...both introduce genes into an organism’s genome (see figure) and to disrupt endogenous genes at the site ...puromycin, hygromycin) and fluorescent protein reporters (GFP, BFP, RFP, no FP). The vectors each contain... fused to a region of the RPL41 gene, to drive the selection/reporter parts of each construct. This new...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 ...

...and a mCherry reporter downstream of the PBAD promoter. To use this plasmid with your gene of interest,...the DNA-binding domain of AraC using a mCherry reporter downstream of a PBAD promoter. While none of the...interest, you would replace mCherry with your gene of interest. pBLADEONLY: This plasmid contains only BLADE ...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...

...popular studies involve using a reporter vector to tag YGOI with lacZ and/or GFP (e.g. pPD80_08) and following...applications used to study gene function. From making transgenic animals, gene targeting and genome modifications...modifications to reporter vectors (e.g. pBV-Luc) and gene silencing (e.g. pLKO.1 - TRC cloning vector). ...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...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...

...capture module (SpyTag-GFP nanobody) assembled on beads. C) Micrograph of H1.8-GFP nucleosomes collected...Prime-editing-Assisted Site-Specific Integrase Gene Editing (PASSIGE), their previously-reported technique for precisely integrating...editing, this epigenetic strategy doesn’t induce DNA damage or permanently alter a gene product; it simply...In MagIC-cryo-EM, target proteins (expressed as a GFP fusion in this proof-of-concept study) are captured...SpyTag-SpyCatcher linkers and spacer proteins ending with a GFP nanobody (Figure 1). This enrichment streamlines ...) 3D model of the obtained structure of the H1.8-GFP-bound nucleosome. Images adapted from Arimura et ...). Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases...

...GCaMP6 family of sensors are commonly used calcium reporters with different kinetic parameters. There are three...ones? Many of the available biosensors use a single GFP or CFP/YFP-derived FRET pair. If you need to free...also available. Detection methods Many calcium reporters use a single fluorescent protein (FP), requiring...unwanted variables. Twitch is a FRET-based calcium reporter derived from troponin C. It is engineered with...for in vivo studies) There are several calcium reporters specially designed for the aims and requirements...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...

...of where the genes were expressed. What’s the use of stable gene expression? Constant gene expression can...Researchers express genes of interest from plasmids in order to study gene function or to engineer cells...input and gene expression is the output. The input, plasmid copy number, directly influences gene expression...buffered against the effects of copy number on reporter gene expression. On the other hand, using a constitutive... varied in size or carried different genes. In these cases, gene expression also remained stable regardless...media and growth stage can both affect gene copy number. Moreover, gene copy number is affected differently... constitutive promoter upstream of GFP increased expression of GFP six-fold, but a stabilized promoter...

...to induce high levels of gene activation for both reporter and endogenous genes. It is highly specific ...anti-FLAG-FB-GFP binding to FLAG-tag H2B for nuclear localization. (c) Applications of anti-FLAG-FB-GFP labeling...between MARCH5 and WSB2 genes using Cas12a’s ability to knock out multiple genes in parallel. Lastly, this...cellular targets Optimized genetic screening technique using Cas12a for multiplexed gene perturbation The Penn...10846-1.   Optimized genetic screening technique using Cas12a for multiplexed gene perturbation by: Gabrielle...many CRISPR-based genetic screening methods have primarily used Cas9 to knock out genes, researchers at ...multiple genes within a single cassette provides increased simplicity for combinatorial genetic screens...

...limits the delivery of larger transgenes, such as base editors which have a gene size of ~5.2 kb. To overcome...Ace-mScarlet voltage indicator plasmids at Addgene! Beck et al., Sci Report 2019. PubMed PMID: 31685893. A MoClo...Article contributed by Jennifer Tsang AAVs are a great gene therapy delivery mechanism, but their small packaging...levels similar to that of a single AAV expressing GFP. The lab tested the base editors in vivo and achieved...dosage comparable to those currently used in human gene therapy trials. They further tested the base editors...voltage indicators, however, fall short compared to GFP-based sensors in terms of response and brightness...expression levels from different promoters, and inhibited gene expression using CRISPRi. The kit functions in the...

...has dubbed these fusions LumiFluor Reporters. The LumiFluor reporter constructs were designed to be packaged... bright enough to detect endogenous tagged genes generated through CRISPR/Cas9 knock-in. Finally, NLuc...imaging tools generate cyan, yellow, green, orange and red fluorescence. The paper reports a wide variety...photoactivatable, optogenetic accuators to depolarize or hyperpolarize membranes. Traditional FRET reporters could...would also activate the optogenetic accuators. The researchers took a FRET reporter and adapted it into a...Fusion Protein, codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase are available...couples NLuc to fluorescent proteins to make better reporters and the second couples NLuc to fluorescent proteins...

...Control Protein Activity with GFP Resources at Addgene.org Find KTR reporter plasmids Find photoactivatable... an ERK kinase translocation reporter with a mRuby2 fluorescent reporter (ERK KTR-mRuby2). The ERK KTR...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...

...and corn. For both β-glucuronidase (GUS) and GFP reporters, as well as multiple transient and transgenic...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...

..., 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...pathway is ubiquitous, with successful recombination reported in all laboratory E. coli strains tested so far...use of this pathway as a cloning tool was first reported over 25 years ago (Douglas and Howard, 1991, Bubeck...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...

... developed a third-generation intensiometric glutamate sensing fluorescent reporter (iGluSnFR3) to study...research.   Abhi Aggarwal: Glutamate sensing reporters Abhi Aggarwal is a postbaccalaureate research ...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...catalyzes the desulfuration of 3-mercaptopyruvate and generates an enzyme-bound hydropersulfide that is then transferred...

...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...cloning plasmids, expression plasmids, gene knock-down plasmids, reporter plasmids, viral plasmids, and genome...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 ...

...include a reporter like GFP, some smaller CRISPR-Cas proteins such as SaCas9, CRISPR gRNAs, or your gene of ...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 ...

...following sucrose treatment. Moreover, several reports have shown that cells treated with sucrose or other...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...

...Figure 1: MTRIA biosensors are generated by inserting a circularly-permuted GFP into an intracellular loop...of interest and co-expressing a cytosolic ZtSH2 reporter, they observe fluorescence localizes to the plasma...And orthogonal sensors with different ITAM/SH2 reporter pairs allow multiplexed measurement of different...10.1101/2022.08.13.503850.  iGECInano NIR calcium reporter Near-infrared (NIR) fluorescent proteins promise...photochemical characteristics than previous NIR calcium reporters iGECI and NIR-GECO2G, and its small size (~1500...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...

...multiple) rAAV-CAG::FLEX-rev:: hM4D-2a-GFP AAV1 Chemogenetics Scott Sternson New viral prep pAAV-CaMKIIa-hM3D...to request? Addgene's Data Hub can help, with over 100 user-submitted viral vector reports. And don't ...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...

...Signals, and GFP. B) Workflow to assess Cas9-PAGE editing with viral sgRNA targeting mCherry reporter (mChe)...-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 .... CAP-Mac also robustly expressed fluorescent reporters in green monkeys and cultured human neurons, with...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 ...

...expression of reporter genes, or to combine reporter expression from one promoter with transgene or siRNA ... 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...stage-specific promoter and design a UAS reporter construct, the reporter will only be expressed where and/or...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...