...NIR FPs can be used similar to GFP-like probes by supplying a single gene to cells. Still in bacteria, ..., which do not produce BV, an additional gene coding for heme oxygenase should be supplied (Baloban et...crosstalk-free three-color imaging with green and red GFP-like FPs (Shcherbakova et al., 2016), as well as ...applications of fluorescent proteins, genetically encoded biosensors, and optogenetic tools. Follow her recent research...-the-art probes, many of which you can find at Addgene. Biliverdin chromophore Near-infrared fluorescent...FPs can be combined with blue-light controlled optogenetics, such as channelrhodopsins and tools based on...reported widely used dimeric iRFPs (Table 1). In general, the monomeric state of a probe leads to less interference...

...ideal for large gene transfer. The M24 promoter provides constitutive, high-level gene expression and ...further customizing gene expression. Sahoo et al. have tested the system with genes up to 4 kb in length...versatile gene expression vector for transient assays as well as stable expression of foreign genes in plants...Scientists have worked to optimize this system for gene transfer, studying the stability of modified Ti plasmids...versatile binary Ti vector for both transient and stable gene expression applications in plants. This smaller,...and many plant scientists want to transfer large genes, smaller binary vectors with a larger cloning capacity.... They also replaced the promoter regulating the gene of interest with a constitutive, robust M24 promoter...

...proteins (FPs) to a yeast gene of interest. These fluorescently tagged genes are expressed under native...sequence and a selectable gene product for isolation of successful transformants. Addgene distributes several...be interested in attaching epitope tags to their genes of interest, allowing for easy capture and detection...modifications in S. Pombe, including full and partial gene deletion, overexpression (by promoter substitution...either the N- or C-terminus (3xHA, 13xMyc, GST, or GFP). Longtine et al.(3) describe a complimentary set...few. Have you used these tools in your own lab? Addgene would love to hear from you, our community, about...

...biosensors To focus on a particular gene or protein, check out our gene-specific biosensors. These constructs...biosensors as an example. Many calcium biosensors include GFP fused to calmodulin (CaM) and the chicken myosin ...Visualization Resources on Addgene.org Find Biosensor Plasmids Find Optogenetics Plasmids Visit the Fluorescent... to the molecule it detects. This change then generates a reporter signal. Reporter signals may be electrochemical... OR enzyme activity. Small molecule biosensors Addgene has many sensors that measure the concentration..., and voltage across membranes. These sensors generally follow the same pattern described above - a sensing...fluorescent molecule   Additional Resources on the Addgene Blog Check out the Rosella Autophagy Sensor Read...

...attach the gene of interest to either the N-or the C-terminus. Even if a researcher’s gene of interest...from Joachim Goedhart: Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection.  ...researchers as we could, and Addgene is a wonderful vehicle for us to do that.” At Addgene, we are equally excited...is available in an easily searchable format on Addgene’s Michael Davidson Fluorescent Protein Collection...collection with over 300 backbones alone,” said Addgene’s Lianna Swanson, who has been working with members...sun, from the standard oldies but goodies (e.g., EGFP and YFP) to the new and improved fruit colors (e.g...the scientific community," says Joanne Kamens, Addgene's Executive Director, "and to promote the great ...

...cells - one containing the gene of interest (pTet-IRES-EGFP, pPRIME-Tet-GFP-FF3) and one with either tTA...the env gene containing the rev response element (RRE), an internal promoter, and the desired gene (transfer...-1 genome containing all viral genes with the exception of the env gene (packaging plasmid), and 3) the...used to express your gene of interest either constitutively or inducibly. If your gene of interest is toxic...expression of your gene. The addition of Dox in the culture will then repress your gene of interest. This... Youichi Suzuki (2011). Gene Regulatable Lentiviral Vector System, Viral Gene Therapy, Dr. Ke Xu (Ed.)...harmful genes into a transfer vector. Lentiviral particles expressing proto-oncogenes or oncogenes for instance...

