...Resources on the Addgene Blog Learn More About Fluorescent Protein Aggregation Catch Up On Your GFP Basics Get...have been around since 2000 when Terskikh et al. reported the production of dsRed E5. This timer predictably...expression time. 2. Timer functionality with a single gene Although it is possible to make a timer system composed...cargoes to FP timer mk-GO (Tsuboi et al.) Monitor gene expression in the developing pancreas by placing...FP timer DsRed-E5 under the control of Neurog3 (a gene controlling pancreatic differentiation) in mouse...(trafficking to a subcellular location, start of gene expression, development of a cell structure, etc...formation of limbs are correlated with changes in gene expression. In a developing embryo, for example,...

... a superfolder GFP fusion to stabilize the protein in mammalian cells. LwaCas13a-msfGFP can mediate cleavage... (Shmakov et al., 2015). They were using Cas1, a gene commonly associated with CRISPR arrays and involved...analysis, they identified 53 potential candidate genes that fell into 3 categories based on the architecture...of interest and an inactivated fluorescent RNA reporter. If the target sequence is present in the pool...activity of Cas13a becomes activated and the RNA reporter will be cleaved resulting in activation of the...expressing 5 gRNAs simultaneously produces comparable gene knockdown to single gRNA expression. Although the...plasmids from this paper at Addgene. Additional Resources on the Addgene Blog Check Out Our CRISPR Featured...

...destination vector contains GFP, enabling you to select cells with high GFP expression. These cells have...sequence between two target sites. Modifying multiple genes at once. Using multiplexed gRNAs can target multiple...plasmids, which contain various promoters and reporters, and subsequently inserted into a Cas9-containing...simultaneous multiplex editing of six different genes. This system doesn’t rely on any additional or host-dependent...-in-one CRISPR/Cas9 vector system.” Scientific Reports 4 (2014): 5400. PubMed PMID: 24954249. PubMed Central...from this paper at Addgene. 9. Yin, Linlin, et al. “Multiplex Conditional Mutagenesis Using Transgenic ...plasmids from this paper at Addgene. Additional Resources on the Addgene Blog Browse Our CRISPR Topic...

...phosphorylation, promoting transcription of a GFP reporter. Subsequent exposure to red light rapidly deactivates...plasmids from this publication at Addgene. Multichromatic control of gene expression in Escherichia coli...optimized by Tabor’s lab, making light-controlled gene expression in E. coli easier and more robust. Synthetic...used to engineer new pathways with sophisticated gene expression controls. Most cell types do not respond... can be coexpressed for sophisticated control of gene expression. An E. coli light-sensitive two-component... was only about 10-fold, precluding the study of gene expression at very high or very low levels. Schmidl...researchers to better fine-tune the level of output gene expression based on the input light intensity. In...

...into contact with than GFP. Similarly, if the viral vector carries an shRNA to a gene essential for cellular...looks like the genetic cargo in one plasmid (the transfer plasmid), the envelope (Env) gene on a separate...with those particles. Since the Env, Gag, and Pol genes are not encoded in the transfer plasmid, the component...familiarize yourself with your organization’s reporting mechanisms before you start an experiment, just...Resources Resources on Addgene.org Addgene viral services Addgene Biosafety Guide Addgene Viral Plasmids ...? That’s okay! In this article we will discuss general viral safety considerations and best practices ...of what is being integrated by the virus. If an oncogene overexpression construct is being delivered, that...

...excised by Cre recombinase. Addgene has several great options: pLV hUbC-dCas9-T2A-GFP for removable dCas9 expression...investigation into the regulation of your favorite gene. But have no fear - I’m here to introduce you to...sigh. The problem is that many of these studies report correlations without clearly testing whether a ...genome – could be affected, leading to unpredicted gene expression changes (Sapozhnikov and Szyf 2021, Charlton...interact with transcriptional machinery to modify local gene expression, confounding any potential conclusions...causal relationship between DNA demethylation and gene expression (Sapozhnikov and Szyf 2021).    ... expressing blasticidin or puromycin resistance genes.  It’s that simple! Good luck and happy editing...

