... from the Structural Genomics Consortium (SGC) Toronto collection...Genomics Consortium Plasmids Browse SGC Toronto collection Structural Genomics Consortium Plasmids Structural...Structural Genomics Consortium Plasmids - Toronto Plasmid Gene/Insert Vector Type Backbone Mutations Tags ...

...Monosynaptic Neuronal Tracing Viral Vector Packaging Service: Tools for Monosynaptic Neuronal Tracing These... tracing from single, genetically targeted neurons. Neuron. 53(5):639-47. PMID: 17329205 Don’t See What...service encoding tools for monosynpatic retrograde neuronal tracing, to be used with modified rabies virus...with modified rabies virus to perform retrograde neuronal tracing. Note : Addgene does not distribute rabies...rabies virus. AAV Vectors for Monosynaptic Neuronal Tracing ID Name Description Serotype PI 52473 pAAV-synP-FLEX-splitTVA-EGFP-B19G...that it could only infect a genetically specified neuronal population. This is done using the envelope protein... pseudotyping the rabies virus with EnvA, only neuronal cells engineered to express TVA will be infected...

...University Stephen Sligar , University of Illinois Ron Vale , University of California, San Francisco Browse...

...Jan-Willem Veening Christopher Voigt Clifford Wang Ron Weiss Synthetic biology describes the rational engineering...plasmids related to intercellular signaling and environmental sensing. Strains Browse Addgene's collection...

...the brain to visualize how neurons are connected. Viral Vectors as Neuronal Tracers Due to their ability...individual neurons they infect. However, they generally are not transmitted to adjacent neurons via synapses...Converting a transsynaptic neuronal tracer into a monosynaptic (first order) neuronal tracer For RABV to crawl...nerve terminals of adjacent neurons (i.e., it is taken up by projection neurons). Research into these phenomenon...order neurons, via entry mediated by the G protein/G-protein receptor. Once in first order neurons, the...most of the projections to these new neurons came from nearby neurons. It wasn’t until later time points...mapping of direct inputs to midbrain dopamine neurons. Neuron. 2012 Jun 7;74(5):858-73. Pubmed PMID 22681690...

...Dopamine? DA is produced by specific dopaminergic neurons in a brain region called the substantia nigra, ..., which is the main source of the body’s DA. Neurons often fire at a certain frequency, and this frequency...understood, the direct effect of DA binding to a target neuron is more mysterious. Thus, scientists have developed...dynamics of optogenetically activated and inhibited neurons in live mice Report modulation of DA signaling ...brain slices Demonstrate DA detection in a single neuron in vivo in Drosophila Detect DA dynamics in response...PMC426888. Patriarchi, Tommaso, et al. "Ultrafast neuronal imaging of dopamine dynamics with designed genetically...

...Limitations of common genetically encoded neuronal tracers Neuronal tracers such as epitope tags and fluorescent...experiment using smFPs and standard neuronal tracers. 2. Visualization of neuronal sub-cellular structures GFP...label somata but not neuronal processes. In contrast, smFPs can label fine neuronal structures with high...functions and others are controlled by collections of neurons that are intricately wired into circuits through...have recently made it much easier to map these neuronal networks. For almost three decades, recombinant... in order to define those that target specific neuronal populations in the brain. The Allen Institute ...quickly find the best AAV serotypes to target the neurons they’re studying. Thanks to this work it is now...

...parts of the brain is mediated via individual neurons. Neurons are composed of a cell body, with dendrites...proximal neuronal cells. In the specific example of episodic memory, a subset of hippocampal neurons projects...axons directly to the prefrontal cortex, but also indirectly via synapses to neurons in other brain regions...using these viruses to mark neuronal connectivity a large majority of neuronal cells are labeled overtime...auditory neuronal circuitry in vivo. A variant of RABV has been developed to only label a neuronal connection...AAV vectors can be equipped with strong promoters for high and neuron-specific transgene expression. Fluorescent...cell types, for example only dopaminergic neurons, neuronal tracing has the added benefit of only labelling...

...available types of degron tags. What is a degron tag and why should I use one?   Degron tags are a class...Finally, some degron tags can do more than just degrade! Read on to find out more. Common degron tags When... comes to degrons, you have options! Below we will review the 4 most common types of degrons and some ...protease is situated between the degron and the gene of interest and the degron domain is terminal. Between...the protease and degron remain attached to the tagged protein. In this event, the degron domain shuttles...In the last decade, the use of degron tags has become increasingly popular for the modulation of endogenously...expressed proteins. Here we will review what advantages degron tags can offer over other protein control methods...

