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...with GeneWeld vectors GeneWeld from Jeffrey Essner’s lab uses CRISPR/Cas9 and a donor vector to create knock-ins... tools for your research! This time: GeneWeld vectors to create knock-ins CasX Drug inducible CRISPR/...proteins, fusion proteins, or transcriptional regulators. In this system, Cas9 generates a double-stranded...cargo (ex: fluorescent protein gene) on the donor vector. Therefore, targeting requires two sgRNAS: one ...site and a universal sgRNA that targets the donor vector sequence. Homology arms that flank the donor plasmid... Read the paper in bioRxiv Find the GeneWeld vector kit CasX, a small Cas protein, is active in E....editing by fusing Cas9 to a mutated human estrogen receptor. This protein fusion stays in the cytoplasm, but...

...populations using combinatorial viral approaches. Current protocols in neuroscience / editorial board, Jacqueline...and for good reason. For the first time in the history of neuroscience research we have a technology that...spent two months in the lab of Antonello Bonci, Director of the National Institutes of Drug Abuse Intramural... electrophysiology data too but that’s what collaborators are for). For example, if you study neural pathways...his optogenetic constructs available through the vector cores of various universities as well as Addgene...transfer agreement, and list of vendors (such as the vector core at the University of North Carolina) that ... Conduct a time course experiment in which you monitor expression at various time points (e.g. every 1...

...recently was invited to attend the first Principal Investigator meeting of the NSF EDGE (Enabling Discovery ...Discovering ways to culture, propagate, transform, store, and share all of these new organisms continues ...continues to be challenging. Organism specific viral vector systems would also be beneficial for many systems....unavailable.” As stated by one of the NSF program directors, this program should “enable the ability to study...parenting strategies and make important ecological indicators since species live in water and on land. Current...that tastes yucky (and is poisonous) for bird predators - birds eat a caterpillar once, vomit, and never...humans learn speech. Finches must learn early, use auditory feedback to learn, babble as baby birds, and have...

...was contributed by Erik Snapp, the Director of Student and Postdoctoral Programs at the Janelia Research...Structural Biology, and now serves as the Director of Student and Postdoctoral Programs at the Janelia Research... payoff for each aim. Learn to communicate your story with only the most salient details. Make the most...your research problem. Creating a coherent unified story and model can help make your ideas easier to understand...Practice presenting your chalk talk Perfect your story and delivery. Design simple figures and diagrams...track. Furthermore, practice getting back to your story. Committee questions can derail your talk. You need...from Oregon Health Sciences University, did his postdoctoral fellowship with Jennifer Lippincott-Schwartz...

...software. The Inkscape website has many tutorials and Lynda has a tutorial as well, which you may have access...professional-grade design software, like Adobe Illustrator, and I didn’t want to pay for these programs ... Vectr is like a pared down version of Adobe Illustrator. There’s both a web and a desktop version of ...layers, which let you lock and hide individual vectors (shapes defined by 2D points connected by lines...Inkscape Inkscape is the most similar to Adobe Illustrator out of the three options for drawing your own...and editing shapes and like Vectr, Inkscape uses vector graphics. There’s a steep learning curve for Inkscape...20,000 icons is designed by professional medical illustrators with input from life scientists. It’s easy to...

...would love to tell such a dramatic story, but it wouldn’t be my story. I love academics. If I didn’t, it... took on TA positions, tutored fifth graders, made a blog and worked as editor-in-chief for the Science...there was even a time when I was too nervous to ask store clerks for help at the mall. In undergrad I was ...thought that I would be best poised to share the stories and difficulties behind great discoveries by tackling...largely my work developing myself as a science communicator that got me here! Thus far being an Outreach... mission to make research easier. I’m hoping my story demonstrates that transitioning from academia doesn...Management for Scienitsts from Addgene Executive Director Joanne Kamens ...

...continuously monitor multiple systems. Ideally this tool would be wireless, able to monitor outcomes of...importantly distortion of the fibers didn’t affect their function (the gut twists and contorts during the...probe due to the lack of tools to continuously monitor it. How to address this? Through the pairing of...Previously, there were very few tools available for monitoring how both the brain and visceral organs interact...harvested after the behavior, or the animals being monitored were physically hooked up to invasive, cumbersome...indicating the system was indeed stable for continuous monitoring. On the gut side of things, the authors optigenetically...studies Channel rhodopsins for optogenetics Viral Vectors 101: Chemogenetics ...