...labeling. Size is also a factor. Bulkier tags, such as GFP, may interfere with protein expression or function...ligands for dopamine D2/D3 receptors. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-78827...fluorescence-based ligand-binding assay. Scientific Reports, 6. https://doi.org/10.1038/srep25769 Grimm, J....mitochondrial dynamics in mammalian cells. Biophysics Reports, 3(4–6), 64–72. https://doi.org/10.1007/s41048-...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...

...contain a toxin gene (regulated by a rhamnose-inducible promoter) and a chromoprotein gene. Allelic exchange...contrast, the widely used green fluorescent protein (GFP) gives a bright green fluorescence when exposed to...the plate. For example, if your plasmid contains a gene encoding a blue chromoprotein, after transforming...3: pTOX plasmids expressing amilCP and tsPurple genes. Image: Lazarus et al., 2019. One such lab is...improved expression in living cells. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-48604...protein family. What sets them apart is the way they generate their color: they strongly absorb visible light...difficult to directly use in bacteria, where most genetic manipulations start. Anthony Forster’s lab used...

...depending on its orientation to your gene. T7lac High levels of gene expression Promoter from T7 bacteriophage...modulating gene expression through varied inducer concentrations. trp High levels of gene expression...human or mouse) General expression mRNA Mammalian promoter from phosphoglycerate kinase gene.  Constitutive...deacetylation. Ubc General expression mRNA Mammalian promoter from the human ubiquitin C gene Constitutive  ... human beta actin General expression mRNA Mammalian promoter from beta actin gene Constitutive  Ubiquitous... Requires the presence of Gal4 gene to activate promoter. Ac5 General expression mRNA Strong insect ...Actin 5c gene Constitutive  Commonly used in expression systems for Drosophila. Polyhedrin General expression...

...the ccdB gene is replaced by the gene of interest. As a result, cells that retain the ccdB gene die off...tool. The plasmid pKSCTMF contains the mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR...The team used this cassette to knock out genes, knock in a GFP cassette, and introduced site-directed mutations...antisense RNA regulates hok gene expression indirectly through the overlapping mok gene." Journal of molecular...associated genes. These systems better ensure that plasmids are passed down through generations (Figure ...application of CcdB is GatewayTM cloning, where the ccdB gene serves as a counterselectable marker in the destination...they tested a lux-mediated bioluminescence reporter in vivo in the absence of selective pressure and found...

...potentially affect function [5]. We and others have reported that FP cysteine residues form inappropriate disulfide...negative bacteria periplasm. Efforts to tag endogenous genes for proteins in these compartments using CRISPR ...tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila...Lindsey M. Costantini, and Erik L. Snapp. "Superfolder GFP is fluorescent in oxidizing environments when targeted...quantitative imaging experiments and cell physiology in general. Pitfalls of Fluorescent Protein Fusions Observing...cellular compartments other than the cytoplasm. Generally, FPs evolved in or were engineered for use in ...N-glycosylation consensus sequences in popular FPs. This generated a palette of inert green, cyan, yellow, and blue...

...an unpublished set of Zika gene plasmids to Addgene’s repository. The genes, from the Zika virus Asian...vectors targeting their genes of interest to generate mAID fusions to endogenous genes. Cell lines with the...targeting a total of 20,121 genes within the human genome (the identity of the genes and target sequences can...expression level for their gene of interest. Although the parts involved in altering gene expression are relatively...promoters and 140 genes, including constructs with unique tissue-specific promoters expressing GFP, tetracycline...contains ~90,000 gRNAs that target ~15,000 genes with 6 gRNAs per gene. The second is a "supplementary" library...library to generate a population of HEK293FT cells expressing a single gRNA targeting a single gene. The gRNA...

...about the Dangers of Using GFP for Protein Localization Resources on Addgene.org Find Plasmids for Mammalian...been known about since 1966 when Stanley Gartler reported that most of the 18 cell lines he had studied ...pharmacology (many of which are represented in Addgene's Special Collections). Arguably, oncology has benefited...examining whether the cell lines had isoenzymes and genetic polymorphisms specific to their origins. This technique...out by the commercial repository. Repositories generally ensure cell line identity and purity making it...to the original as possible and so any results generated are reproducible and reliable. When you order ...Abcam Cell Culture Guidelines Resources on the Addgene Blog Learn All about Mammalian Vectors Read up...

