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...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....

...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...

...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...

...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...

...Despite its relatively recent development, the history of optogenetics begins almost 50 years ago with...Wick and Krook-Magnuson, 2018). Optogenetic FLEx vectors contain a strong promoter upstream of an opsin ...Cre-dependent viruses, such as the optogenetic FLEx vectors in combination with transgenic animals or rAAVs...example, Taylor and colleagues used rAAV FLEx-based vectors to study the neural circuitry behind the mechanisms...paper, de Jong and colleagues used rAAV FLEx-based vectors to answer this question, mapping and characterizing...spatio-temporal precision and reversible modulation of FLEx vectors combined with the use of several Cre transgenic...motivated behaviors (de Jong et al., 2019). FLEx vectors represent, therefore, an ideal partner to optogenetics...

... learning. 1. Create a peer mentoring group We don’t all get the mentoring support we need, and most of...to access great mentorship is by recruiting your own peers and classmates! Your mentors don’t need to be...Own Peer Mentoring Group: A How-To Guide for Scientists.   Once in-person, peer-mentoring groups ... of us could use more mentors to listen, guide and support us during training and throughout our careers...goals.  I’ve been surprised to see that virtual mentoring groups seem to be as successful as in person groups...? You could do a talk virtually for your peer mentoring group (see above). However, now that we are all...initiative. In fact, hear from Addgene’s former blog editor Tyler Ford on how initiative landed him at Addgene...

...transcriptional regulator to modulate expression at that locus. The use of regulators allows a greater...shifted in recent years toward tinkering with regulatory networks and multiplex gene control. However,...approaches are often met with the challenges of regulatory bottlenecks and their scope is limited by the...Far removed from the bio-industry’s vision of ‘biofactories’, most successes in metabolic engineering have...us one step closer to the vision of metabolic biofactories by demonstrating the use of CRISPR-based RNA...scaffolds that can recruit various transcriptional regulators (Zalatan et al. 2015).  These scaffolds are stretches...levels dependent upon the type of transcriptional regulator or the number of designed scaffold binding sites...