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We narrowed to 1,076 results for: TOR;

Showing: 301 - 330 of 1076 results

  1. Hot Plasmids May 2018 - Optogenetics, Decaffeination, Biosensors, and Fluorescent Protein Tools

    Type
    Blog Post
    ...proteins for use as FLIM-FRET acceptors  Listen to the FLIM-FRET acceptor podcast segment Article contributed...FLIM-FRET acceptors here. See Addgene's FRET reference page for many other common FRET vectors. Murakoshi...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles...termied soCoChR, by fusing a fragment of the kainate receptor subunit to the C-terminus of the protein. Unlike...soCoChR-expressing cells. Finally, using synaptic receptor blockers to distinguish real and artifactual postsynaptic...placesTagRFP-T and Dronpa in close proximity. By monitoring changes in TagRFP-T fluorescence intensity at...Murakoshi lab has developed two new FLIM-FRET acceptors, ShadowG, a novel dark green fluorescent protein...

  2. Tips for Titering Your Lentiviral Preps

    Type
    Blog Post
    ...primers that target the common features of viral vector backbones; once validated these universal primers...higher than for functional assays depending on the vector backbone. When using qPCR based methods for viral...plasmid which can over-inflate results. A packing vector deficient in reverse transcriptase will produce...the number of target cells that are positive for vector encoded transgene expression following transduction... prep. This method is particularly useful when vectors carrying fluorescent markers are used as it eliminates...significantly higher than the same prep that has been stored in the freezer. If you plan to freeze a batch of...titration of pseudotyped HIV-1-based lentiviral vectors."Nature protocols 4.4 (2009): 495-505. PubMed PMID...

  3. Your Top Requested Plasmid in 2015!

    Type
    Blog Post
    ...We’ve dug into the data from our repository to find our most requested plasmid in 2015. In line with ...that the plasmid most requested from the Addgene repository in 2015 was... lentiCRISPR V2   LentiCRISPR...fantastic research tool has been requested from the repository nearly 1,000 times in 2015! If you haven’t already...suggests, lentiCRISPR v2 is a lentiviral transfer vector used to deliver both Cas9 and your gRNA of interest...lentiviral titer as compared to previous lentiviral vectors. It can be used both to knock-out single genes ...is available from Addgene both as a single empty vector and in pooled libraries containing thousands of... has already been used for includes work probing mTOR signaling in cancer cells, deciphering the mechanism...

  4. Cloning Mammalian Cells with the Agarose Method

    Type
    Blog Post
    ...recently we have also been using Addgene CRISPR vectors to generate cell lines with knockouts of specific...specific genes. Many years ago, people in the laboratory became frustrated with using glass cloning rings ...sterile truncated pipet tips. Indeed, an experienced operator can pick 48 clones from a 10 cm dish in 15- 20...medium containing serum in each well. A 200 ul pipettor is set to 200 ul, wiped down with 70% ethanol,...individually punched out, gently sucked up into the pipettor, placed into wells, and partially dislodged from...YouTube video. Before returning the plate to the incubator, additional growth medium is added to about halfway.... We highly recommend the use of an electronic pipettor with variable spacing (such as the Viaflo from...

  5. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    ...variant can then be fused to effector domains such as transcriptional activators or repressors. These fusion...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 ...hairpin, that is capable of recruiting additional effector domains (4). Taken together, these minor gRNA ... 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...

  6. Top Requested AAV of 2017: pmSyn1-EBFP-CRE

    Type
    Blog Post
    ... and other viral vector tools in the years to come. We’ll make many more viral vector tools and resources... Service Page Browse Additional Viral Vectors Find Viral Vector Protocols ... retrograde (the most popular form), this viral vector is used to deliver DNA encoding Cre recombinase...be sure to check back often! To find more viral vectors available through Addgene, be sure to check out...out our viral service page and browse our viral vector blog posts for many more tips and ideas on how to...thanks to Hongkui Zeng’s lab for providing this AAV vector! Additional Resources AAV Titers: Where do they...for Gene Expression in Mammals Subscribe to Viral Vector Blog Posts Additional Resources on Addgene.org...

  7. Isolating Genomic Regions of Interest with the CRISPR System

    Type
    Blog Post
    ...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...interest, the promoter region of the transcription factor IRF-1. Immunoprecipitation, using an antibody targeting...lab is CRISPR affinity purification in situ of regulatory elements (CAPTURE) (Liu et al., 2017). This system...miR483) that binds to the promoter of the growth factor IGF2 open reading frame to relax imprinting (Zhang...