...blot showing GFP expression in cells that have been transfected with a plasmid encoding GFP. Actin, a common...which come from naturally-occurring immunoglobulin genes, recombinant antibodies are plasmid-based. Their... What is an Antibody? Resources on Addgene.org: Browse Addgene’s Antibody Plasmid Collection Find antibodies...cascades that clear these infections. Anything that generates an antibody response in the immune system is referred...in the DNA of the hybridoma can occur, known as genetic drift, meaning that over time, the antibody you...monoclonal antibodies, recombinant antibodies are a homogeneous population. However, unlike monoclonal antibodies...antigen binding, the variable regions alone can be generated as a fusion protein. An ScFv is made up of the...

...both introduce genes into an organism’s genome (see figure) and to disrupt endogenous genes at the site ...first-generation transposase (as determined by transposition assays of antibiotic-resistance genes conducted...). In their Nature Genetics paper, the lab showed that efficiencies of stable gene transfer using SB100x... the rapid generation of stable transgenic cell lines which can robustly express your gene-of-interest... exciting tool for producing specific genetic mutations and gene disruptions without the use of viral ...transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009 Jun;41(6):753-61. Epub 2009... et al. A new approach to gene therapy using Sleeping Beauty to genetically modify clinical-grade T cells...

...read-through transcripts of the effector gene. Effector genes, any gene that you want to control the expression...chemical inducers of gene expression (del Valle Rodríguez et al., 2011).  Genetics of SPARC Figure...Learn more about controlling gene expression in flies Resources on Addgene.org Find fluorescent protein...truncated attP sites to control recombination of the gene construct. A predictable number of cells will express...binds to the UAS binding motif, driving downstream gene expression. attP and attB, recombination sites. ...side of a stop cassette, upstream of an effector gene. In cells where the stop cassette is removed during...expression of either a stop cassette or an effector gene. HDVR (2X Hepatitis Delta Virus Ribozyme), a self-cleaving...

...typically include a gene that is advantageous for survival, such as an antibiotic resistance gene. In contrast...the target gene. Vital component for expression vectors: determines which cell types the gene is expressed...scientists use plasmids? Generally, scientists use plasmids to manipulate gene expression in target cells...replication machinery proteins Antibiotic Resistance Gene Allows for selection of plasmid-containing bacteria...MCS is often downstream from a promoter. Insert Gene, promoter or other DNA fragment cloned into the ...obtained. Selectable Marker The antibiotic resistance gene allows for selection in bacteria. However, many ...referred to as “vectors” or “constructs.” To insert a gene of interest into a vector, scientists may utilize...

...CRISPR-Cas9 constructs from Addgene to gene-edit mutations into XP genes and other genes involved in photosensitivity...researchers identified the gene responsible for the disease as ATP1A3. The gene encodes a sodium-potassium...Syndrome, a devastating genetic disease caused by mutations in the OCRL1 gene that leads to the early ...studies on OCRL1 already underway in his lab. The gene appears to play a role in many cell processes involving... learned there was a disease associated with the gene, it piqued their interest. They obtained cells from... University with David Goldstein’s lab, will use GFP-ATP1A3, FLAG-ATP1A3, and ATP1B3 plasmids to explore...McSweeney says they now think that mutations in the gene may prevent neurons from returning to basal conditions...

...this plasmid with your gene of interest, you would replace mCherry with your gene of interest. pBLADEONLY...already have a pBAD33-based plasmid expressing your gene of interest, all you have to do is co-transform ...co-transform pBLADEONLY. That way, a previously cloned gene of interest, under the pBAD promoter, can be controlled...strain transformed with pBLADE expressing superfolder GFP instead of mCherry under the PBAD promoter. They ... Resources on Addgene.org Check out the Molecular Biology Reference Download Addgene's Plasmids 101 eBook...simply more precise. “We show that you have less heterogeneity in the population and you can do all of these...permission.   BLADE is just one of the several optogenetic tools that Di Ventura’s lab and Khammash's lab...

...reporter like GFP, some smaller CRISPR-Cas proteins such as SaCas9, CRISPR gRNAs, or your gene of interest...through this challenging sequence. Transgene The transgene is the gene that will be delivered by the AAV...expression of the AAV transgene. On a plasmid map, promoters are upstream or 5’ of the gene they control. Promoters...plasmid which contains the structural and packaging genes, the adenoviral helper plasmid which contains the...for the successful and specific expression of your gene of interest. References Haery L, Deverman BE, ...Terminal Repeats of Adeno-Associated Virus. Human Gene Therapy Methods 30:206–213 . https://doi.org/10.1089...control transgene expression. Lox sites are directional 34 bp sequences that can flank the transgene and ...

