...? Depositing lab: The name of the Principal Investigator (PI) in whose lab the construct was made. The...

...Voltage indicators are voltmeters for cells! In this blog we will review how these indicators work, their...fluorescence or voltage indicators.   Viral Vectors and Voltage Indicators As previously mentioned...their use as biological tools. Why Use Voltage Indicators? Electrical signals are integral to many facets...within a cell are genetically encoded voltage indicators (GEVIs) and chemigenetic sensors. These tools...describe in more detail below. Types of Voltage Indicators GEVIs can be divided up structurally into 2 main...structurally separates the two components of the indicator, which can make the kinetics of voltage sensing...Factors like photobleaching, lack of red-shifted indicators, and brightness of fluorescence all have room...

...Neuromodulators like dopamine and norepinephrine have important functions in the brain but have been ...developing and depositing two new norepinephrine indicators: nLightG and nLightR (Kagiampaki et al., 2023...White insets indicate surface expression of the indicators over white dashed lines. Scale bars, 10 μm (HEK293T...trialed in two-photon in vivo experiments. nLight indicators are more sensitive to and selective for norepinephrine...They can, however, be studied with the nLight indicators. Speed One of the major advantages of this next...… Patriarchi, T. (2023). Sensitive multicolor indicators for monitoring norepinephrine in vivo. Nature...

...plasmids use Rho-independent terminators, which include naturally occurring terminators, such as T7 and rrnB,...engineered terminators come close (>95%). For most purposes, however, any common terminator will suffice...double terminators to reduce unwanted translation of downstream elements. A high affinity terminator may ...of yeast terminators of varying strength. A discussion of the effects of various terminators in the context...termination and polyadenylation? The role of the terminator, a sequence-based element, is to define the end...synthesized RNA from the transcription machinery. Terminators are found downstream of the gene to be transcribed...transcribed, and typically occur directly after any 3’ regulatory elements, such as the polyadenylation or poly...

...California Institute of Technology Cold Spring Harbor Laboratory  Dana-Farber Cancer Institute  Fred Hutchinson...

... download the PEI calculator for AAV packaging Use the PEI transfection calculator for AAV packaging ...corresponding column in the calculator spreadsheet: We hope that this calculator will make it easier to plan...for AAV packaging, considering using this PEI Calculator. AAV packaging typically requires transfecting...right, you need to do a few calculations. This calculator can do the math for you and it’s simple to make...whole experiment And here’s the equation the calculator uses to give you the μl needed of each plasmid...experiments. Let us know what you think about the calculator in the comments section below. References 1.... transfection calculator...

...with us. Tom McFadden is a YouTuber and science educator looking for creative ways to implement the next...

...isoforms, to help researchers understand this regulatory network. The transcription atlas and library...deliver multiple TFs to each cell, allowing for combinatorial overexpression programs on cellular processes...

... an easy-to-use tool to analyze the complex combinatorial genetic networks underlying your favorite biological...screening are vastly useful for mapping the combinatorial genetics that underlie complex regulation in...barcoded gRNA libraries that can be used to combinatorially modify the genome, screen for a particular ...successfully applied to functionally characterize combinatorial gene knockouts generated via multiplexed gRNA...expression (Wong et al., 2016), in addition to the combinatorial expression of other genetic elements including... Figure 2: Strategy for assembling barcoded combinatorial gRNA libraries. (Adapted from Wong et al., 2016...antibiotic-resistant bacteria enabled by massively parallel combinatorial genetics." Proceedings of the National Academy...

...we introduce you to the Journal of Emerging Investigators, an open-access journal that enables high school... the interview with the Journal of Emerging Investigators Podcast breakdown Intro Chapter 1 - Introduction...Olivia Ho-Shing, and the Journal of Emerging Investigators. Chapter 2 - Getting high school students and...

... plasmid toolkit, the Broad Institute Target Accelerator Plasmid Collection, that we hope researchers ... For many years, we’ve hoped that large scale laboratory-based functional experiments that classify which...and cancer phenotypes. Broad Institute Target Accelerator Plasmid Collections To overcome this first challenge..., we’ve developed the Broad Institute Target Accelerator Plasmid Collections, now widely available to ...Collection). In addition, we worked with many collaborators to aggregate a sequence-confirmed series of ...

