...Additional Resources on the Addgene Blog Treating Muscular Dystrophy with CRISPR Gene Editing Learn to Use ...activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that can produce HIV-1 ...Cas9 have also been used to create mutations in genes essential to HIV-1 replication. However, this approach...modification targeting either the LTRs or essential genes. Many of these escape mutations were located near...Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing. Sci Rep. 2016 Mar 4;6:22555. PubMed PMID...Screens Additional Resources onf Addgene.org Check out Addgene’s newly updated CRISPR Guide! Find CRISPR...Activation Mediators (SAM) system available from Addgene. This system uses the dCas9-VP64 fusion commonly...

...may be the hot new way to manipulate gene expression, but other gene manipulation systems remain valuable...important ways to spatially and temporally control gene expression, especially in in vivo models, and new...-2.1 construct can express one of four colors (n-GFP, RFP, YFP or M-CFP.) The construct contains two tandem...mOrange2, jellyfish EGFP, and sea anemone mKate2.) They then successfully generated custom antibodies to...-2. The general structure of Brainbow-1.0 is retained in Brainbow-3.0, but with mOrange2, EGFP and mKate2...at least six generations. In addition to the neuronal pThy1-Brainbow constructs, Addgene also has two ...Brainbow plasmids available from Addgene! Find Brainbow Plasmids @Addgene: Brainbow 1.0, 1.1, 2.1 plasmids...

...Figure 1: MTRIA biosensors are generated by inserting a circularly-permuted GFP into an intracellular loop...new to Addgene this year. Feel the love with MTRIA sensors One of the latest additions to Addgene’s catalog...imaging. Vladislav Verkhusha’s lab has generated a new genetically encoded calcium indicator using small...on the Addgene blog Fluorescent Proteins 101: Fluorescent Biosensors Resources on Addgene.org Fluorescent...Here at Addgene’s headquarters, the skies are getting dark and wintry with the end of year approaching...localized for synaptic glutamate response. They generated variants optimized for different applications ...St-Pierre lab and colleagues have developed a new genetically-encoded voltage indicator (GEVI) called JEDI-...

... transcription of a target gene, it therefore determines the timing of gene expression and largely defines...These permit exquisite control of gene expression and tracing of gene expression across development. One...of herpes simplex virus immediate early gene expression. Genes Dev. 1988 Jun;2(6):718-29. doi: 10.1101...systems are genetic tools that consist of two parts, both of which must be present for a gene of interest...placing UAS upstream of a transgene permits directed expression of that gene in cells that also express...expression of reporter genes, or to combine reporter expression from one promoter with transgene or siRNA expression...together for complex genetic analysis in Drosophila or for precise control over multiple genes in synthetic systems...

... bright enough to detect endogenous tagged genes generated through CRISPR/Cas9 knock-in. Finally, NLuc...Fusion Protein, codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase are available...fluorescent protein that could be co-imaged with GFP using the same wavelength for excitation. The result...such as the dynamics of subcellular structures and gene expression. You must see the movies demonstrating...al. (2016) CRISPaint allows modular base-specific gene tagging using a ligase-4-dependent mechanism. Nature...when employing optogenetic regulators to initiate the event being monitored. Optogenetic regulators respond...wavelengths overlap with optogenetic initiation wavelengths, excitation of the optogenetic regulator leads to...

...protein, permeabilized cells  Cy5NTAc 32 25-45 GFP (fluorescein is similar)   Lumio Red Tc: CCPGCC 48...in setting up a FRET-based assay or screen (the Addgene blog has an excellent primer on FRET and how to...Fluorescent proteins (FPs) are the gold standard for genetically-encoded FRET donors (Fig. 1). Scores of FP variants...presented (Fig. 1) which can be carried out using Addgene plasmids. His-tag labeling reagents You may already...Gradia Lab has deposited a gateway vector with Addgene, where you can insert your protein of interest,...PubMed Central PMCID: PMC4380784. Resources on the Addgene Blog Get Tips for Using FRET in Your Experiments...Your Fluorescent Protein Basics Resources on Addgene.org Find Plasmids for FRET Browse Empty Plasmids...

...labeling. Size is also a factor. Bulkier tags, such as GFP, may interfere with protein expression or function...the dye and the endogenous target, and thus no genetically encoded tag is required. These dyes can also ...be SNAP-tag® and HaloTag®. These consist of a genetically encoded enzyme variant tag that covalently reacts...maintained through mechanisms like fission, fusion, biogenesis, and mitophagy, can be tracked using dyes like...Keller, P. J., Ji, N., & Lavis, L. D. (2017). A general method to fine-tune fluorophores for live-cell ....1016/j.bbamem.2013.09.005 More resources from Addgene Fluorescent protein plasmids and resourcesA practical...

