Enzymology
Lisa Landvogt, Jan Andreas Ruland, Christian Montellese, Jan Peter Siebrasse, Ulrike Kutay, Ulrich Kubitscheck
Ribosomes are formed of a small and a large subunit (SSU/LSU), both consisting of rRNA and a plethora of accessory proteins. While biochemical and genetic studies identified most of the involved proteins and deciphered the ribosomal synthesis steps, our knowledge of the molecular dynamics of the different ribosomal subunits and also of the kinetics of their intracellular trafficking is still limited. Adopting a labelling strategy initially used to study mRNA export we were able to fluorescently stain the SSU in vivo...
September 6, 2018: Methods: a Companion to Methods in Enzymology
Dominique Förster, Anna Kramer, Herwig Baier, Fumi Kubo
All-optical methods enable the control and monitoring of neuronal activity with minimal perturbation of the system. Although imaging and optogenetic manipulations can be performed at cellular resolution, the morphology of single cells in a dense neuronal population has often remained unresolvable. Here we describe in detail two recently established optogenetic protocols for systematic description of function and morphology of single neurons in zebrafish. First, the Optobow toolbox allows unbiased mapping of excitatory functional connectivity...
September 5, 2018: Methods: a Companion to Methods in Enzymology
Xiufeng Wu, Stephen J Demarest
Bispecific antibodies (BsAbs), which target two antigens or epitopes, incorporate the specificities and properties of two distinct monoclonal antibodies (mAbs) into a single molecule. As such, BsAbs can elicit synergistic activities and provide the capacity for enhanced therapeutic efficacy and/or safety compared to what can be achieved with conventional monospecific IgGs. There are many building block formats to generate BsAbs and Trispecific antibodies (TsAbs) based on combining the antigen recognition domains of monoclonal antibodies (mAbs)...
August 30, 2018: Methods: a Companion to Methods in Enzymology
Leonid A Serebryannyy, David A Ball, Tatiana S Karpova, Tom Misteli
The nuclear envelope (NE) is an essential cellular structure that contributes to nuclear stability, organization, and function. Mutations in NE-associated proteins result in a myriad of pathologies with widely diverse clinical manifestations, ages of onsets, and affected tissues. Notably, several hundred disease-causing mutations have been mapped to the LMNA gene, which encodes the intermediate filament proteins lamin A and C, two of the major architectural components of the nuclear envelope. However, how NE dysfunction leads to the highly variable pathologies observed in patient cells and tissues remains poorly understood...
August 24, 2018: Methods: a Companion to Methods in Enzymology
Junpeng Qi, Shih-Shih Chen, Nicholas Chiorazzi, Christoph Rader
Bispecific antibodies (biAb) targeting two different antigens or two distinct epitopes on the same antigen have demonstrated broad therapeutic utility. CD52 and CD20 are co-expressed on the cell surface of malignant B cells in B-cell non-Hodgkin lymphoma (B-NHL) and chronic lymphocytic leukemia (CLL) and increased expression of both antigens is detected on dividing or recently divided cells ("proliferative fraction") in CLL. The CD52-targeting monoclonal antibody (mAb) alemtuzumab (atz) not only depletes malignant B cells but also healthy CD52+ B and T lymphocytes and monocytes, causing severe immunosuppression...
August 24, 2018: Methods: a Companion to Methods in Enzymology
A Maxwell Burroughs, Yoshinari Ando
Over the past decade, modifications to microRNAs (miRNAs) via 3' end nucleotide addition have gone from a deep-sequencing curiosity to experimentally confirmed drivers of a range of regulatory activities. Here we overview the methods that have been deployed by researchers seeking to untangle these diverse functional roles and include characterizing not only the nucleotidyl transferases catalyzing the additions but also the nucleotides being added, and the timing of their addition during the miRNA pathway. These methods and their further development are key to clarifying the diverse and sometimes contradictory functional findings presently attributed to these nucleotide additions...
August 20, 2018: Methods: a Companion to Methods in Enzymology
Yichen Li, Samuel L Junod, Andrew Ruba, Joseph M Kelich, Weidong Yang
The nuclear exit of messenger RNA (mRNA) molecules through the nuclear pore complex (NPC) is an essential step in the translation process of all proteins. The current limitations of conventional fluorescence and electron microscopy have prevented elucidation of how mRNA exports through the NPCs of live cells. In the recent years, various single-molecule fluorescence (SMF) microscopy techniques have been developed to improve the temporal and spatial resolutions of live-cell imaging allowing a more comprehensive understanding of the dynamics of mRNA export through native NPCs...
August 18, 2018: Methods: a Companion to Methods in Enzymology
Mabel Guzman-Rodriguez, Julie A K McDonald, Richard Hyde, Emma Allen-Vercoe, Erika C Claud, Prameet M Sheth, Elaine O Petrof
The study of complex microbial communities has become a major research focus as mounting evidence suggests the pivotal role microbial communities play in host health and disease. Microbial communities of the gastrointestinal tract, known as the gut microbiota, have been implicated in aiding the host with vitamin biosynthesis, regulation of host energy metabolism, immune system development, and resistance to pathogen invasion. Conversely, disruptions of the gut microbiota have been linked to host morbidity, including the development of inflammatory diseases, metabolic disorders, increased cardiovascular risk, and increased risk of infectious diseases...
