... developed an improved voltage indicator (GEVI) called JEDI-2P. Using a custom, high-throughput screening... the protein sequence of the predecessor GEVIs (called ASAP 1 and 2), they found an indicator that is ...approach and outcomes of the screen for improved genetically-encoded voltage indicators (GEVIs) based on the...optogenetic silencer in experiments with mouse cortical neurons, far out-performing previous attempts ...selective for K+ over other Na+ and other cations. B) Cortical slice of HcKCR1-EYFP and tdTomato expressed layer...

...also have a lipid bilayer surrounding the capsid, called an envelope. Viruses without an envelope are referred...non-enveloped, while viruses with an envelope are called enveloped viruses (Figure 1). Like bacteria, only...individual viral particles. A single viral particle is called a virion. Finally, viral vectors, which are used...labs. Viral vectors are viruses that have been genetically modified to either limit or completely eliminate...Viral Applications Viral Vectors 101: Viruses as Biological Tools Viral Vectors 101: Viral Vector Elements...

...constant regions are identical for antibodies within certain classification groups called isotypes.  Why does...that of a different group (Fig. 1). This process, called isotope conversion, gives users the flexibility...antibodies are plasmid-based and are created synthetically or derived from hybridoma antibody sequences...antibodies affects binding properties. In Journal of Biological Chemistry (Vol. 269, Issue 17, pp. 13048–13055...

...antibodies in your lab. Perhaps you’re even in that mythical place where antibodies are well-organized, documented... sample prep. This involves using heat and/or chemicals to break some of the bonds keeping the protein...epitope (right). Created withBioRender.com.   Biological Processes  If you’re feeling relieved that you...the available epitopes could be affected by, or critical to, the assay.   Next, look for antibodies that... also working for a Western because they both typically use denatured protein samples. If, however, you...

...reproducibility and an inability to improve upon these critical research tools. Addgene is excited to announce...reagent resource for the neuroscience community called NABOR (Neuroscience AntiBody Open Resource). We...partnered with Dr. James Trimmer of the University of California Davis School of Medicine on this grant. Dr. Trimmer...in neuroscience research today, but there is a critical unmet need for an open-access, molecularly defined...such as plasmids and viral vectors. However, it typically takes a few years for a new project to reach a...

...hereditary determinant”. His proposal, however, was basically ignored. A separate term, “episome”, defined as... plasmids have greatly impacted many areas of biological research and have been key in advancing our knowledge...Joshua. "Cell Genetics and Hereditary Symbiosis" Physiological Reviews 32.4 (1952) 403-430. Pubmed PMID: 13003535...PMC2134509. Cohen, Stanley N. et al. “Construction of Biologically Functional Bacterial Plasmids In Vitro.” Proceedings...Nomenclature for Bacterial Plasmids: A Proposal.”Bacteriological Reviews 40.1 (1976): 168–189. PubMed PMID: ...

...Harvard Medical School and runs everything science (CSO). Clinton is a cost analyst at Dow Chemical (a Fortune...a company in the health sciences without those critical three letters after your name (P-H-D).  Finally...two projects, including one led by a talented biomedical engineering team from MIT.   I would like to ...connections and finding mentors -- well, it's time you call upon these people for guidance.  Before even beginning...

...has a maximum of one gene knocked out. In this genetically heterogeneous population, cells behaving differently...knockout mice for (all of the genes involved as I recall). The lab then used these knockout mice to show...cells we study are migratory, and their exact localization is important for their function. Their migration... you could majorly accelerate progression into clinical trials. Secondary screens  If you have generated...genes you’d like to test for phenotypic effects. Typically the phenotype would be studied in vitro, but there...The different labs use different algorithms to calculate which would be the best gRNAs. Interestingly, ...

...to identify and reference documents, files, or physical reagents. What are persistent identifiers? Perhaps...persistent and exist online indefinitely. PIDs historically referred to objects that exist online, but this...approach has been adapted for the identification of physical materials that we use in our labs, including plasmids...Bourges for editing this post and sharing her technical expertise in persistent identifiers. Additional...Identification Initiative Compact identifiers for biomedical data How Addgene works to enhance scientific ...

...might not have to do the calculation yourself as many fluorometers will calculate your sample concentration...than 1.5. Then, using the A260 reading, you can calculate the DNA concentration. Generally, A260 of 1.0 ... start by pouring your gel containing a DNA intercalating dye (ex: ethidium bromide) and choosing a DNA...quantified over time using a fluorescent dye that intercalates into the DNA such that smaller fragments are...