...repression of your target gene, activation and repression of different genes in a single cell, and localization...Favorite Fluorescent Protein Oligomerizes in Cells GFP oligomerization can lead to misleading artefacts ...replication and transcription as well as reporter genes. To learn how you can use minigenomes in your research... them in action. AAV: A Versatile Viral Tool for Gene Expression in Mammals Adeno-associated viruses (... (AAVs) are commonly used to deliver a genes of interest to a variety of cell types in-vivo. These are...localization of a fluorescent signal to your target gene. Read this post to find out more about how the Casilio...are widely used by the optogenetics and chemogenetics communities to activate neurons and neuronal pathways...

...applications used to study gene function. From making transgenic animals, gene targeting and genome modifications...These vectors allow you to test the function of Your Gene Of Interest (YGOI) in a controlled environment under...design. You can create a new strain with a disrupted gene by transforming wild-type yeast with an integrating...using a reporter vector to tag YGOI with lacZ and/or GFP (e.g. pPD80_08) and following the expression pattern...modifications to reporter vectors (e.g. pBV-Luc) and gene silencing (e.g. pLKO.1 - TRC cloning vector). Check...visualize YGOI in vivo (try pcDNA3-EGFP). If you can get away with generating transiently transfected cells... More Plasmid Resources from Addgene: Plasmids 101 blog series Addgene's Molecular Cloning Guide & Other...

...Versatile Tool for Gene Expression in Mammals Additional Resources on Addgene.org Overview of Viral ...pooled libraries. Whether you’d like to knockout a gene, replace it, modulate its expression, or perform...also have lentivirus for delivering control shRNAs, GFP, and rtTA from the Tet-on system. The first two can...while rtTA can be used to activate expression of a gene under the control of the tet operator. AAV AAVs ...associated virus (AAV, primarily chemogenetics tools for now but with optogenetic tools coming soon). The viral...excited that Addgene is now offering virion preparations to the scientific community. Addgene's AAV preparations...posts on the blog and pages on the Addgene website. blog.addgene.org The blog provides tips for working...

...in applying gene technology to bacteria and plants. Her research involves cloning cry genes from the soil...using a bacterial expression system such as the pET GFP LIC cloning vector and the pOKD plasmids. The recombinant... shared mission of both Seeding Labs and Addgene. The Addgene-Seeding Labs plasmid grant is a collaborative...of the Addgene-Seeding Labs Plasmid Grant awardees' projects progress. Subscribe to the Addgene blog for.... Additional Resources on the Addgene Blog Learn More About the Addgene-Seeding Labs Plasmid Grant Check...introduce the 5 scientists who were awarded the Addgene-Seeding Labs Plasmid grant this year. These scientists...plant biology, she coordinates the Network for the Genetic Improvement of Cowpea, a knowledge database for...

...experiment. PITChing MMEJ as an Alternative Route for Gene Editing Michohomology-mediated end joining (MMEJ...PubMed Central PMCID: PMC5495063 Kan et al. use a GFP-BFP conversion system to assess precision genome ...template to increase efficiency of insertion and gene replacement. Care to share your favorite CRISPR ...Blugene and I represented Addgene at the recent Keystone meeting on Precision Genome Editing with Programmable...hopes to evaluate genome editing pipelines and generate a common lexicon for genome editing studies. These...desired edit, and I want to make sure that I (and Addgene) don’t forget this in the future. Here’s a list... to keep current with the latest and greatest! Addgene resources 3 Tips to Improve HDR Efficiency for ...

...endocytosis of a fluorescent dye and transduction of GFP-expressing AAV particles we did not observe any difference...during adeno-associated virus manufacturing." Human gene therapy21.10 (2010): 1251-1257. PMID: 20649475. ...have probably heard about Addgene’s viral service but did you know that Addgene also performs independent...101 eBook Additional resources on Addgene.org Learn about Addgene's viral service Learn about viral production...research? Since launching the viral service in 2016 Addgene’s research team has been actively investigating ...PMCID: PMC2957237. Additional resources of the Addgene blog Read our beginner's guide to viral vectors...