...School and Paratore is the General Auditor at Tufts Health Plan. The addition of Katz and Paratore onto the...Interactive. Learn more about Carl Paratore, MBA, CPA Mr. Paratore serves as the General Auditor at Tufts...re excited to welcome Karen Katz, Esq and Carl Paratore, MBA, CPA to the Addgene Board of Directors. Katz...that Addgene was looking for new board members. Paratore: Kristen Darby, who’s also a board member, reached...necessary information that’s required to do research. Paratore: What I’ve been most impressed with is the level...potentially also be very helpful to the scientific community.Paratore: I’ve certainly had a front row seat watching...be, an interesting legal and professional challenge.Paratore: The promise there is the ability share the...

...potent transcriptional activators (p65 and Rta). These transcriptional activators work in tandem to recruit...CRISPRa methods vary in their transcriptional activators: some methods rely on fusion proteins while others...generally thought of as the “first generation” CRISPR activator. While it requires a relatively simple construct...modest gene activation. Synergistic Activation Mediator (SAM) Description SAM uses specially engineered...levels of gene activation compared to other CRISPR activators, making it a popular method for gene activation... SunTag performs better than first generation activators while showing lower activation levels than SAM...transcription. This complex consists of the VP64 activator used in other CRISPR activation methods, as well...

...voltage indicator Archon Article contributed by Shreya Vedantam  Listen to the voltage indicator podcast... genetically encoded voltage indicators. To expand the voltage indicator toolbox, Ed Boyden’s lab developed...Archon1, a novel opsin-based fluorescent voltage indicator. Archon1 can embed itself into the cell membrane...

... for a curated list of coregulators). Coregulators can function as coactivators and corepressors depending...complexes with secondary coregulator (or co-coregulator) partners. The coregulator ‘core’ complex is composed...feature afforded by coactivator complexes suggests that coactivators may be integrators of multiple cell ...illnesses, and the metabolic syndrome. Coregulators Coregulators constitute a group of some 400 molecules...that coregulators belong to distinct complexes may explain how more than 300 different coactivator proteins...manner. Because coregulators exist as multiprotein complexes, a member of a single coactivator complex can... W. (2002). Combinatorial control of gene expression by nuclear receptors and coregulators. Cell 108, ...

...Choose a moderator with a backbone. It is nice to have a subject area expert, but the moderator is not ...LinkedIn profiles of all panelists to the moderator. The moderator and organizers should develop a list of...(lapel mics are best). The moderator takes control It is the moderator's job to prevent anyone from ...effortless to the audience, but the organizers and moderator should have a well-planned approach to making ... of 4-6 pre-event interview questions. The moderator (possibly with an organizer) should set up a 30 minute...to schedule. In the pre-event interviews, the moderator should be taking good notes and discovering interesting...opinions that each panelist can contribute. The moderator should identify the funny, provocative or interesting...

... the perfect voltage indicator and optogenetic actuator pair. Ideally, indicators should possess fast ...lab wished to improve on the Arch indicator and their collaborators in Robert Campbell’s lab carried out...kinetics. By combining both the voltage indicator, QuasAr, and actuator, CheRiff, in the bicistronic Optopatch...activation of specific genes using dCas9-VP64 activator fragments (dCas9(C)-FKBP-2xNLS-VP64 and dCas9(...62885 & 62886) Rapamycin-inducible dCas9-VP64 activator set (Addgene plasmids 62887 & 62888) Zetsche... which will ultimately co-localize the VP64 transactivator with a dCas9 that has been targeted to a very...tool for activating endogenous genes Moritoshi Sato's Lab has engineered CRISPR-Cas9 to create a photoactivatable...