...measurable through an epitope tag such as FLAG/His/GFP. Fluorescent protein tags, which correlate closely...procedure and application to the major ALS/FTD disease gene C9ORF72. eLife, 8, e48363. https://doi.org/10.7554...101: Validation Resources on addgene.org Addgene's Antibody Guide Addgene's Antibody Protocols... outlined below. Knockout cell line approach Genetic knockout-based validation can conclusively prove...you are working with cells that are amenable to genetic knockout, this represents a very robust approach...to start with a screening procedure in easily genetically modified cell types (top). The specific approach...10.1038/s41598-019-55133-7 More resources from the Addgene blog Introduction to Gating in Flow Cytometry Antibodies...

...reprogramming transgenes and the up-regulation of corresponding endogenous pluripotency genes. Retroviral...disrupt the expression of tumour suppressor genes and/or oncogenes, potentially leading to the presentation... Shao, L., & Wu, W. (2010). Gene-delivery systems for iPS cell generation. Expert opinion on biological...a single polycistronic vector or multiple single-gene vectors can be used to package reprogramming factors... the transposase. The donor plasmid contains the gene(s) of interest flanked by terminal repeat sequences...cassette comprised of OCT4, SOX2, LIN28, NANOG, and a GFP reporter. Minicircles are removed from cells by dilution...demethylation and expression of endogenous pluripotency genes (i.e. Oct4) are other measures of reprogramming....

...understanding of B. subtilis gene functions and interactions. The single-gene deletion library is derived...studying the phenotype of an essential gene, the CRISPRi Essential gene Knockdown library was constructed ...Bacillus subtilis libraries with Addgene – the B. subtilis Single Gene Deletion Library—Kanamycin and the...compatible cloning sites for easy cloning of your genes of interest. In order to improve the study of specific...cassette. However, it was unknown whether variations in gene expression from these polycistronic cassettes could... stoichiometry impacted reprogramming and global gene expression patterns. Polycistronic cassettes that...-positive assay) as well as stabilization (Nanog-GFP reporter activation and silencing of factor-linked...

... this gene. Should you cut it at the beginning of the coding sequence to delete the entire gene? Perhaps... in vitro, and using it in mouse embryos to generate gene modified mouse lines. Hopefully this post will...they have been generated (more on that later). Find Validated gRNAs to Target Mouse Genes To begin the ...chances of gene modification in vivo. Find Plasmids for Testing Your gRNAs in vitro at Addgene CRISPR/Cas9...decide to do a gene modification experiment in mice to the day you have your F0 generation (barring life...Haoyi, et al. "One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated ...blogger Samantha Young. The use of CRISPR/Cas9 for gene editing has expanded since its adaptation for use...

...neurotransmission. But with approximately 950 different human genes encoding GPCRs (Takeda S et al., 2002) and many ...2002) Identification of G protein-coupled receptor genes from the human genome sequence. FEBS Letters 520... acceptor GFP2-Gγ chimeras, and two untagged Gβ constructs. For the Gγ constructs, the GFP2 tag is fused... Gα-Rluc8 and Gγ-GFP2 will be in close association, resulting in a high level of GFP2 emission. Following... exhaustive refinement, it was not possible to generate an optimal Rluc8-Gα construct for two of the 16...interest, a Gα-Rluc8 plasmid, and the appropriate Gγ-GFP2 and Gβ constructs. Cells are then treated with the...complex will disassociate, resulting in a decreased GFP2 emission. The activation state can be measured by...

...6 letter representation of the gene (or DNA sequence). pBACKBONE-hGene Tip: Often researchers will add...pBACKBONE-Flag-hGene(Q295A)-EGFP  Wildtype version: pBACKBONE-Flag-hGene-EGFP or pBACKBONE-Flag-hGene(wt)-EGFP...would list it first. pBACKBONE-Flag-hGene If there was also an EGFP fused to the C-terminal of your insert...would list it after the insert. pBACKBONE-Flag-hGene-EGFP Other considerations  If your insert contains...be included in the plasmid name. Mutations are generally listed as the amino acid change and not a nucleotide...

...magnetic responses: EGFP, mScarlet, and AsLOV2. After several rounds of semi-random mutagenesis and screening.../nn.4265.  Additional resources on addgene.org Find Optogenetics Plasmids Find Biosensor Plasmids Visit... al., 2024). They deposited their plasmid with Addgene, hoping to inspire others to explore the phenomenon...Although a wide range of fluorescent proteins and optogenetic tools exist, humans (like many organisms) are...magnetoresponsive fluorescent protein. Controlling an optogenetic or fluorescent tool with magnets would enable... molecules like proteins with magnetic fields generally appears to be weak — if they exist at all. Andrew...protein they tested — E. coli expressing good ol’ EGFP — showed a slight but detectable change in fluorescence...

