We narrowed to 1,082 results for: TOR

...employing optogenetic regulators to initiate the event being monitored. Optogenetic regulators respond to specific...fluorescent protein (acceptor) in the pair. The higher the emission from the acceptor, the more powerful...and NanoLuc® Luciferase are available from the repository. We encourage people to go to Addgene to get ...furimazine by Promega’s Chemistry group is a good story (1). From a deep sea shrimp to a compact powerhouse...applications of the Enhanced Nano-Lanterns including monitoring multiple cellular events such as the dynamics...initiation wavelengths, excitation of the optogenetic regulator leads to spurious activation of the FRET sensor...still transfer energy to the fluorescent protein acceptor. NLuc is an excellent BRET donor due to its bright...

...restriction enzymes or vector purchases (Table 1). Restriction enzymes and vector purchases can raise the...use of both when making plasmids. Use repositories Repositories such as Addgene help share materials quickly...After the great Plasmid Debate of ’18, Blue Flame depositor John Doench calculated the time to make a plasmid...for the simple insertion of a PCR product into a vector. The time it takes to make a plasmid could increase.... This cost varies hugely based on a number of factors, but we can assume it’s at least equal to the $155...in a 96-well (or 384-well) plate! How much do repositories help?   Designing precise calculations for any...any of the numbers in this post is a mathematical story well beyond my ability to tell. But even from these...

...knock out plasmids? Check out our Cre vector and Murine LoxP vector.   Turn it off for a little while Transient...? Check out our Retro expression vector and Lenti expression vector. Turn it on for just a little while...CRISPR for that, too! CRISPR activators (CRISPRa) are transcriptional activators fused to a dead Cas9. The...out Addgene's Lentiviral dCas9 SAM vector and Piggybac dCas9 SAM vector! On/off control all the time If ...Addgene's Lentiviral dTAG cloning vector and SMASh degron tagging vector! Destabilizing domains Destabilizing...increase expression. There are several flavors of activators which can be used with this approach - read more...them on our CRISPRa blog! Fig. 1 CRISPR activator and repressor mechanisms Want to transiently...

...begun to use more precise genetic tools: activators, inhibitors, sensors, recombinases, and more. With ...by using cis regulatory elements such as promoters and enhancers, multicistronic vectors, and post-translational...Addgene blog Download the viral vectors 101 eBook Read our viral vector related blog posts Learn more about...With the help of adeno-associated viral vectors (AAV), neuroscientists can combine classical genetic tools... how these changes affect the phenotype. Viral vectors and genetic approaches are continuously refined...Education and Development workshop aimed at brainstorming strategies for using combinations of AAV-based...designing experiments with AAV: 1. Getting your viral vector into your cell of interest: AAV tropism and route...

... of stimulation to receptors. Optogenetics offers better control over these factors as the light source...more about optogenetics New to viral vectors? Browse our Viral Vectors 101 blog posts Learn more about precise...Addgene.org Browse all viral vectors available at Addgene Visit our viral vectors guide Find our chemogenetics...different advantages and limitations.  The four main factors in which chemogenetics and optogenetics differ ...offers a high degree of temporal resolution as receptors can be turned on and off simply with the presence...diffusion of drugs throughout the body, the onset of receptor activation takes minutes. As a result, it is not...response or track the exact time of activation. Receptor activity is also drawn out as drugs can remain...

... they monitor. Gene traps Gene traps are promotor-less constructs that contain splice acceptors. They...below), but fundamentally the reporter construct monitors the expression of a nearby gene. The reporter ...high/low) and patterns (tissue/timing) can be monitored under various conditions without necessarily modifying...stage you could infer that the gene which GFP was monitoring was being expressed in the same pattern. These...throughout an organism’s genome. These reporters have historically been used extensively in fly, mouse, and yeast...result but will still allow for expression pattern monitoring. Enhancer traps Like promoter traps, enhancer...traps, it will still likely disrupt the gene it monitors due to transcriptional fusion.   Examples...