...upstream of an effector/reporter gene. In cells where the stop cassette is removed, the effector is expressed...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles...cells with sgRNAiBAR library containing lentiviral vectors the iBARs can be detected by next generation sequencing...including Drosophila, fish, and mice, by replacing the effector with a gene of interest in the cells you are studying...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ...screening, calcineurin dynamics measurements, or monitoring in vivo. To overcome these technical issues, ...4X CDRE repeat. In addition to showing that this vector is suitable for flow cytometry and fluorescence...

...co-founder Melina Fan and former Associate Director of Viral Vectors Karen Guerin for the journal Molecular... in Gene Therapy, Director of the Grousbeck Gene Therapy Center, Associate Director of the Ocular Genomics...Addgene welcomes three new members to the Board of Directors: Khandan Baradaran, Frank Stegmeier, and Luk Vandenberghe...Baradaran’s expertise in viral safety, gene therapy vector development, vaccine development, and assay technologies...funded,” she says. Baradaran met Addgene Executive Director Joanne Kamens several years ago through the Healthcare...Officer at KSQ Therapeutics and is an Addgene depositor. KSQ uses a CRISPR-Cas9 platform to systematically...yeast and did a genetic screen to identify the factors that regulate cell division. And that really got...

... plasmids in the repository, each verified by sequencing, which makes the repository a convenient source...content and rare codon percentage were not good predictors. There is not much variation in GC content between...for GC content. Rare codons also are not good predictors because they are just that: rare. “Nature has...says. Percent sequence identity was a different story - the team found that the percent sequence identity... a synthetic version of that gene was a clear predictor of whether the gene is synthetic or natural. They...most common expression system within the Addgene repository is mammalian, but the largest source of unique...is from Proteobacteria to mammalian expression vectors. And more broadly, their data revealed that the...

...in a mammalian expression vector (pcDNA3) and a bacterial expression vector (pRSETB). Fabritius A, et...achieve this they created a lentiviral vector system that monitors both APOBEC- and Cas9-mediated Editing...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles...blue light. This tool has now been modified by Torsten Wittmann’s lab at UCSF to modulate cellular dynamics...New tools for developing new FPs - the mCarmine story Article contributed by Caroline LaManna  Listen ...segment The increasing popularity of single base-editors has resulted in the rapid expansion of the genome...rapid fluorescence-based quantification of base editors that utilize dCas9 fused to cytidine deaminases...

...It’s nearing the end of #DepositorWeek at Addgene and we’d like to show our gratitude on the blog by ...highlighting some of the great posts written by Addgene depositors. Below you’ll find brief summaries of several...several excellent guest posts from our depositors. Interested in writing your own post for the Addgene Blog...Tools In this post, Jessica Polka, formerly a postdoctoral researcher in the Silver Lab at Harvard University...them. CRISPR Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening CombiGEM is a technology that... organism. In this post, Chris Richardson, a postdoctoral researcher in Jacob Corn’s lab, describes how...Albert Cheng and Mark Wanner from the Jackson Laboratory for Genomic Medicine described Casilio, a system...

... introduced, stochasticity will result in a combinatorial expression of these three genes with different...inherently low. This is because the stochastic and combinatorial expression of fluorescent proteins is only possible...Tetbow In modern neuroscience, plasmid or viral vector-mediated gene delivery is more common than conventional...conventional transgenic strategies. In such vector-based gene delivery systems, the stochastic expression...original method, we used the Tetracycline trans-activator system to boost the expression levels, and we ...we named the system Tetbow (Tetracycline trans-activator Brainbow) (Sakaguchi et al., 2018) (Figure 1)....the functional and anatomical maturation of the olfactory system.      Takeshi Imai is a professor at Kyushu...