...Roth New serotype pAAV-EF1α-F-FLEX-mNaChBac-T2A-tdTomato AAV8 Molecular tool Massimo Scanziani New viral...Optogenetics Jonathan Ting New viral prep pAAV_BiSSTe10_dTomato_nlsdTomato AAV-PHPeB Control Gordon Fishell New...Optogenetics Gordon Fishell New viral prep pAAV_BiCHATe27_dTomato_nlsdTomato AAV-PHPeB Control Gordon Fishell New...Optogenetics Gordon Fishell New viral prep pAAV_BiVIPe4_dTomato_nlsdTomato AAV-PHPeB Control Gordon Fishell New...Gordon Fishell New viral prep pAAV_BiLAMP5e3_dTomato_nlsdTomato_nlsdTomato AAV-PHPeB Control Gordon Fishell...Optogenetics Gordon Fishell New viral prep pAAV_BiPVe4_dTomato_nlsdTomato AAV-PHPeB Control Gordon Fishell New...Optogenetics Gordon Fishell New viral prep pAAV_BiPVe3_dTomato_nlsdTomato AAV-PHPeB Control Gordon Fishell New...

...cheaper to use Cas9 activators. This larger scale of activation allows for exploratory-based research as...of the gene, Cas9 activators efficiently solve these problems.   Which Cas9 activator should I use? There...significant work on constructing Cas9 transcriptional activators. These constructs allow for the upregulation ...genome-wide studies One of the best uses for Cas9 activators is in genetic screening. gRNAs targeting every... using oligo library synthesis. Prior to Cas9 activators, similar tools were made using other DNA binding...to easily change the sequence targeted by the activator by simply providing a new gRNA rather than engineering...organism, have been used in a similar manner to Cas9 activators. However, these can be difficult to construct...

...Repressible: An activator protein is bound, turning transcription ON. When a repressor binds the activator protein...introduced the second generation QF2 activators. These activators solve the problem of broad QF expression-induced...protein, the activator protein cannot bind the promoter sequence anymore and transcription is turned OFF....protein, the repressor protein can bind to the operator. The bound repressor then prevents transcription... repressor (TetR) can bind to the tetracycline operator sequences (TetO), preventing transcription. In... Bujard created the tetracycline-controlled transactivator (tTA). They fused TetR with the transcriptional...by Gossen and Bujard, both the promoter and transactivator components of the system have been engineered...

... of robust CRISPR activators We previously reported on a novel Cas9-based activator developed by the Church...method, called STARR-seq (self-transcribing active regulatory region sequencing) that tests enhancer sequences...promoters, RBS’, coding sequences (CDS), and terminators of varying strengths. These can be easily combined...both standard Gateway cloning and the FNLCR Combinatorial Cloning Platform (CCP); the latter of which ...pooled libraries, but thanks to a deposit from the laboratory of Dr. Ji-Long Liu at Oxford University, we are...and context-dependent human fitness genes, the laboratory of Jason Moffat at the University of Toronto ...effectiveness of their library design, Moffat laboratory members conducted multiple screens in different...

...the activator - the CRY2 photolyase homology region (CRY2PHR) fused to a transcriptional activator domain...control transcription. Two separate labs, Moritoshi Sato’s lab at the University of Tokyo and Charles Gersbach...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. ...fusion of full-length CRY2 and the transcriptional activator domain VP64. Using this system in HEK293T cells...NHEJ and HDR using split systems Later in 2015, the Sato lab unveiled a photoactivatable system to cleave...DSBs (non detectable). Like their prior work, the Sato lab also showed that the paCas9-2 system could be...

...Suntag” Cas9 activator that recruits up to twenty copies of the p65-HSF1 transcriptional activator to each ...and engineered combinations of transcriptional activators to induce gene transcription at genomic sites... vitro settings due to the large size of Cas9 activators, which largely precludes delivery by adeno-associated...will fit). Cre-dependent expression of the SPH activator paired with AAV sgRNA delivery make the SPH mouse...calmodulin-dependent kinase II alpha (aCaMKII; excitatory neurons) and Synapsin-1 (pan-neuronal) promoters...Neuron-optimized viral vectors Alternatively, Cas9 activators can be delivered to the CNS by lentivirus, which...expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church Lab. Neuron-specific expression...