...inheritance. CRISPR activators and repressors are thought to be reversible once the effector is inactivated...promoters. Other researchers additionally fused a TAL effector to LSD1 histone demethylase to demethylate enhancer...methylation and activate transcription. A lentiviral vector with the same modifier, Fuw-dCas9-Tet1CD, is available...and repair pathways work downstream of Tet1 to restore the DNA after cytosine removal. Recently, Albert...alone, or coupled with DNA oxidation and repair factors that allow for increased gene activation at the... targeted enhancers. Although the lab used this vector in chick embryos, it also functions in mammalian...and this fusion allows for packaging into viral vectors and targeted delivery into hard-to-manipulate cell...

...bacterial prey. Currently, receptor binding proteins (RBPs, the host recognition factors of phages) constitute...Therefore, in addition to their utility in the health sector, phages represent a reservoir of carbohydrate binding...function of digestion time. For phage DNA that is refractory to enzymatic digestion, however, a physical fragmentation...a hose to a nitrogen gas tank with a pressure regulator. Other methods for physical shearing exist as ... blunt ends) and ligate into a bluntly digested vector designed for protein overexpression in E. coli,..., such as pET30a. If working with DNA that is refractory to enzymatic manipulation, amplifying phage DNA...primers bordering the multiple cloning site of the vector. In order to reduce the chances of some clones ...

...Existing methods for AAV quality control Historically, AAV vector QC has been sparse including only titration... procedures and Python scripts with laboratories and viral vector cores routinely producing AAV. We believe... our viral vectors related blog posts Read our blog post on AAV titers Browse viral vectors protocols ... platform to accommodate adeno-associated viral vector (AAV) samples and created a simplified process,... of quality control is sufficient but for viral vector core facilities producing a large number of viral...demonstrated that NGS is an effective tool to confirm AAV vector genome identity. These studies used SMRT sequencing... simplified approach, we sequenced over 100 AAV vector preparations and showed that VGS can quickly and...

...Louis provides a combinatorial indexing strategy that simultaneously tracks clonal history and cell identity...embryonic fibroblasts (MEFs) into induced endoderm progenitors (iEPs), a self-renewing cell type that can differentiate...iEPs by retroviral expression of transcription factors Foxa1 and Hnf4α, a process that like other reprogramming...CellTagMEF), at the end of the three-day transcription factor reprogramming treatment (CellTagD3), and 13 days... Morris Lab found that cells took one of two trajectories: a route towards successful iEP conversion or... The CellTags revealed that cells with common ancestors tended to have similar reprogramming outcomes....2019). Key takeaways of CellTag CellTag is a combinatory cell-indexing tool that combines scRNA-seq and...

...and money producing virus from select vectors in the repository yourself, you can now order ready-to-use...As some of the most popular lentiviral vectors in the repository are used to produce CRISPR tools, we’re...Expanding the viral vector knowledge base: You can help! We’ve been expanding our viral vector educational resources... production process while still ordering viral vectors. Virus production can be a frustrating process ...delivery of Cas9 endonuclease, Cas9 nickase, Cas9 activators, Cas9 repressors, select gRNAs, and CRISPR pooled...expression of a gene under the control of the tet operator. AAV AAVs are the workhorses of both chemo- and... work with virus. You can find all of our viral vector blog posts here. If you have virus expertise you...

... AAV vectors expressing RGB proteins under the control of an inducer, i.e. tet-off transactivator (tTA...Subscribe to Viral Vector Blog Posts Additional Resources on Addgene.org Search for AAV Vectors to use with...   Adeno-associated viral (AAV) vectors are the most frequently used gene-transfer tools in the study...infect the PNS or the CNS much better than the AAV vectors they were derived from. Additionally, AAV.PHP.eB... of AAV.PHP.eB and AAV.PHP.S to their parental vectors. To quantify this comparison, capsids were packed...AAV-PHP.eB and AAV-PHP.S versus their parental AAV vectors. What can these AAV’s be used for?  1. Multi-color...multi-color labeling is to inject a mix of AAV vectors that constitutively express a red, green, or blue...

