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The efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two (TKIT) guides as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labeling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.
Pubmed ID: 33689678
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Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
View all literature mentionsNIH genetic sequence database that provides annotated collection of all publicly available DNA sequences for almost 280 000 formally described species (Jan 2014) .These sequences are obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects, including whole-genome shotgun (WGS) and environmental sampling projects. Most submissions are made using web-based BankIt or standalone Sequin programs, and GenBank staff assigns accession numbers upon data receipt. It is part of International Nucleotide Sequence Database Collaboration and daily data exchange with European Nucleotide Archive (ENA) and DNA Data Bank of Japan (DDBJ) ensures worldwide coverage. GenBank is accessible through NCBI Entrez retrieval system, which integrates data from major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of GenBank database are available by FTP.
View all literature mentionsA national mouse monoclonal antibody generating resource for biochemical and immunohistochemical applications in mammalian brain. NeuroMabs are generated from mice immunized with synthetic and recombinant immunogens corresponding to components of the neuronal proteome as predicted from genomic and other large-scale cloning efforts. Comprehensive biochemical and immunohistochemical analyses of human, primate and non-primate mammalian brain are incorporated into the initial NeuroMab screening procedure. This yields a subset of mouse mAbs that are optimized for use in brain (i.e. NeuroMabs): for immunocytochemical-based imaging studies of protein localization in adult, developing and pathological brain samples, for biochemical analyses of subunit composition and post-translational modifications of native brain proteins, and for proteomic analyses of native brain protein networks. The NeuroMab facility was initially funded with a five-year U24 cooperative grant from NINDS and NIMH. The initial goal of the facility for this funding period is to generate a library of novel NeuroMabs against neuronal proteins, initially focusing on membrane proteins (receptors/channels/transporters), synaptic proteins, other neuronal signaling molecules, and proteins with established links to disease states. The scope of the facility was expanded with supplements from the NIH Blueprint for Neuroscience Research to include neurodevelopmental targets, the NIH Roadmap for Medical Research to include epigenetics targets, and NIH Office of Rare Diseases Research to include rare disease targets. These NeuroMabs will then be produced on a large scale and made available to the neuroscience research community on an inexpensive basis as tissue culture supernatants or purified immunoglobulin by Antibodies Inc. The UC Davis/NIH NeuroMab Facility makes NeuroMabs available directly to end users and is unable to accommodate sales to distributors for third party distribution. Note, NeuroMab antibodies are now offered through antibodiesinc.
View all literature mentionsA commercial antibody supplier which supplies primary and secondary antibodies, biochemicals, proteins, peptides, lysates, immunoassays and other kits.
View all literature mentionsAn Antibody supplier and subset of ThermoFisher Scientific which provides fluorescence reagents for various experiments and methods.
View all literature mentionslaboratory mouse with name C57BL/6N from MGI.
View all literature mentionsMus musculus with name C57BL/6J from IMSR.
View all literature mentionsThis polyclonal targets NMDAR2A&B
View all literature mentionsThis monoclonal targets GABA A Receptor beta 23 Chain extracellular domain of the beta2 & beta3 subunits of the GABA A receptor clone BD17
View all literature mentionsThis monoclonal targets PSD-95 MAGUK scaffolding protein
View all literature mentionsThis monoclonal targets HA-Tag
View all literature mentionsThis monoclonal targets Gephyrin
View all literature mentionsThis polyclonal targets Green Fluorescent Protein (GFP)
View all literature mentionsThis monoclonal targets NR1 glutamate receptor
View all literature mentionsThis polyclonal targets GFP
View all literature mentionsAn online resource which helps researchers manage and organize labs and experimental results by supplying molecular biology software tools for experimental design and data analysis. Benchling provides tools for functions such as primer design and colony counting as well as CRISPR guide design and automated Gibson and Golden Gate cloning. Users can take notes in line with data, link data across entries, keep files and data in one place, and manage and keep track of team progress. An enterprise version of Benchling is available for scientists working within an organization with additional administrative, compliance, and security protocols.
View all literature mentionsMus musculus with name B6.129-Igs2tm1(CAG-cas9*)Mmw/J from IMSR.
View all literature mentionsMus musculus with name B6.Cg-Tg(Camk2a-cre)T29-1Stl/J from IMSR.
View all literature mentionsSoftware package as distribution of ImageJ and ImageJ2 together with Java, Java3D and plugins organized into coherent menu structure. Used to assist research in life sciences.
View all literature mentionsStatistical analysis software that combines scientific graphing, comprehensive curve fitting (nonlinear regression), understandable statistics, and data organization. Designed for biological research applications in pharmacology, physiology, and other biological fields for data analysis, hypothesis testing, and modeling.
View all literature mentionsMus musculus with name B6;129P2-Pvalbtm1(cre)Arbr/J from IMSR.
View all literature mentionsImaris provides range of capabilities for working with three dimensional images. Uses flexible editing and processing functions, such as interactive surface rendering and object slicing capabilities. And output to standard TIFF, Quicktime and AVI formats. Imaris accepts virtually all image formats that are used in confocal microscopy and many of those used in wide-field image acquisition.
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