...editing assemblies are sensor-actuators, either transcriptional regulators or chromatin-modifying enzymes...s chromatin status into account (Fig 1a). The laboratory of Karmella Haynes at Emory recently contributed...with examples of potential synthetic chromatin mediators and probes. Image from Priode et al., 2021. ...flexibility of the single-pot system will allow investigators to rapidly create multiple synthetic chromatin-modifying...interactions By Jennifer Tsang GFP-based Touching Nexus (G-baToN) is a new tool developed by Rui Tang in Monte Winslow...label cells after cell-cell interactions. Using G-baToN, “sender” cells express GFP on the surface and “...receiver cell. Image from Tang et al., 2020.   G-baToN can also be applied to other fluorophore/antibody...

...single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies for neurodegeneration...lab recently "broke the PE efficiency barrier in tomatoes" by combining several approaches (Vu et al., 2024...chaperone, RT: reverse transcriptase. Right: T1 tomato plants that inherited an edited gene to confer ...the team was able to drive prime editing in both tomatoes and Arabidopsis, suggesting the improvements may... species. In addition, they generated offspring tomato plants that inherited the genomic edit but not ...prime editing enables heritable desired edits in tomato and Arabidopsis. Nature Plants, 10(10), 1502–1513...2024.12.20.629623.  A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic...

...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...

...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...monster, a meiotic arrest gene required for spermatogenesis One of our smaller conference rooms is named... meiotic arrest gene that is necessary for spermatogenesis. Flies that lack cookie monster are sterile...

...doi: 10.1038/s41592-022-01597-x. New glutamate indicator iGluSnFR3 Improving on the glutamate sensor iGluSnFR...PDGFR.codonopt    Aggarwal A, et al. Glutamate indicators with improved activation kinetics and localization...have developed a new genetically-encoded voltage indicator (GEVI) called JEDI-2P (Liu et al. 2022) optimized...Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Cell. 2022 ...has generated a new genetically encoded calcium indicator using small monomeric NIR fluorescent proteins... of a Small Near-Infrared Fluorescent Calcium Indicator. Front Cell Dev Biol. 2022 Jun 29;10:880107. doi...they developed a family of fluorescent potassium indicators with a range of binding affinities (Torres Cabán...

...genes. We’ve created enough regulatory elements, CRISPRs, repressors, activators. We’ve been putting together...CRISPR/Cas genetic circuits are linked to the regulatory networks within E. coli host cells. ...in building genetic circuits is getting enough regulatory proteins that don’t interfere with each other...programmable that you can make a very large number of regulators. That basically means larger genetic circuits...that could conceivably be as large as natural regulatory networks. Addgene: What kinds of things could... pattern, shape. All of that is controlled by regulatory networks in the plant. If we want to access that...CRISPR is how much easier it is to work with than regulatory proteins. There are still things we haven’t solved...

...In-Fusion cloning molar ratio calculators Lab math calculators These calculators can help you with common...lab. Molarity calculator Promega's Biomath Calculators Primer resuspension calculator (sign-in required...Golden Gate Reactions Cloning ligation reaction calculators These links can help you calculate how much insert...assembly, or In-Fusion cloning.  NEB Ligation Calculator: This tool works for any cloning method. You ...required.)  DailyCalcs Science Calculator app for iPhone Reagent and Data Repositories These links can help...

...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...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...with distinct programs controlled by each dCas9 regulatory switch. By no means limited to the field of metabolic...scaffolds can recruit various transcriptional regulators to alter gene expression at the targeted site...

...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 ...involved in the Ras pathway alone and thousands of investigators studying them, how do we ensure that the research...

...target genes using dCas9-based activators The simplest of the dCas9 activators consists of dCas9 fused to ... dCas9 and antibody-activator fusion proteins. The Synergistic Activation Mediator complex, generally ...exploited to localize factors such as transcriptional activators and repressors to target genes. Repressing target...activation domain - VP64.  Later generations of activators include the SunTag system which facilitates recruitment...generally the strongest activator, consists of a dCas9-VP64 fusion and a modified gRNA that is capable of interacting...with an additional RNA-binding transcriptional activator. Plasmids for activating target genes in a variety...fusion, both targeted to the promoter region. Cas9 activators include Cas9 fused to VP64, VP64 along with other...