...posts related to viral vector protocols and tips Read all of our viral vectors blog posts Resources on...Since Addgene began producing AAV vector preparations, we have used quantitative PCR to evaluate the ...titrating AAV by qPCR, viral DNA is amplified and monitored in real time. When the PCR is complete, the results...issue with qPCR is that the results can vary by a factor of 2. This means that if you set up two identical.... This partitioning reduces the number of PCR inhibitors per reaction and allows for enhanced detection...up with poor droplet quality. Using a droplet generator, the sample and the droplet generation oil are... = Volume of virus in reaction mix D = Dilution factor of virus Most droplet readers have a few channels...

... plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles. Today, it'... in this post:  New compact, efficient prime editors A versatile and precise system to improve on-target...proximity labeling and TransitID Tracking cellular history with protein ticker tape Teaching an old antibody...antibody new tricks   New compact, efficient prime editors By Susanna Stroik The Liu lab recently used phage-assisted...protein engineering to generate seven new prime editors, PE6a–g, that overcome some current prime limitations... and PE6b are 500–800 bp smaller than previous editors, but just as effective and with no additional off-target...protein engineering yield compact, efficient prime editors. Cell, 186(18), 3983–4002.e26. doi: 10.1016/j.cell...

...the activator protein cannot bind. After an inducer binds to the activator protein, the activator protein...In this system, the activator rtTA (reverse tetracycline-controlled transactivator) is normally inactive...the binding of RNA polymerase and transcription factors. Since the promoter region drives transcription...agents, temperature, and light are all examples of factors that can lead to the induction of a promoter. Below...inducible system is found in many bacterial expression vectors. Negative inducible promoter pBad is another popular...protein purification. When arabinose is absent, regulatory protein AraC binds O and I1 sites upstream of...addition to arabinose, cAMP complexed with cAMP activator protein (CAP) can also stimulate AraC binding ...

...zinc finger proteins, and transcription activator-like effectors (TALEs), with fifteen total variants deposited... plasmids, antibodies, and viral preps in our repository through our Hot Plasmids articles. Here's what...systems for specific applications! Delivering prime editors (PEs) via engineered virus-like particles (eVLPs... contain no genetic material, which ensures the editor will only be transiently present in transduced ...site-specific serine recombinase. First, the prime editor installs the recombinase “landing sites,” then ...landing sites on opposite DNA strands. When the prime editor releases the DNA, the two 3’ flaps containing the...-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo. Nature Biotechnology...

...G-protein-coated RABV infects cells via the G receptor. In neurons, the G receptor is restricted to presynaptic nerve...Control Gene Expression with FLEx Vectors Resources on Addgene.org AAV Vector for TVA Expression AAV for Rabies...housed in specific locations and give rise to progenitors during growth and development or that blood flows...to visualize how neurons are connected. Viral Vectors as Neuronal Tracers Due to their ability to efficiently...through their interactions with target cell surface receptors (Callaway 2008). Based on this, cell type specificity...EnvA-pseudotyped RABV, and only expressing the EnvA receptor (TVA) in user-defined cells. For example, a researcher... via entry mediated by the G protein/G-protein receptor. Once in first order neurons, the G-deleted RABV...

...acquire during their PhD and postdoctoral training. Addgene executive director Joanne Kamens has written ...were many changes to the  Board of Directors and the Executive Director changed twice. I had to learn how... to report to the Board of Directors and liaise with the Executive Director. As members changed, I had...management-for-scientists-managing-vs-leading. The National Postdoctoral Association (NPA) outlines 6 Core Competencies... and Management skills”. The NPA states that Postdoctoral scholars should have the skills and techniques... levels. These skills will also help the person mentor others more successfully. But how does one gain... leadership experience). I was involved with postdoctoral issues first at Brigham and Women’s Hospital...

...the vector backbone you cloned your insert into and fill out the appropriate boxes with the vector backbone...backbone manufacturer, vector backbone size, total size of your vector + insert, any modifications you ...standard conditions (37 °C, LB, antibiotics, etc.) and stored as glycerol stocks We will need information on...provide any full or partial plasmid sequences and any vector maps. The more sequence data available, the better... was a part of the insert (when cloned into the vector backbone). If your plasmid has more than one insert...you made to the backbone, and the vector type. Pro Tip! Reference the Addgene ID in the "Backbone Manufacturer...enter "Feng Zhang (Addgene plasmid #52961)".   The vector type should describe how your plasmid is intended...