...Topoisomerase I is released from the vector when the PCR product and vector ligate; however, it can potentially...this sequence. The energy from this breakage is stored as a covalent bond between the cleaved 3′ DNA strand... days commercially available TOPO kits provide vectors or cloning arms with overhanging 3´ deoxythymidine...residues that are covalently linked to topoisomerase. Vectors in these kits also often have the topoisomerase...screening after transformation - self joining of the vector ends results in the production of blue colonies...Reaction: Mix together the PCR product and TOPO Vector.  3. Incubate 5 Minutes at Room Temperature: You...are planning to transform right away OR you can store the reaction at -20C overnight. 4. Transform TOPO...

...characteristics that make them ideal for studying in a laboratory environment. We’ve covered five model organisms...are five more model organisms that have left a historical impact on the scientific community. (And, check...have been specifically developed to thrive in laboratory settings. One of its most attractive features...Level 1 microbe makes it a perfect candidate for introductory teaching and research environments (Idalia &...  The E. coli genome has an extensive research history and characterization. Many proteins and processes...studies. Elly Tanaka’s lab has constructed several vectors for use in the axolotl including plasmids for use...organisms in the world. With its extensive scientific history, highly characterized genome, and widespread utilization...

...molecule integrin inhibitors and natural ligands, such as fibronectin, to block receptor function, e.g.,...technologies and laboratory techniques related to antibody discovery. You can find cell surface receptor biology...Integrins are essential cell surface receptors involved in complex signaling pathways and the linkage...Despite their ubiquity and significance, these receptors have proved challenging to study. Cells often ...integrin antibodies available through Addgene’s repository. The collection includes 26 unique antibodies...from its closed to its open state, activating the receptor. Ligands can bind to integrins in their closed...subunit. Using these affinity reagents, the investigators inhibited other RGD-binding integrins, individually...

...deaminase-mediated base editing (cytosine base editor or adenine base editor) and reverse transcriptase-mediated ... et al., 2019). Both cytosine base editors and adenine base editors have been developed for plants and...CRISPR has taken the genome engineering world by storm owing to its ease of use and utility in a wide variety... al., 2013). Nuclease dead Cas9 (dCas9) based activators (such as dCas9-VP64) or repressors (dCas9-KRAB...variety of Agrobacterium-compatible, Cas9 containing vectors ready for Golden Gate mediated cloning of your ... the Patron Lab.  As with all plasmids in the repository, we highly recommend reading the associated publications...Z, Wang Y, Wang X (2019) Rescue of a plant cytorhabdovirus as versatile expression platforms for planthopper...

...contributed by guest blogger Natalie Niemi, a postdoctoral fellow at the Morgridge Institute for Research...phosphorylation sites. If knowledge of a candidate regulatory kinase is known, it’s possible to generate non-phosphorylated...Often, however, researchers lack knowledge of the regulatory kinase, access to active kinase for the reaction...tRNA with phosphoserine A specialized elongation factor that allows proper extension and translation of...synthetase, and EF-Sep – an optimized elongation factor that accepts Sep-charged tRNAs during translation...This modification allowed the deletion of release factor 1, the protein responsible for terminating translation...TOPO E17TAG GFP and pGEX-6P-1. The pCRT7 NT TOPO vector contains a mutant pSer-containing GFP that can ...

...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles...activation, using expression of differentiation factors as the screen readout. This retroviral mouse CRISPR...library can be used for screening of cells that are refractory to lentiviral transduction, now enabling forward...lipoproteins, shuttles lipoproteins through the circulatory systems, but can also lead to the formation of...as both a powerful tool for generating complex regulatory systems and further the study of natural σ54-... PMID: 31451697. Genetically encoded voltage indicators for use in worms Article contributed by Brook...Brook Pyhtila Genetically encoded voltage indicators (GEVIs) change fluorescence in real time when neurons...

...the abundance of a protein variant library is monitored by fusing the variants to eGFP followed by an ... is an enzyme critical for activating clotting factors and is the target of the anticoagulant drug warfarin...serotonin sensor The goal of Elizabeth Unger’s postdoctoral work in Lin Tian’s lab at UC Davis is to generate...serotonin is a well-known but poorly studied neuromodulator, and evidence suggests that it is incredibly...sensor). In preliminary studies with the lab’s collaborators, they were able to use the serotonin sensor ...activity. Unger also plans to use the serotonin receptor to study how single nucleotide polymorphisms present...and dose response. Selective serotonin reuptake inhibitors are frequently used to treat depression and anxiety...