...these proteins are FDA-approved or in clinical trials or preclinical studies already. We are following up...expand to other cell types, including more physiologically-relevant systems, to catch all the important... Nsp3 and Nsp16, and includes an additional catalytically dead version of the viral protease, Nsp5. Our...Melanie Brewer (right) from the University of California, San Francisco. Manon Eckhardt and Melanie Brewer...

...both perform an optimization-scale infection and at least one screening-scale experiment achieving the gRNA...Chavez, researcher at the Wyss Institute for Biologically Inspired Engineering and member of the Addgene... studying. You need to make sure your assay is scalable and robust. What you don't need to do is spend...particular, the Church lab at the Wyss Institute for Biologically Inspired Engineering and the Zhang and Doench...

...understood what I was going through. (Parenthetically, when I called the lab to ask my benchmate to take...within a few days or a week) so they can easily recall the encounter. As a side note, my guess is that...; focus on the nice people who do respond. Don’t call or email multiple times if you don’t get a response...years ago using a now defunct (but fun) plug-in called Maps.  Figure 1: See a starfish? This is...

...several treatments are currently being tested in clinical trials. It is speeding the process of the discovery...saw these two papers,” said the University of California Berkeley’s Jennifer Doudna. “They are the first...continue to be a primary preoccupation of her California lab and many others. “Those off-target effects...cleavage sites are repaired.” Those insights will be critical for scientists’ ability to influence editing efficiency...Application in Genetic Diseases: A Review.” Current pharmaceutical biotechnology. https://doi.org/10.2174/1389201021666200621161610...

...four proteins Oct4, Sox2, Klf4, and cMyc (the so called OSKM factors). As the Yamanaka lab's primary Addgene...field has exploded so quickly, citing the first clinical trial with iPSCs with his collaborator Takahashi...picture perspective: Dr. Yamanaka talked about his clinical trial patients with advanced macular degeneration...Eggan presented work that is leading to promising clinical trials for ALS.   I have an uncle with Parkinson's...the first stem cell line that could be used in clinical trials. The quality control process alone takes...

...nearly 50%, showing proof-of-concept for pre-clinical and clinical applications of dCasRx. The identification... with CasRx and AdmCas13d fused to a nuclear localization signal (NLS). In a HEK293 mCherry reporter assay...target essentially any RNA in a cell. Since catalytically dead dCasRx maintains its RNA-binding properties...enhancers. After AAV-mediated delivery to iPS-derived cortical neurons, dCasRx-mediated exon skipping improved...

...plasmids can be used to carry out any biochemical and biophysical study involving protein stability or ...degradation. Despite how easy these experiments are to physically set up in the lab, RNAi is often completely ineffective... most scientists use mutant animals that are genetically altered to overexpress endogenous RNAi pathways...facilitate protein-protein interactions and mediate biochemical processes. One of the largest and best characterized...

...robust, with an eNL-based calcium sensor displaying a 500% signal change upon calcium binding. Suzuki et al...around these issues, but this solution wasn’t practical due to the low intensity of luminescent proteins... to previous publications. They also developed calcium, cAMP, and ATP sensors based on Nano-lantern, showing...not alter cellular physiology in your system. Optical sectioning cannot be conducted with luminescence...

...plasmid with the delivered cargo (often a gene) is called the transfer plasmid while the other plasmids with... with packaging or replication components are called helper plasmids. Below, we’ll breakdown the vector...viral experiments is to deliver a genetic cargo, typically an expression construct, to cells/organisms. This...yield dsDNA. Unlike LTRs, ITRs are perfectly symmetrical to allow for this pairing to occur. Like LTRs...gene, making it replication deficient. This is typically supplied by the production cell line (HEK-293)...

...made of chemicals, which have an unfortunate tendency to react with other chemicals and biological structures...antibody out on the bench overnight. Do not panic! Typically, antibodies are okay if they’re left out overnight... fluorophores, which work not by reacting to a chemical, but by emitting a specific wavelength of light...

...Bryan Roth’s lab created an open source resource called PRESTO-TANGO (parallel receptorome expression and...activated downstream of GPCRs. This collection, called TRUPATH, allows researchers to identify specific...neurons and specific behaviors. Chemogenetics uses genetically engineered receptors to interact with small molecules... to optogenetics but instead of light, these genetically modified receptors are activated by small molecules...some GPCRs further to respond to specific, pharmacologically-inert, small molecules. These new receptors...