... — but critical — factor for successful experiments or therapies. Using AAV vectors with enhancers that... plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles. Here's what...what you'll find in this post: Glutamate indicators with increased sensitivity and tailored deactivation...viral preps targeting interneurons Glutamate indicators with increased sensitivity and tailored deactivation...abundant neurotransmitters, but it is challenging to monitor because only a small number of glutamate molecules...and they are rapidly taken up by postsynaptic receptors and transporters. The intensity-based Glutamate-Sensing...fusions for membrane display, they recommend the NGR vectors for most experiments. These new tools promise to...

...Comparison of TnpB (A) and Cas9 (B) genome editors. Effector nucleases are shown bound to the target DNA...AMPA.FingR (AF) that binds to AMPA receptor subunit GluA1 found at excitatory synapses (Rivera & Huang, et ...plasmids, antibodies, viral preps, and more in the repository through our Hot Plasmids articles. Here's what...components (nucleus, endosomes, lysosome, golgi, ER, inhibitory synapses, and cytoskeleton components) and in... household name, the search for smaller genome editors is heating up and TnpB has found itself in the ...but TnpB is small enough to be carried by viral vectors (~400 amino acids, less than a third of the size... (2025). Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis...

...point will be those that have excised the vector backbone. Vectors pTOX4, pTOX5, and pTOX6 express the YhaV...Find the allelic exchange vectors here! ShadowR, a novel FLIM-FRET acceptor Figure 4: ShadowR is...levels. Chromoproteins in nature Compared to the history of fluorescent proteins in molecular biology research...research, chromoproteins have a shorter history. In 1987, scientists described an unusual blue-colored... selection markers for allelic exchange. These vectors contain a toxin gene (regulated by a rhamnose-inducible...leads to the desired mutation and excision of the vector backbone. To do this, rhamnose is added which induces...respectively, and all encode the amilCP chromoprotein. Vectors pTOX7, pTOX8, and pTOX9 express the YhaV, MqsR,...

... you want the works: organizing reagent storage and inventory, managing orders, tracking students and ....io beta platform for sharing, annotating, and storing life science protocols. Since we began sharing ...website in December 2006, the pLKO.1-TRC cloning vector (deposited by David Root of the Broad Institute...Institute) has consistently been one of the repository’s most frequently requested plasmids. I wondered how many... and you decide to share them using Addgene’s repository, you can also share your associated protocols...listed under “Supplemental Document”: gRNA_Cloning Vector (deposited by George Church’s lab). Addgene has...the software aims to be a life science protocol repository. The software can be used with the ZappyLabs ...

... insert and vector are first PCR amplified. The forward primer used to amplify the vector is located in...your vector and insert. Once you’ve confirmed that the PCR was successful, mix together the vector and ... PCR with vector specific primers to confirm that the products are the correct size (vector + insert)....specific sequences for base pair overhangs, insert and vector purification, and, most importantly, excessive ..., it does not require purification of insert or vector products, nor does it require the use of specific...process in brief In FastCloning, you PCR both the vector and the insert in separate reactions and then combine...bases that are complementary to the overlapping vector ends. See an overview of the PCR process and the...

...Professional to mentor and coach you. There are many grant consultants who offer grant training, mentoring and coaching...to late-career grant professionals have similar stories about a time when their passion for an organization...their new career path.  A part of the career path story for most established grant professionals is learning...commitments to and proof of compliance with the grantor’s requirements as well as state and federal regulations...amount of grant funds available and the increasing regulatory process for grants has fostered the demand for...professional. Dr. Cook served as a principal investigator for several research grants. One of the benefits...benefits of being a principal investigator is that you are asked to review grant proposals from others. ...

...guide to viral vectors Find all of our viral vector blog posts Download our viral vectors 101 eBook Additional...that this approach can be adapted by vector cores and laboratories alike as an easy and inexpensive method...pressure on the production of retroviral vectors: Enhancement in vector stability." Biotechnology and bioengineering...been actively investigating ways to improve viral vector production. Whether redesigning viral packaging...membrane When packaging adeno-associated viral vectors (AAV) not all serotypes are equal; while some serotypes...cells by treating the cells with the endocytosis inhibitor sucrose. Previous work by Stoneham et al. demonstrated...less time and resources resupplying our current inventory and more time providing new materials to the research...