... (FLAG, Myc, etc.) and fluorescent protein tags (GFP, mCherry, etc.). Fluorescent tags are primarily used...Solubility Enhancer. In P. E. Vaillancourt, E. coli Gene Expression Protocols (Vol. 205, pp. 99–118). Humana...-59259-301-1:99 Resources on Addgene.org Addgene antibodies Addgene’s antibody guide Antibody protocols...having solubility issues!). Need a GST antibody? Addgene has you covered! His His tags, often called polyhistidine...Purification of His fusion proteins from E. coli is generally more straightforward and of a higher purity than...and other qualities of each tag class, they are generally best suited for their primary application. Ready...protocols  Resources on the Addgene blog Plasmids 101: Protein tags Antibodies 101: Epitope tags To Each...

...multiple) rAAV-CAG::FLEX-rev:: hM4D-2a-GFP AAV1 Chemogenetics Scott Sternson New viral prep pAAV-CaMKIIa-hM3D...mCherry AAV1 Chemogenetics Bryan Roth New serotype pAAV-CaMKIIa-hM4D(Gi)-mCherry AAV1 Chemogenetics Bryan ...mCherry AAV1 Chemogenetics Bryan Roth New serotype pAAV-hSyn-hM4D(Gi)-mCherry AAV1 Chemogenetics Bryan Roth...AAV-PHPeB Optogenetics Jonathan Ting New viral prep AiP14033: pAAV-AiE0779m_3xC2-minBG-CoChR-EGFP-WPRE3-...AAV-PHPeB Optogenetics Jonathan Ting New viral prep AiP14035: pAAV-AiE0452h_3xC2-minBG-CoChR-EGFP-WPRE3-...pAAV-AiE0873m_3xC2-minBG-CoChR-EGFP-WPRE3-BGHpA (Alias: CN5140) AAV-PHPeB Optogenetics Jonathan Ting New viral... Brian O'Neill.  More resources on the Addgene blog Addgene's Data Hub What is a Virus (and a Viral Vector...

...highlighted back in 2015, where sdAbs were used to create GFP scaffolds for transcriptional activation.  sdAbs ...from addgene.org  Plasmids encoding nanobodies at Addgene Sybody Generation Toolbox from Addgene     ...only antibodies. (A) Overview of genetic modifications made to generate the nanomouse. (VHH = variable ...The UN General Assembly has declared 2024 the International Year of Camelids. The declaration is intended...to human populations around the world. Here at Addgene, we love camelids too, though not just for the ...chromobodies,” which are single-domain antibodies genetically fused to fluorescent proteins and used to visualize... synthetic single-domain antibody libraries to generate new sdAbs. There has also been some interesting...

... Fig. 3: Image of Addgene’s western blot for the myc-tagged protein GFP-smFP-myc expressed from ...C-terminal region of the human proto-oncogene c-Myc. Since the generation of the first anti-c-Myc [9E10] (...high-throughput screening platform to apply saturating mutagenesis on 21 positions in the protein sequence of the...approach and outcomes of the screen for improved genetically-encoded voltage indicators (GEVIs) based on the...kinetics. They demonstrate the use of HcKCR1 as an optogenetic silencer in experiments with mouse cortical neurons... HcKCR2, making it more suitable for in vivo optogenetic use.   Fig. 2: A) HcKCR1 is highly selective...control K+ gradients and can be used as a potent optogenetic silencer. The current work also proposes promising...

...contains circularly permuted green fluorescent protein (GFP), and nLightR uses a red fluorescent protein, mApple...Imaging Tools Resources on Addgene.org Find Biosensor Plasmids Find Optogenetics Plasmids Visit the Fluorescent... visualize their activity. In 2019, the first generation of norepinephrine sensors was developed, named...norepinephrine (Feng et al., 2019). But this first generation of tools had limited sensitivity and speed and... models of nLightG (left) and nLightR (right) generated using ColabFold (Mirdita et al., 2022). B, Representative...Speed One of the major advantages of this next generation of norepinephrine sensors is their speed. nLightG...GRABNE were more sensitive than nLightR, both generating a measurable signal after electrical stimulation...