  8. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

    Type
    Blog Post
    ...catalytic protein and its cofactor VP35. VP30 is a transcription enhancement factor. When used to study minigenomes...Addgene.org Browse Our Viral Vector Plasmids Read Our Lentiviral Vector Guide Read Our AAV Guide  ...producing infectious virus (similar to lentiviral vectors) because they do not encode all of the necessary...changing the minigenome cis-acting sequences and monitoring effects on genome replication and transcription...Microbiology, National Emerging Infectious Disease Laboratories.  She is interested in genome replication and...Karl-Klaus Conzelmann. "Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is...on the Addgene Blog Read Other Posts on Viral Vectors Check out Our Scientist's Guide to Ebola Contribute...

  9. What's New in CRISPR - September 2019

    Type
    Blog Post
    ...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...

  10. A Primer on Optogenetics: Introduction and Opsin Delivery

    Type
    Blog Post
    ...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...

  11. What's Your Organism? Expanding Genomic Tools via the NSF EDGE Program

    Type
    Blog Post
    ...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...

  12. Designing Your Chalk Talk for the Academic Job Interview

    Type
    Blog Post
    ...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...

  13. Early Career Researcher Toolbox: Free Tools for Making Scientific Graphics

    Type
    Blog Post
    ...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...

  14. Walking beside Academia and giving it a high five: My transition from graduate work to a position at Addgene

    Type
    Blog Post
    ...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 ...

  15. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

    Type
    Blog Post
    ...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...

  16. Meet the New Addgene Board Members!

    Type
    Blog Post
    ...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...

  17. Tetbow: Bright Multicolor Labeling for Neuronal Tracing

    Type
    Blog Post
    ... 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...

  18. Hot Plasmids September 2018 - Optogenetics, RNA Localization, Fluorescent Protein, and Base-editing Tools

    Type
    Blog Post
    ...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...

  19. Multifunctional Neural Fibers: Modulation from Gut to Brain

    Type
    Blog Post
    ...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 ...

  20. Natural or synthetic? How Addgene’s dataset reveals trends in biological innovation

    Type
    Blog Post
    ... 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...

  21. 10 Great Guest Posts We're Thankful For

    Type
    Blog Post
    ...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...

  22. Plasmids 101: TOPO Cloning

    Type
    Blog Post
    ...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...

  23. Five Popular Model Organisms, Part 2

    Type
    Blog Post
    ...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...

  24. An Integrin Antibody Toolkit from IPI

    Type
    Blog Post
    ...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...

  25. CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9

    Type
    Blog Post
    ...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...

  26. Using Phosphoserine to Study Protein Phosphorylation

    Type
    Blog Post
    ...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 ...

  27. Fluorescent Protein Travel Awards - Protein Variants, a Serotonin Sensor, and an Artificial Leaf Replica System

    Type
    Blog Post
    ...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...

  28. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

    Type
    Blog Post
    ...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...

  29. The Crispr Babies Saga Shows the Need for Action, Not More Delays

    Type
    Blog Post
    ...upon a moratorium until another meeting, three branches – a private funder, government regulators, and ...there was a generally agreed-upon international moratorium on utilizing CRISPR/Cas9 in humans. This topic...mtDNA transfer options being investigated; A UK regulatory body tasked two separate non-governmental groups...technology to research centers engaged in long-term, mandatory follow-ups and observational studies of the technique...whether or not scientists can recognize so-called bad actors among them and “self-police” or whether the media...to argue otherwise). To be sure, throughout the history of medical research, there have always been people...stop punting decisions on scientific, ethical, regulatory, and general public consensus to the next meeting...

  30. Synthetic Photobiology: Optogenetics for E. coli

    Type
    Blog Post
    ... fields, including synthetic biology. Addgene depositors Christopher Voigt and Jeffrey Tabor have been...and inactivate biological processes. Chemical effectors are suboptimal for a number of reasons, including...cross-reactivity with other pathways. Since these effectors are diffusible, they’re also not suitable for ...histidine kinase that phosphorylates a response regulator (RR). TCS-induced responses are varied; one outcome...conditions, the chromophore activates the response regulator via phosphorylation, promoting transcription of...spread across three plasmids, with the response regulator encoded in the genome. To make the system less...The utility of these TCSs was limited by a few factors. These bulky systems were spread over a number ...
Showing: 301 - 330 of 1076 results