...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...Catalysis by TEV protease allows a transcription factor to enter the nucleus and modulate transcription... a cell. The Goedhart lab, Gadella lab, and collaborators have an ongoing effort to improve genetically...pMAK backbone by modifying pMAL-T-Avi-His/BirA, a vector commonly used for in vivo biotinylation via BirA...Institute developed fluorescently labeled piggyBac vectors for uniform and sustained expression of single ...multiplexed gRNAs and of dCas9 fusions. The dCas-KRAB activator and the dCas-VPR repressor are introduced into...an EGFP reporter. Expression of the dCas9-KRAB activator or dCas9-VPR repressor in hPSCs is also driven...

...into small, well defined chunks, called stories, and work on stories during short periods of time called ...clarifications lead to the story being scrapped or broken up into smaller stories. Since this occurs before...plasmids from the moment a deposit kit is sent to a depositor, to streamlining the order fulfillment process... sprints. The team is mindful of the number of stories we commit to for each sprint, to ensure we will...process for working on a new feature. We start with a story that describes the feature and what the criteria...accepting the feature as done correctly. These initial stories are usually a result of collaborations between ...stakeholders (other Addgenies). Once we begin work on a story, we continue to collaborate closely with each other...

...joined Elsevier after her postdoctoral studies, and has held various editorial roles on review and primary... of the Cell Press editorial department, a team of around 100 professional editors who produce a broad...contributed by Deborah Sweet, Vice President of Editorial at Cell Press. Almost everyone who works in a ...filtration system, environmental stability in an incubator), but there’s a lot you can do to help other scientists...shoulders of journals and publishers. As journal editors, we often find ourselves having to ask authors ...paper off their desk and move on. Nevertheless, editors can and do play this role, and we take our responsibility...Press. Deborah Sweet is the Vice President of Editorial at Cell Press.  She trained originally in cell...

...expression vectors which are often more difficult to transfect or package into viral vectors. Cas9 multiplexing...multiplexing vectors are larger because they require regulatory sequences to allow for expression of multiple... 1) Most rely on transfection of more than one vector to express the gRNAs and Cas9. Co-transfections...Yamamoto Lab Golden Gate Assembly transfection One vector expresses Cas9 and up to 7 gRNAs Each gRNA requires...Expression Cassettes lentiviral transduction One vector expresses Cas9, eGFP, and up to 4 gRNAs Each gRNA...plasmids resulting in smaller, more streamlined vectors which are easier to use across multiple expression...In most of Zetsche et al’s experiments, a single vector co-expressing Cpf1 and a crRNA array were used....

...Modular Assembly method Nuclease Expression Vectors - Cloning vectors for expressing zinc finger FokI nuclease...proteins for distribution to academic and nonprofit laboratories for research and educational use. Links to additional...

...stimuli are processed by a network of excitatory and inhibitory neurons whose function and connectivity...She fused fluorescent protein tags to the receptors to monitor their localization and expression. Qiu saw... are working on developing new surface display vectors to improve expression and trafficking of these ... that is then transferred to thiol-containing acceptors. These hydropersulfides modulate protein activity...have important roles during oxidative stress. To monitor this activity, Pedre Pérez’s biosensor fuses MPST...can also be used along other thiol-containing acceptors in competition assays. “I believe that improved...where she engineers genetically encoded voltage indicators. Tian developed a video-based high-throughput...

...express in a broad number of cell types, such as inhibitory neurons, rods, cones, or retinal ganglion. Rather...repeating mouse cell type specific transcription factor binding sites interspaced with random sequences...: transcription start site. TFBS: transcription factor binding site. Image from Jüttner et al., 2019 with...types (ex: rods or cones) or cell classes (ex: photoreceptors which encompassed both rods and cones).  Targeted... to target a cell class in mice is not a good predictor for targeting the same cell class in NHPs (R=0.38... to target a cell class in monkeys was a good predictor for targeting the same cell class in humans (R...also expressed. If Cre-mCherry expresses in photoreceptors and horizontal cells, and Cre-dependent-GFP...