August 11, 2018: Methods: a Companion to Methods in Enzymology
N Dimasi, R Fleming, H Wu, C Gao
In recent years, bispecific antibodies (BisAbs) have emerged as novel pharmaceutical candidates owing to their ability to engage two disease mediators simultaneously, thus providing a possible alternative therapeutic approach in complex diseases such as cancer and inflammation. Here we provide an overview of the molecular design, recombinant expression in mammalian cells and purification of BisAbs based on full-length IgG-scFv formats. Practical considerations and strategies to optimize transient expression and purification are also discussed...
August 11, 2018: Methods: a Companion to Methods in Enzymology
Irina Oganesyan, Cristina Lento, Derek J Wilson
No abstract text is available yet for this article.
August 10, 2018: Methods: a Companion to Methods in Enzymology
Alexander Johnson-Buck, Jieming Li, Muneesh Tewari, Nils G Walter
Conventional methods for detecting small quantities of nucleic acids require amplification by the polymerase chain reaction (PCR), which necessitates prior purification and introduces copying errors. While amplification-free methods do not have these shortcomings, they are generally orders of magnitude less sensitive and specific than PCR-based methods. In this review, we provide a practical guide to a novel amplification-free method, single-molecule recognition through equilibrium Poisson sampling (SiMREPS), that provides both single-molecule sensitivity and single-base selectivity by monitoring the repetitive interactions of fluorescent probes to immobilized targets...
August 10, 2018: Methods: a Companion to Methods in Enzymology
Nicholas Cummins, Alice Baird, Björn W Schuller
Due to the complex and intricate nature associated with their production, the acoustic-prosodic properties of a speech signal are modulated with a range of health related effects. There is an active and growing area of machine learning research in this speech and health domain, focusing on developing paradigms to objectively extract and measure such effects. Concurrently, deep learning is transforming intelligent signal analysis, such that machines are now reaching near human capabilities in a range of recognition and analysis tasks...
August 10, 2018: Methods: a Companion to Methods in Enzymology
Shiyong Gong, Chengbin Wu
Bispecific antibody (BsAb) has become an important trend in developing next generation biologics therapies. By simultaneously engaging two molecular targets, BsAbs show distinctive mechanism of actions that could lead to clinical benefits unattainable by conventional monoclonal antibodies (mAbs). Successful launch provided clinical validation and encourage more BsAb development in the pipeline of pharmaceutical companies. Fabs-in-tandem immunoglobulin (FIT-IgTM ) format was initially described in 2017. This unique design provides a symmetrical and tetravalent IgG-like bispecific molecule with correct association of 2 sets of VH/VL pairs, where two Fabs are fused directly in a crisscross orientation without any mutations or use of peptide linkers...
August 3, 2018: Methods: a Companion to Methods in Enzymology
Lina Bartels, Hidde L Ploegh, Hergen Spits, Koen Wagner
Historically, bispecific antibodies have been constructed through the genetic fusion of additional binding domains to the constant domains of the antibody heavy- or light chains. We present an alternative method for the introduction of additional functional domains to an antibody: site-specific chemo-enzymatic conjugation. This method relies on the combination of site-specific transpeptidases and bioorthogonal chemistry. Transpeptidases are used to site-specifically introduce chemical handles, which can then be used to couple new functional groups by means of a bioorthogonal chemical reaction...
August 3, 2018: Methods: a Companion to Methods in Enzymology
Wei Qin, Xiaochan Lu, Shuo Lin
The use of CRISPR/Cas9 to knockout genes in zebrafish has been well established. However, to better model many human diseases that are caused by point mutations, a robust methodology for generating desirable DNA base changes is still needed. Recently, Cas9-linked cytidine deaminases (base editors) evolved as a strategy to introduce single base mutations in model organisms. They have been used to convert cytidine to thymine at specific genomic loci. Here we describe a protocol for using the base editing system in zebrafish and its application to reproduce a single base mutation observed in human Ablepharon-Macrostomia Syndrome...
August 2, 2018: Methods: a Companion to Methods in Enzymology
Le A Trinh, Vanessa Chong-Morrison, Tatjana Sauka-Spengler
Interrogation of gene regulatory circuits in complex organisms requires precise and robust methods to label cell-types for profiling of target proteins in a tissue-specific fashion as well as data analysis to understand interconnections within the circuits. There are several strategies for obtaining cell-type and subcellular specific genome-wide data. We have developed a methodology, termed "biotagging" that uses tissue-specific, genetically encoded components to biotinylate target proteins, enabling in depth genome-wide profiling in zebrafish...
August 2, 2018: Methods: a Companion to Methods in Enzymology
Brandon W Simone, Gabriel Martínez-Gálvez, Zachary WareJoncas, Stephen C Ekker
The rapid growth of the field of gene editing can largely be attributed to the discovery and optimization of designer endonucleases. These include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regular interspersed short palindromic repeat (CRISPR) systems including Cas9, Cas12a, and structure-guided nucleases. Zebrafish (Danio rerio) have proven to be a powerful model system for genome engineering testing and applications due to their external development, high fecundity, and ease of housing...
August 2, 2018: Methods: a Companion to Methods in Enzymology
Chiso Nwokafor, Robert H Singer, Hyungsik Lim
We describe a method for visualizing mRNAs in living mouse. Nascent transcripts and cytoplasmic mRNAs were labeled via lentiviral expression of MS2 coat protein (MCP) tagged with fluorescent protein (MCP-XFP) in knock-in mice whose β-actin mRNAs contained MCP binding stem loops (MBS). Then the mRNA molecules were imaged in the live cerebral cortex through an optical cranial window by intravital two-photon microscopy. By means of the controlled expression of MCP-XFP, single mRNA particles could be detected differentially in the nucleus and cytoplasm of a specific cell type...
July 29, 2018: Methods: a Companion to Methods in Enzymology
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