...phosphatase-positive assay) as well as stabilization (Nanog-GFP reporter activation and silencing of factor-linked mCherry...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 ... in this work are available at Addgene. Kim, et al. Stem Cell Reports. 2015. PubMed PMID: 25772473 ...elements and combined it with a variety of reporters (i.e. eGFP) and effectors (i.e. DREADDs). The team ...Bacillus subtilis libraries with Addgene – the B. subtilis Single Gene Deletion Library—Kanamycin and the...their useful reagents shared via Addgene. Narayan S, et al. Cell Reports. 2017. PubMed PMID: 28813671 ...

...ACE) in real-time. The reporter constitutively expresses eGFP to indicate reporter abundance, and mCherry...quantifying multiple gene editing activities. Click here to find the ACE reporter plasmid! Martin et al...and N177F mutations). Its excitation maximum is reported as 603 nm, while the emission maximum is 675 nm... ACE system: A versatile in vivo fluorescent reporter for rapid quantification of base-editing and Cas9...editing tools, the Reuben Harris lab has deposited a reporter plasmid that allows for rapid fluorescence-based... out a frame-shift mutation within the mCherry reporter. Quantification of editing efficiencies simply...containing rat APOBEC1. Finally, they show that the ACE reporter can also quantify Cas9 cleavage, thus exhibiting...

...comprised of OCT4, SOX2, LIN28, NANOG, and a GFP reporter. Minicircles are removed from cells by dilution...started the field of iPSCs Find gene-specific plasmids (including reporters and RNAi) for:  NANOG  OCT4 ...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...high is based on the reprogramming efficiencies reported in González et al. A percentage is not listed ...a single polycistronic vector or multiple single-gene vectors can be used to package reprogramming factors...

...aptamers able to bind to a small molecule derivative of GFP called 3,5-difluoro-4-hydroxybenzylidene imidazolinone...growth as RNA biology, we are looking forward to reports on the next additions to aptamer soup! References...If you’re been following Addgene on #souptwitter or if you just enjoy a warm meal on a winter day, you...molecule into the cell. The fluorescence that is generated allows for qualitative and quantitative measurement...placing the aptamer downstream of a known promoter. Addgene’s Plasmids 101: Aptamer Fluorophores describes the... we’ll take a deeper dive into the content of Addgene’s aptamer soup by exploring the development and ...and applications of fluorescent RNA aptamers in Addgene’s collection. Green fluorescent aptamers Spinach...

...along with a reporter, such as a fluorescence gene or a resistance gene. These reporters allow you to easily...you could replace one of the reporter genes above with your gene or genes of interest. Some of the plasmids...the reporter gene). Addgene's collection offers a variety of plasmids to express two or more genes, some... that are expressing your gene, especially if you are studying a novel gene, is not always a straightforward... your gene of interest, scientists have instead developed novel methods to co-express your gene along ...express more than two genes at a time using IRES elements. Further, scientists have reported lower expression...multicistronic element and reporter cloned in. In these plasmids you would simply clone your gene of interest into...

...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...ten colors (see table to the right); in various reports, Sanes and Lichtman have observed from 90-160 distinct...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 ...

...Figure 1). All viral genes are removed and replaced by a single (non-viral) reporter gene (e.g. firefly luciferase...transcribe the reporter gene (e.g. firefly luciferase). The polymerase recognizes the gene start signal ...transcription of the reporter gene. The polymerase will elongate the mRNA until it reaches the gene end signal,...luciferase or eGFP) flanked by the EBOV-specific gene start and gene end sequences. Thus, the minigenome...replication and viral gene transcription (6). The EBOV genome contains seven viral genes, each flanked by ...sequence (gene start) and a transcription termination/polyadenylation signal sequence (gene end). A minigenome...necessary to direct minigenome replication and reporter gene transcription by the EBOV polymerase (reviewed...