...professors, and mentors. Ask for specific guidance on grammatical errors, sentence structures, and overall format...support.        5. Application submission  Set a calendar reminder for every deadline and shoot to submit...Pierre is currently a doctoral candidate in the Chemical Biology Program at Harvard. Roo wants to simplify...simplify access to academic resources among economically disadvantaged populations. Roo is the co-founder...resources that can help their children thrive academically. Rose is the co-founder of STEAMid.     Additional...

...transcription factors, starting with an established method called ChIP-seq. ChIP-seq relies on antibodies to recognize...We’ve been doing that for years using this assay called ChIP-seq, but it’s really hard for lots of transcription... the second plasmid inserts a small epitope tag called FLAG at the end of the transcription factor. It...have good antibodies. The results look almost identical. Addgene: What advantages does your system have...happy to provide them, that never works on a large scale. The world is too big now. Mendenhall: So many groups...

...missing or damaged nucleotides that are common to biological sources of DSBs, and which need to be processed...generated in the course of repair by NHEJ are typically small (1-10 bp) but extremely heterogeneous. There.... NHEJ can also be engaged by variants of the canonical Cas9 approach. A pair of CRISPR guides that flank...Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining. Molecular Cell 55:829...CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens        ...

...present in the cell, it can also introduce non-physiological RNA-protein interactions due to sample processing...its rate of biotinylation. The improved enzyme, called BASU, is derived from a BirA* homolog found in ... interactions in events that occur on a short timescale, such as during drug action or cell signaling....t be used to study endogenous RNAs at their physiological expression levels; however, knock-in of BoxB... by 90%, suggesting that QKI is a host protein critical to Zika replication. Human genetic disease. RaPID...

...and success. Customer and technical support Our customer and technical service brands continue to ... We score ~99.9% helpful and the customer and technical service teams put 100% effort into helping our...positive, supportive culture, we are attractive to the local employee pool and we get a lot of successful candidates...connections and even via Twitter. We love working with local organizations like Just-a-Start, the Northeastern...

... department at the University of Massachusetts Medical School. We are working with postdocs at other institutions... and all 6,000+ postdocs at the University of California, who had a contract - negotiated by their union... December 1st. Salary inconsistencies across geographical regions When we originally started gathering...salaries. Postdocs at the Salk Institute in La Jolla California are not getting a raise. Meanwhile, in less densely...changes in science. Follow him on Twitter @BiophysicalFrog and Future of Research @FORsymp.   Additional...

...ribonucleoprotein, and AAV.  Read the paper in Nature Chemical Biology Find the AsCas12f1 plasmids   Selective... Mike Lacy The retroviral-like protein PEG10 specifically binds its own mRNA to facilitate its packaging...of cargo sequences in the capsid, in an approach called Selective Endogenous eNcapsidation for cellular... the prime editor, yielding PEmax, to work synergistically with epegRNAs and PE4 and PE5. These enhanced...systems improve gene editing performance at therapeutically relevant gene targets and expand the application...

...donating and receiving labs)” — Jim Woodgett, biomedical researcher at Lunenfeld-Tanenbaum Research Institute...reagents and protocols so easy!” — Nina Jain, Harvard Medical School. We want to give a special thanks to the...decade. We did this the old-fashioned way: through physical outreach, visiting organizations, knocking on ...standardized agreements and electronic signatures to dramatically speed up MTA approvals. In 2013 we were thrilled...for being Addgene. You guys rock." — Andrew York, Calico Life Sciences. Most of all, we’re incredibly thankful...

...requisite for a functioning lab, but it is for a scaleable lab. When you need to track the location, quality...our second example we’ve added some simple but critical logic. Some steps fail “hard”, they reset the ...interchange them easily as needed. In this case, the physical considerations of the laboratory make object composition...science_name`. You'll need to decide given the technical details of the problem you're solving, but consistency...interesting biotechnology code.There are valid technical reasons to choose a non-Python stack, but if you're...

...sequence gives the loxP site directionality, and the canonical loxP sequence is ATAACTTCGTATA-GCATACAT-TATACGAAGTTAT...the two strands are ligated, resulting in the reciprocal crossover event. This event can have three general... features with loxP sites on either side (often called a “lox-stop-lox” or “LSL” cassette) upstream of...putting the loxP sites on either side of a gene (called “floxing”, for “flanked by loxP”), will permit ...where "N" indicates which bases may vary from the canonical sequence. Among these are loxN (GtATACcT), lox2272...