...targeting a gene associated with diphtheria toxin lethality and were sorted into single wells to generate clonal...instance, it’s much more difficult to introduce a gene of interest into primary cells, so most primary ...Neuronal stem cells transduced with iTOP resulting in GFP expression. Image courtesy of Diego D'Astolfo. ... risk of viral integration causing mutations and gene expression changes elsewhere in the genome. Second...j.cell.2015.03.028. PubMed. Find the Plasmids @Addgene: iTOP-tested proteins and reporters Previous...

...collection is now complete! pAAV-CAG-GFP (plasmid 37825) and pAAV-hSyn-EGFP (plasmid 50465) are now available...include: EGFP-expressing AAV for serotype testing Calcium sensors and GABA sensors Optogenetics Recombinases... pAAV.hSynap.iGABASnFR (112159-AAV1) Optogenetics Optogenetics continues to be a useful method for manipulating...viral vectors 101 eBook Resources on Addgene.org Learn about Addgene's viral service Find viral protocols... testing AAV, which are small (20 ul) samples of EGFP-expressing AAV packaged in various serotypes. This...recently released these controls: pAAV-hSyn-DIO-EGFP (50457-AAV5): The Synapsin promoter directs broad...broad, neuronal expression. AAV pCAG-FLEX-EGFP-WPRE (51502-AAV5): The CAG promoter directs ubiquitous expression...

...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...simultaneous multiplex editing of six different genes. This system doesn’t rely on any additional or host-dependent...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...add multiple gRNAs on one plasmid. Sound good? Addgene has many tools to help you multiplex - we’ll use...multiplex gRNAs, some being: Using dual nickases to generate a knockout or edit. This can help reduce off-target...

...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...via phosphorylation, promoting transcription of a GFP reporter. Subsequent exposure to red light rapidly... 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...

...test for your gene of interest may be another method to assess transfection success. GFP is often used...lines and are not used for general plasmid selection. Instead, reporter genes are often employed to easily...conjunction with positive selection to ensure your gene gets targeted to a specific location within the ...used selection markers are listed below: Name Gene Conferring Resistance Cell Types* Mode of Action...use G418. This can be confusing since the neo/kan gene confers G418 resistance; however, like gentamicin...Read More: Plasmids 101: Antibiotic Resistance Genes Plasmids 101: Origin of Replication All Plasmids...the genetic traits of the foreign material and will subsequently pass it on to future generations. Stably...

... for gene expression, turning transcription on and off by binding proximal to their target genes. Since...Since gene expression patterns determine everything from stem cell differentiation fate to tumor suppression...library comes either with or without mCherry and GFP fluorescent controls. Alternatively, you can order...from the library to further investigate specific genes and TF isoforms. Due to the size of the collection...use to generate the relevant disease model. Accurate modeling of a disease depends on both genetic and expression... not yet available, just ask an Addgene scientist at help@addgene.org! References and Resources  References...scientific community. Recently, the Zhang lab generated a TF library (aka the MORF Collection) and an ...

...that control cell cycle gene expression.  Additional Resources on the Addgene Blog Visit the fluorescent...page Get the basics on GFP Find microbiology blog posts Resources on Addgene.org Find fluorescent protein...work in Lin Tian’s lab at UC Davis is to generate a genetically encoded fluorescent sensor for serotonin...sequence an individual’s genome. But when they find genetic variants, what does that mean? Previous research...of Washington sought to address the effects of genetic variants in a high throughput manner. She helped...variant library is monitored by fusing the variants to eGFP followed by an internal ribosomal entry site and...a variant has wild-type levels of abundance, the eGFP will be stable and fluoresce. But if the variant...

... also includes a GFP construct as a control. For easier portability, all viral genes are flanked by EcoRI...coronavirus or COVID-19 research, please email Addgene at help@addgene.org with your order number and so it can .... Sign up for email alerts on the Krogan lab's Addgene page to get notified!) Attacking coronavirus, one...reasons, the QCRG is delighted to be partnering with Addgene, who will be taking over the distribution to expand... coronavirus related plasmids and resources at Addgene* Many thanks to our guest bloggers Manon Eckhardt...