...comparing gene activation when enhancers were active or silent, they could identify the target genes of previously...efficient. In general, epigenetic chromatin modifications are correlated with alterations in gene expression...updated Jun 24, 2020. Epigenetic modifications are an additional layer of control over gene expression that ...proteins that help package DNA, were associated with gene expression or silencing. To alter DNA modifications...oxidation and repair factors that allow for increased gene activation at the targeted site compared to other...the only CRISPR-based technology designed to alter gene expression. Fusing dCas9 to a transcriptional activator...a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nature Biotechnology...

...include: Optogenetics AAV Control AAV Biosensor AAV And more! Optogenetics Optogenetics continues ...Nuc-flox(mCherry)-EGFP 27056 AAVrg pAAV-Ef1a-DIO EYFP 50457 AAV2 pAAV-hSyn-DIO-EGFP   Biosensor AAV...pOTTC1032 - pAAV EF1a Nuc-flox(mCherry)-EGFP 50457 AAV2 pAAV-hSyn-DIO-EGFP Recombinases Plasmid Serotype...viral vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols...you pick the best AAV for your experiment. The Addgene viral service has now expanded to offer many of... Plasmid Serotype Name 51502 AAV5 pCAG-FLEX-EGFP-WPRE 114471 AAV1, AAV5 pAAV-Ef1a-fDIO mCherry ...pEF1a-DIO-FLPo-WPRE-hGHpA    Additional resources on the Addgene blog Read all of our viral vectors blog posts ...

...AAV9 Optogenetics Adesnik New viral vector pAAV-nEF-Con/Foff DREADD Gi-mCherry AAV8 Chemogenetics Deisseroth... pENN.AAV.EF1a.eGFP.WPRE.rBG AAV PHP.eB Controls Wilson New serotype pAAV-CaMKIIa-EGFP AAV PHP.eB Controls...latest in Addgene's viral vectors services and resources!   More resources on the Addgene blog Addgene's...What's new in Addgene's ready-to-use viral vectors repository? Quite a bit! In this post, we'll share...Table 1: New ready-to-use viral vectors in the Addgene repository  Name Serotype Category Depositor...Deisseroth New viral vector pAAV.CMV.PI.EGFP.WPRE.bGH  AAV PHP.eB Controls Wilson New viral vector ...the full viral services collection or check out Addgene's Data Hub to view viral vector reports from other...

...victoria, AvGFP forms a homodimer. In corals, the red fluorescent proteins form tetramers. In general, fluorescent... develops genetically encoded FRET based biosensors.     Additional Resources on the Addgene Blog Which...detect weak dimerization tendency, as is present in EGFP. Constantini et al, (2012) developed a cell-based... properties of new fusions can be compared with mEGFP or mTurquoise2, which are established true monomeric...Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes. You can follow him... Protein Biosensors Additional Resources on Addgene.org Find Empty Backbones with Fluorescent Tags Find...

...for doing gene targeting, reporter generation, controllable gene knockouts, and inducible gene inactivation...Alec Kimmelman targets mouse metabolic genes with ~6 gRNAs per gene.  CRISPR-Act3.0, developed by Yiping... Shirley Liu's lab targets all mouse genes with 10 gRNAs per gene. The guides are optimized to maximize... to develop CRISPR-based approaches for studying gene function in these systems. As a part of this effort...Yiping Qi's lab, allows simultaneous gene activation in plants.  FnCas12a, unlike other Cas12a nucleases identified...opportunities for investigating genetic mechanisms related to morphogenesis, cell fate specification, and...Lab have compiled a new toolkit for performing genetics studies in organoids. The Organoid EasyTag System...

...provide the Brunello (targeting human genes) and Brie (targeting mouse genes) pooled libraries from the labs...targeting EGFP, BRAF, and MAP3K4. The gRNAs targeting EGFP can be used as positive controls for gene editing...editing. You can also enhance expression of your gene of interest with a variety of Cas9 activators available...editing or gene activation, while the BRAF and MAP3K4 gRNAs allow you to target these widely studied signalling...pooled gRNA libraries that can be used to screen for genes involved in a wide variety of cellular processes...80,000 gRNAs to target around 20,000 individual genes. We provide enough lentivirus for you to both perform...and member of the Addgene Advisory Board) says: "When you're setting up to do a genetic screen, you need...