...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...

... 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...

...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...

...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...

...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...

...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...

...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...

...exactly how postdoctoral salaries are changing. Transparent salary information for postdoctoral positions...institutions set their postdoctoral salary scales to the NRSA scale (Figure 21, National Postdoctoral Association...was contributed by Future of Research Executive Director, Gary McDowell. On Dec 1st, the threshold at which...when the ruling was issued, a joint blog post by Director of the NIH Francis Collins and Secretary of Labor...paid. Institutional salary policies are one indicatator of how salaries may changes due to the FLSA. ...as having postdocs, representing ~85% of the postdoctoral workforce. Based on our communications with ...according to the NSF data, account for 18% of the postdoctoral workforce in the sciences. The Dana Farber Cancer...

... at some of the stories behind the names. We also have many plasmids in our repository that you can use...to a specific set of DNA-binding transcription factors to repress the expression of genes primarily involved...Cancer Institute! Sonic Hedgehog, an important regulator of embryonic development The largest conference...the hedgehog signalling pathway, which is a key regulator of all bilateral animal development. SHH plays...publication, Ivics et al 1997. Spaghetti Squash, a regulator of cellular contraction You may think that a gene...vessels). Spaghetti squash (Sqh) is the Drosophila Regulatory Light Chain (RMLC) of nonmuscle myosin II (Karess... Jordan and Karess, 1997), which contains the regulatory regions that are necessary for myosin activity...

...the activator - the CRY2 photolyase homology region (CRY2PHR) fused to a transcriptional activator domain...directed by the guide RNA (gRNA), while the CRY2PHR-activator fusion floats freely, depicted in the figure below...and CIB1 proteins heterodimerize and move the activator into position to activate gene transcription. ...manner. With their light-activated CRISPR/Cas9 effector (LACE) system, the Gersbach lab utilized a similar...fusion of full-length CRY2 and the transcriptional activator domain VP64. Using this system in HEK293T cells...photoswitchable proteins derived from the fungal photoreceptor, Vivid (VVD, N. crassa) (Kawano et al., 2015...transcription, they fused BldD to the transcriptional activators p65 and VP64 which in turn transcribes N-Cas9...

... and progenitor cells. Now with the DAP array, it is possible to use the state-of-art base editor, prime...prime editor, or any CRISPR-based gene editors, for correcting mutations or installing functional genes...thought to enable MBE by using Cas12a fused base editors (dCas12a-BE). However, the poor editing efficiencies...they found led them back towards Cas9 fused base editors (nCas9-BE), as Cas9 is mechanistically more favored...number of gRNAs that can be packaged into viral vectors for potential in vivo applications or multiplex...array.7 Using DAP arrays with a variety of base editors, the Gao Lab successfully demonstrated up to 31...unprecedented number of genes with a single viral vector.   Fig. 1: Schematic of multiplex gene ...

...adjacent fragment or vector. 5′ overhangs are generated on all the fragments and the vector, specifying the... together through transformation, so the cloned vector must be ligated. But in LIC, longer single-stranded...due to the absence of the other dNTPs. Once the vector and insert are (separately) digested to create ...received an important update, courtesy of Addgene depositor Stephen Elledge. His new method, named sequence...homologous recombination. SLIC inserts and linearized vectors must have homologous DNA sequences (20–60 bp) on...complete plasmid. Besides being compatible with any vector, the SLIC protocol itself is flexible. For one ...digestion (no dNTPs needed), allows both insert and vector to be digested in the same tube. Alternatively,...

...calcium indicator (Shemetov et al., 2021). In addition to FRET, intensiometric calcium indicators NIR-GECOs...proteins (NIR FPs) were derived from natural photoreceptors incorporating biliverdin IXα (BV) autocatalytically... derived from bacterial phytochrome (BphPs) photoreceptors have higher apparent cellular brightness than...were derived from cyanobacteriochrome (CBCR) photoreceptors (Oliinyk et al., 2019). They were engineered...performance, and applications of near-infrared calcium indicators in this recent review (Shcherbakova et al., 2021...encoded near-infrared fluorescent calcium ion indicator. Nat Methods 16, 171-174 (2019). Matlashov, M..... A near-infrared genetically encoded calcium indicator for in vivo imaging. Nat Biotechnol 39, 368-377...