...developed four acoustic reporter gene (ARG) plasmids to better monitor gene expression in vivo using ultrasound...non-immunogenic violet-excited GFP variant Vex. To understand the impact of this gene editing tool on the immune...plasmids at Addgene. Liu Q et al., Neuron. 2019. PubMed PMID: 30704911. Acoustic reporter genes for noninvasive...by Lukas Morgan  Listen to the acoustic reporter gene podcast segment In recent years, scientists are ...through a host organism, the expression of some genes can change depending on the location. Current methods...of microbes rely heavily on fluorescent protein genes. These optical reporters have limited deep-tissue...ultrasound. These ARG plasmids contain genes for “gas vesicles,” a gas-filled protein structure that water-bound...

...They could also switch from FLAG to GFP or a different epitope. Addgene: Do you have any additional tips,...homology arm as a gBlocks® Gene Fragment from Integrated DNA technologies. Generally speaking, anyone using... an effort to track down every last one of those gene regulatory proteins and the places in the genome...together on several projects and this is one related to gene expression. One goal of the ENCODE project is to...with detailed protocols, go to www.addgene.org/crispr/tagging. Addgene: What motivated you to adapt CRISPR...tagging system, which they’ve now made available at Addgene. For more details on the plasmids that are currently...can now look at many more transcription factors. Addgene: How does the system work? Mendenhall: The system...

...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...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... and the same holds true for types of viruses generated in the lab. The four most common types of viruses...

...ones? Many of the available biosensors use a single GFP or CFP/YFP-derived FRET pair. If you need to free...Resources on Addgene.org Addgene’s AAV Guide Fluorescent Proteins: Biosensors Addgene’s Viral Services...calcium sensors. Selecting a calcium sensor Genetically encoded calcium indicators (GECIs) are fluorescent...neuronal cell body or the axon (GCaMP6 only). The genetic elements required for Drosophila studies have been...chemical biology. 2021 May;17(5):509-18. Inoue M. Genetically encoded calcium indicators to probe complex brain...Aug 1;169:2-8. Suzuki J, Kanemaru K, Iino M. Genetically encoded fluorescent indicators for organellar...SA, Looger LL. Imaging neuronal activity with genetically encoded calcium indicators. Cold Spring Harbor...

...line, Hep3B. From left to right: Mitotracker Red, GFP for the biosensor, Hoechst, and overlay).   ...(DKFZ) who has developed new redox sensitive GFP2 (roGFP2)-based biosensors for live cells. His work focuses..., Pedre Pérez’s biosensor fuses MPST to roGFP2. Because roGFP2 contains two cysteines near the chromophore...developed a dual virus system using both optogenetics and chemogenetics to study the activity of parvalbumin...in the lab. He has recently developed a third-generation intensiometric glutamate sensing fluorescent ...catalyzes the desulfuration of 3-mercaptopyruvate and generates an enzyme-bound hydropersulfide that is then transferred...the MPST hydrosulfide can be transferred to the roGFP2 cysteine thiols and change its fluorescence properties...

...Cre-lox Control Protein Activity with GFP Resources at Addgene.org Find KTR reporter plasmids Find photoactivatable...spatiotemporal resolution than pharmacological or genetic approaches, but only a few such methods exist and...Solution: In Zhou et al, the Lin lab present a generic design for creating photoswitchable kinases and...possible to create other photoswitchable kinases? The general design of psMEK1 also worked for creating three...PMCID: PMC4097317. Additional Resources on the Addgene Blog Learn More about Photoactivatable Fluorescent...

...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...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...Vogelstein B (1983) Hypomethylation distinguishes genes of some human cancers from their normal counterparts...

... enriched relative to controls using Split-BioID-GFP and BioID run on six unrelated proteins. One caveat...study binary protein interactions. However, FRET generally only tells you if two proteins interact and cannot...with your research? Plasmids are available from Addgene! References Schopp, I.M., Ramirez, C.C., Debeljak...Central PMCID: 5053326. Additional Resources on the Addgene Blog Learn about techniques for visualizing protein...-DNA Interactions with CUT&RUN Resources at Addgene.org Find plasmids from the Bethune Lab Find Plasmids...