... voltage indicator: JEDI-2P  by: Brian O'Neill  The François St-Pierre lab and collaborators have successfully...what you'll find in this post: Improved voltage indicator: JEDI-2P Novel class of light-gated potassium...successfully developed an improved voltage indicator (GEVI) called JEDI-2P. Using a custom, high-throughput...predecessor GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...screen for improved genetically-encoded voltage indicators (GEVIs) based on the ASAP sensors. The resultant...Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Cell, 18, 3408... cations. B) Cortical slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action...

...massive amount of information regarding which regulatory proteins, signaling molecules, mutations, and...paper (1). Lastly, it is possible to build gene regulatory RSMs (GRSMs, the subset of RSMs that regulate...also demonstrates that genetic parts (genes, terminators, and promoters) may be interweaved onto a GRSM...parts used in this paper (recognition sites, terminators, promoters, and genes) were built with flanking...them. We recommend using strong, directional terminators and promoters with buffer sequences on their ...ends (for insulation). A list of promoters and terminators used in our GRSM registers can be found in Table... parts (recognition sites, promoters, genes, terminators, etc) are composed. Fear not – the state machine...

...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...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 ...research. Promoter Subtype Promoter Example Activator Addgene Plasmid/Kit Alcohol-regulated AlcA...

...cut either strand of DNA. Traditional Cas9 regulators In addition to its utility in making targeted...to DNA, Cas9 can be reprogrammed to serve as a regulator of gene expression. Its catalytic domains can ...fused to effector domains such as transcriptional activators or repressors. These fusion proteins retain the... generate a potent Cas9-based transcriptional regulator without making any functional changes to Cas9....adapter capable of recruiting a transcriptional activator, robust regulation of gene expression is observed...the truncated gRNA recruits a transcriptional activator to the Cas9-gRNA complex. When this complex is...of the Addgene Scientific Advisory Board. His laboratory at Columbia University is focused advancing scientific...

...carrying a synthetic gene drive were to escape the laboratory, the drive could eventually spread through the...mistake would necessarily extend far beyond the laboratory and seriously damage public trust in scientists... can spread through populations of transgenic laboratory organisms but not wild organisms. For example...synthetic sequence will only spread in transgenic laboratory populations with that sequence2. Both methods...Reproductive confinement involves working with laboratory organisms that can't reproduce with wild ones...confinement seeks to keep the organisms in the laboratory. It varies by organism, but your local biosafety...population and immunizes it against the first drive. Laboratories interested in building candidate gene drives...

...the Tapscott Lab identified regulators of DUX4, a transcriptional regulator that when inappropriately expressed...activation using a fusion to a transcriptional activator such as VP64 (Gilbert et al., 2013), or repression...region to inhibit association of transcriptional activators (Qi et al., 2013). However, the fusion of dCas9...specific genomic region In 2013, the Hodaka Fujii Laboratory first described a method to purify a specific...lab is CRISPR affinity purification in situ of regulatory elements (CAPTURE) (Liu et al., 2017). This system...additional research conducted in the Hodaka Fujii Laboratory, please also see our interview with Dr. Fujii...immunoprecipitation identifies miR483 as an epigenetic modulator of IGF2 imprinting in tumors." Oncotarget 8.21...

...  Javier García-Nafría is an investigator scientist at the MRC Laboratory of Molecular Biology. He is ... Watson and Javier García-Nafría from the MRC Laboratory of Molecular Biology. Plasmid cloning is an essential...with successful recombination reported in all laboratory E. coli strains tested so far. The use of this...low cost and can be immediately adopted by any laboratory. All procedures are performed using the same...the homologous region (calculated using Oligo Calculator). Insertions - To insert short sequences of ... Watson and Javier García-Nafría from the MRC Laboratory of Molecular Biology. Jake Watson is a postdoctoral...postdoctoral researcher in neurophysiology at the MRC Laboratory of Molecular Biology. He is currently investigating...