...are expressed and purified from plasmids in our repository....summarize antibody production and storage techniques, and explain common applications. View...some of our other resources to help you choose, store, or use your antibody. Antibody Blog Posts Recipient...

...fluorophores can “spill over” into detectors that are not the primary detector for that fluorophore. For example...single (primary) detector to identify a fluorophore, spectral flow cytometers use all detectors for all fluorophores...panels: five laser spectral flow machines have 64 detectors, allowing for the use of up to 64 markers in a...emitted by a fluorophore is guided to a single detector using a series of mirrors and filters. Importantly...Importantly, conventional flow cytometry uses one detector to detect one fluorophore and therefore one cellular... FITC signal is spilling over into the primary detector for PE. In order to correct for this, compensation...the fluorescent molecules can be quite broad, detectors will usually only pick up a small portion of that...

... plasmids, antibodies, and viral preps in the repository through our Hot Plasmids articles.  Here's what...single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies for neurodegeneration...order to study the conformational dynamics of the motor protein kinesin-1, the Twelvetrees Lab took advantage...2024.12.20.629623.  A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic...synaptic events By Brian O’Neill The Lin Lab and collaborators recently created ASAP5, a faster and more responsive...responsive genetically-encoded voltage indicator (GEVI) than previous versions. ASAP5 can be imaged using... and subthreshold events, including miniature excitatory postsynaptic potentials (mEPSPs) (Hao and Lee...

...to their laboratory. A single mouse click on a gene of interest will direct the investigator to a list... cancer are yielding promising results in the laboratory, and Addgene is accelerating this research by...proto-oncogenes and tumor suppressors to the accelerator and brake pedal of a vehicle, respectively. In... (now an oncogene), this is analogous to the accelerator of a vehicle being stuck down. Similarly, a mutation...cells still need to successfully infiltrate the circulatory or lymph systems to spread, or metastasize. Many...Many cancerous cells will die trying, as the circulatory and lymph systems are not ideal environments ...Clone Collection 2.0. The collection is a set of vectors for use with the Gateway Cloning Platform (Life...

...in HEK293T cells. Laboratory tools Numerous plasmids for the production of laboratory enzymes have also...to be cloned into the pSNAP-tag (m) Vector or pCLIP-tag (m) Vector to generate a tagged fusion protein...bacterial stab format. Viral vector production Plasmids for producing AAV vectors are also available to industry...Throughout its 20-year history, Addgene’s mission has been to empower scientists to advance discoveries...the transfer of a plasmid between two academic laboratories. While Addgene is well known for facilitating..., we shipped our first plasmid to an industry laboratory, and today we have almost ten thousand unique...EGFP-Tubulin-6.  CRISPR OpenCRISPR-1, an AI-generated gene editor deposited by Profluent Bio, maintains Cas9 architecture...

...from a GFP variant and a DA receptor such that GFP fluoresces when the receptor is bound by DA. Two variants... variant bound to a DA receptor, such that the GFP fluoresces upon DA-receptor binding (Sun et al., 2018...Find dopamine and other biosensors in our AAV vector inventory. References Darvesh, Altaf S., et al. "In... of our viral vectors blog posts Find tips for preparing AAVs Download our viral vectors 101 eBook Resources...in reinforcement learning, decision making, and motor control.  What do we understand about Dopamine?...ms on and 0.7 or 2.5 s off, for mouse or human receptors, respectively). Using GRABDA sensors in experiments...rat brain, and its similarity to the “dopamine receptor”." Proceedings of the National Academy of Sciences...

...finally decide the LEGO laboratory was good enough. There are no pre-made LEGO laboratory sets so we had to ...to show people the very human struggles in the laboratory routine. We hope that sharing and laughing at...the LEGOs were mirrored by the nearly infinite regulatory capabilities of gene pathways just waiting to...was creating a LEGO stage that resembles a real laboratory. It only took one night to build the first model...use our imaginations to mold it all into a full laboratory. It is priceless to see our “toys” gaining life...working as scientists in a custom made, colorful laboratory. When we started, the hardest part of the process...building each scenario, I go “WOW, now I have my own laboratory” (the main character is much more than a piece...