...-invasive systemic gene delivery in non-human primate (NHP) brains.  Systemic gene delivery capsids previously...Monomeric StayGold variants AAV.CAP-Mac for systemic gene transfer in primate brains Endothelial-specific ..., Myc and SV40 Nuclear Localization Signals, and GFP. B) Workflow to assess Cas9-PAGE editing with viral...Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain....plasmid here! Chen, X., et al. (2023). Functional gene delivery to and across brain vasculature of systemic...past decade, primary cells remain difficult to genetically edit due to toxicity from electroporation or ... under CC-BY license.   The Miyawaki lab generated a series of StayGold mutants and identified a ...

...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...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...eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, meffBlue, aeBlue, amilCP, tsPurple...

...under-expressing clones. If GFP-encoding cDNA is co-transfected at a favorable ratio to the gene of interest (or,...overexpress genes of interest; more recently we have also been using Addgene CRISPR vectors to generate cell ...cell lines with knockouts of specific genes. Many years ago, people in the laboratory became frustrated with...with different levels of expression of our gene of interest. Advantages of the agarose cloning method ...figure 1.   Find Additional Useful Protocols on Addgene's Protocol Pages Agarose cloning method procedure...-well cell plate to a 96-well assay plate. We generally freeze at least 2 ampules each of 4 clones with...som.umaryland.edu.   Additional Resources on the Addgene Blog Tips for a 1st Time CRISPR User (by a 1st...

...Vectors Read about Reporter Gene like Luciferase and GFP Resources on Addgene.org Find Plasmids for Your...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 ...

...sequence (UAS) of its target gene, in this case a reporter gene (e.g. luciferase or GFP), regardless of whether...promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection...reporter gene (3, 4), there are now many variations, including many that use auxotrophic rescue genes as reporters...Shapira S.K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in Escherichia coli and ...hybrid system (also called the interaction trap), genes encoding proteins of interest(POI’s) are fused to... in a yeast strain that also contains a reporter gene cloned downstream of a promoter containing the target... the “bait”, Figure 2b) cannot activate reporter gene expression on its own, but, if the two POI’s interact...

...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...toxin-antitoxin system to construct a stable synthetic gene circuit that can be used over prolonged periods....

...Express a gene of interest fused to the C-terminus of SCFP3A Amber N1 Yellow Mammalian Express a gene of interest...Express a gene of interest fused to the C-terminus of Amber mVenus N1 Yellow Mammalian Express a gene of interest...Clover (a GFP variant) commonly used with mRuby2 pLSSmOrange-N1 Orange Mammalian Express a gene of interest...your choice of fluorescent fusion protein with your gene of interest: Plasmid Color Expression Description...Aquamarine mCerulean N1 Cyan Mammalian Express a gene of interest fused to the N-terminus of monomeric...Cerulean mCerulean C1 Cyan Mammalian Express a gene of interest fused to the C-terminus of monomeric...monomeric Venus mVenus C1 Yellow Mammalian Express a gene of interest fused to the C-terminus of monomeric...

...Resources on the Addgene Blog Learn More About Fluorescent Protein Aggregation Catch Up On Your GFP Basics Get...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,...long-lived transcription factors control gross changes in gene expression as new body sections are formed. In synthetic...

..., or a mutation can be introduced in a gene, while adding a GFP-tag (Figure 3). Achieving multiple modifications...insertions, deletions, and point mutagenesis to complex assembly of multiple genes. Primer design for each type...amplified, restriction digested or even a synthesized gene. PCR is the primary method to produce the linear...included in a single PCR, for example for subcloning genes from different templates. All PCRs are run as an... 5’ end of either primer. Subcloning - To move a gene from one vector to another simply amplify the vector... to the 5’ end of the reverse primer. Mutagenesis - Mutagenesis is performed by designing primers astride...using the same three step protocol:  Fragment generation - Any linear DNA fragments can be assembled in...

... 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...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...27, 2020. CRISPR, and specifically Cas9 from S. pyogenes (SpCas9), is truly an exceptional genome engineering... PMC5127784. Find plasmids from this paper at Addgene. 2. East-Seletsky, Alexandra, et al. "Two distinct...