...and on self-propagation by reproduction. Both physiological processes are controlled by deliberate and compulsory...receptors (NRs), their coregulators, and their physiological endocrine ligands. Nuclear receptors and their...the diet. Additional members of this family are called orphan receptors because their ligands remain unknown...circadian regulation, as well as in important physiological states such as adipogenesis and macrophage activation...widely used today.  The sheer abundance of these critical research tools is a double-edged sword, however...

...the transfection method (i.e. polyethyleneimine, calcium phosphate or liposomal methods), the volume of ...unconcentrated prep. For example, at Addgene we typically concentrate 100-fold meaning that a 10 cm2 dish...experiment requires a certain volume of virus, consider scaling up by either increasing the number of 10 cm2 dishes...be optimized prior to starting. We recommend calculating the ratio of the surface area of the larger vessel...lentivirus is the cell line that is used for titering. Typically researchers will choose a relatively permissive...

...expression observed with wild type CoChR, soCoChR localizes primarily to the cell body with very little signal...engineered microbes, used coffee grounds could be economically repurposed as starting material for the bioproduction... FLINC biosensors for live-cell imaging of biochemical activity Article contributed by Angela Holmes... live-cell imaging of the dynamic changes in biochemical activity within the cell. Their Fluorescence ...peak of 519 nm. These proteins have improved photophysical properties and can be used in quenching, FRET...

... from E.coli, named TadA7.10 in ABE7.10)  a catalytically impaired Cas protein (dCas or Cas nickase)  ... the eighth generation of adenine base editors, called ABE8 (Gaudelli et al., 2020). The generated ABE8s...accidental insertions and deletions (indel) by the catalytically impaired D10A nickase from ABE7.10 can be a ...the authors created ABE8 constructs using a catalytically “dead” S. pyogenes Cas9 (dC9-ABE8.x-m/d). The...even in adult patients. In order to analyze the clinical use of ABE8 for primary human cell editing, the...

... staying on top of the ever expanding body of biomedical literature! PubMed PubMed is the classic search...interest.  Best uses Accessing the vast body of biomedical research papers. Twitter Scientists often use...papers, and more. Saving papers There isn’t technically a way to save papers on Twitter, but you can ... contain a short summary of the topic and a chronological list of highly cited papers that contain your...also just follow feeds curated by Meta. Feeds automatically update as papers are published, similar to Facebook...

...past decade, primary cells remain difficult to genetically edit due to toxicity from electroporation or ...some cases) and minimal toxicity, including in clinically-relevant lines such as CAR-T cells. Plus, this...Cas9, fused to HIV TAT, Myc and SV40 Nuclear Localization Signals, and GFP. B) Workflow to assess Cas9...microscopy in HeLa cells expressing COX8a-mStayGold. Scale bars 10 μm (main), 1 μm (inset). Image reused from...versions. Thanks to Christophe Leterrier, whose FocalPlanes piece on mStayGold inspired and informed this...

...methyltransferase DNMT3A allows researchers to easily add biochemical marks to genetic regions from which where they...eventually, develop treatments, we need tools to specifically edit methylation sites.  Step aside, transcriptionally...interact with transcriptional machinery to modify local gene expression, confounding any potential conclusions... DNA. When dCas9 is bound to a target CpG, it physically blocks DNMT1 from that site. So, when DNMT1 methylates...Szyf 2021.   Let me leave you with a few practical considerations: Design your gRNA to overlap the...

...can contain different neurotransmitters. Using genetically encoded fluorescent proteins coupled with Fibronectin... endogenous synaptic proteins allowing them to localize to the synapse and minimize extraneous fluorescence...Jerome Goudeau, Cynthia Kenyon, and colleagues at Calico Life Sciences. Like many split FPs, split-wrmScarlet... genes whose protein products have distinct localizations. Figure 2: Split-wrmScarlet labeling ...AAV5. Find the AAV prep here The red-shifted calcium sensor jRGECO1a is now available as AAV-PHPeb. ...

...using either loxP or lox511 sites, leaving two identical sites on one end of the DNA fragment (Figure 1...recombination event then excises the DNA between the identical loxP or lox511 sites (Figure 1, bottom). Since...presence of Cre recombinase. This plasmid now specifically drives expression of mCherry instead of BFP.... rescue with in the antisense orientation (let’s call the gene “GeneR”). The key to this experiment is...information. References: 1. Schnütgen F, Doerflinger N, Calléja C, Wendling O, Chambon P, and Ghyselinck NB. A ...