...protein, permeabilized cells  Cy5NTAc 32 25-45 GFP (fluorescein is similar)   Lumio Red Tc: CCPGCC 48...protein kinase dynamics in living cells with FRET reporters." Methods 40.3 (2006): 279-286. PubMed PMID: 16908183...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...

...other labs have reproduced their EGFP findings since the initial report in 2023.)     Figure 1:...Andrew and Maria were encouraged by previous reports that described magnetic responses of cryptochrome ...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...

...a gRNA designed to target a mutated gene. In a Scientific Reports paper introducing the delivery method...been used for many years for in vivo delivery and gene therapy and represent one of the most studied viruses...CRISPR-Cas. Figure 1: Schematic of targeted gene knock-out (left) and targeted DNA knock-in (right...binds to target DNA. In the final step of targeted gene knock-out (left), a fragment is deleted from two... This capacity is large enough to carry the Cas9 gene and a gRNA expression cassette in one single viral...Joining (NHEJ) DNA repair mechanism to knock out genes as a result of an insertion, a deletion or a frameshift...method in 2014, Manuel Goncalves lab report that AdV-mediated transduction of gRNA:Cas9 ribonucleoprotein...

...services collection or check out Addgene's Data Hub to see viral vector reports from other users. And don't...promoter. Use this for doxycycline inducible (tet-off) gene expression! We've also added AAV1 and AAV5 preparations...variants of a new optogenetic tool called PdCO, deposited by the Yizhar lab. Addgene is now offering AAV1... pAAV_hSyn-SIO-stChrimsonR-EGFP-P2A-PdCO-miniWPRE AAV1, AAV5 Optogenetics Yizhar New viral service with...AAV1, AAV5 Optogenetics Yizhar New viral service with multiple serotypes pAAV_hSyn-PdCO-EGFP-WPRE AAV1... Brian O'Neill.  More resources on the Addgene blog Addgene's Data Hub What is a Virus (and a Viral Vector...What's new in Addgene's ready-to-use viral vectors repository? Quite a bit! In this post, we'll share...

...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... specific detection of human TRPA1. Scientific Reports, 9(1), 18500. https://doi.org/10.1038/s41598-019...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...

...services collection or check out Addgene's Data Hub to view viral vector reports from other users. And don't...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 ...

...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...

...research. But be aware that the fluorescent proteins reported in publications are only characterized under a...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...

...of posts explains how to critically assess the reported properties of fluorescent proteins, how to do ...observed in mammalian cells is to tag an endogenous gene. By imaging cells (or tissues) that produce an endogenous...fluorescent proteins or using endogenous tagged genes) can be used to verify performance under different... develops genetically encoded FRET based biosensors.     Additional Resources on the Addgene Blog Which... QY (EC*QY, sometimes normalized to the value of EGFP). The higher the number, the higher the theoretical...mVenus as a reference protein is available from Addgene. Another way to dodge cellular variation observed...Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes. You can follow him...

... 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...

... Figure 1: In conventional gene targeting (A), you must first generate your desired mutation in mouse...References Yang, Hui, et al. "One-step generation of mice carrying reporter and conditional alleles by CRISPR...mutations, the 8-10 month process of conventional gene targeting is lowered to about 2-3 months. But certain...example of inserting a 2A-FlpO cassette into the Fgf8 gene. Miura et al. (2018) suggest using a 50-80 bp sequence... mouse model generation easi-er! Generating mice before CRISPR The process of generating knock-in mice...process of mouse model generation simpler, as described in an earlier post on generating CRISPR mouse models...after editing. How to use Addgene plasmids for Easi-CRISPR You can use Addgene plasmids to create a repair...

...in neurons. (b) Genetic cargo can be customized to control transgene expression. Gene regulatory elements... relatively even distribution of target gene expression (EGFP, in the depositing manuscript) across the....     Figure 1: AAV toolkit for targeted gene delivery to the brain. The AAV (a) capsid, (b) cargo...Abbreviations: AAV, adeno-associated virus; GRE, gene regulatory element; NHP, non-human primate. ...Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nature Protocols;14, 379–414...2017). Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems...Gradinaru, V. (2022). Engineered AAVs for non-invasive gene delivery to rodent and non-human primate nervous...