...bidirectional promoters, or the creation of bicistronic or multicistronic vectors. Unlike promoters which will...upstream cistron will have a few additional residues added to the end, while the downstream cistron will ...transcripts for each gene that is expressed, multicistronic vectors simultaneously express two or more ... so in this blog post we’ll cover basics of multicistronic vectors: why they are useful, how they work... and how to get started with them. Why Use multicistronic vectors? Detecting cells that are expressing... selectable markers from a unique promoter, multicistronic plasmids ensure that any cells that are positive...derived from the same transcript. Of course multicistronic vectors do not have to exclusively be used ...

...backstage team. Meet the microenvironment, a tumor’s backstage team. Microenvironments are cellular regions...Tumors are surrounded by microenvironments and what encircles a disease is highly prone to affect it. While...type in the tumor microenvironment is the immune cells. The contradiction is strong in this one. After...cancer microenvironment Ever since Stephen Paget’s “seed and soil” theory in 1889, microenvironments have...Isaac P. "The tumor microenvironment: the making of a paradigm." Cancer Microenvironment 2.1 (2009): 9-17...associated fibroblasts (CAFs) in tumor microenvironment." Frontiers in bioscience: a journal and virtual...precisely that for breast cancer. What are tumor microenvironments made of?  A tumor’s genesis is divided into...

...solution for discriminating between neurons for light microscopy-based neuronal reconstruction. To achieve stochastic... et al., 2016, 2018).   How to use Tetbow for neuronal labeling To use Tetbow, you can introduce the ... is interested in developing genetic tools for neuronal circuit tracing.     Marcus Leiwe is an assistant...Meng-Tsen, et al. "Super-resolution mapping of neuronal circuitry with an index-optimized clearing agent...and Takeshi Imai. "Bright multicolor labeling of neuronal circuits with fluorescent proteins and chemical...Read about Brainbow Learn about using AAVs for neuronal tracing Visit the viral vectors blog topic page...

...that your project is an innovative solution with a strong impact, or a journalist does not understand the...

...thrive, in the face of some of Earth’s harshest environments: corrosive pH ranges, crushing pressures, near-melting...folding at low temperatures, since mesophilic chaperones would not function well under suboptimal conditions...engineering a plasmid which contained cold tolerant chaperones (the Cpn 10/60 isolated from Oleispira antarctica...the figure below shows, E. coli expressing the chaperones protein resulted in higher levels of growth compared...help foster a more productive and collaborative environment for research.  We’ve also involved ourselves ...

...and developed the SciNote Electronic Lab Notebook. Implementing an electronic lab notebook starts with ...should have functionalities such as: electronic signatures, electronic witnessing and audit trails. How ...write things down when doing scientific research? Electronic lab notebooks and researcher's needs The familiar...traceability and reproducibility of research. Electronic lab notebooks (ELNs) are here to make lab work...explored the barriers to adoption within academic environments to better understand the issues labs are facing...traceable. However, switching from paper to an electronic lab notebook requires time and knowledge. In ...recent article, we translated our knowledge of electronic lab notebooks into 9 questions that will help...

... the brain, a connectome that would detail how neurons form circuits and where they synapse. View the ...Golgi staining, injection of diffusible labels, or electron microscopy of stained sections, were limited due...inversion of the construct. Sanes and Lichtman used pronuclear injection to insert these Brainbow constructs...vast array of colors needed to mark individual neurons, the system also suffered from a number of limitations...the tendency of fluorophores to aggregate in the neurons’ somata. Second, the system did not permit analysis... disproportionately expressed by a majority of neurons, limiting the number of distinct colors that could...through at least six generations. In addition to the neuronal pThy1-Brainbow constructs, Addgene also has two...

...Nicola Patron is Head of Synthetic Biology at the Sainsbury Laboratory, where she often feels more like...that go on between plants and their pathogens. As Patron explains it, her work has always been focused on...Modular Cloning (MoClo) Kit. What does it include? Patron: Sainsbury has a mandate to share and educate and...Addgene: Will the MoClo tools work in any plant? Patron: The MoClo kit is entirely for dicots, not grasses...tips for those looking to get started with MoClo? Patron: The parts will be familiar to most plant scientists...there other tools you are working on for plants? Patron: Our lab and others at the Sainsbury are working... of engaging about science in that way for you? Patron: I started my Twitter account recently inspired...

...neuroscience with many brain regions, neuronal networks, and specific neuronal populations to consider. To tease...Making use of AAV’s selectivity for neurons: targeting defined neuronal populations using viral strategies... in neurons that project to different regions of the brain. A more locally defined set of neurons can ...tease apart how different neurons impact a certain output, neuroscientists have begun to use more precise...genetic tools with the manipulation of specific neuronal subtypes and determine how these changes affect...combinations of AAV-based molecular tools for targeted neuronal manipulation. The goal of the workshop was to ...practical guide which was recently published in Frontiers in Neuroanatomy.  Here are the three main takeaways...

...Elementary and Middle Schools   Image courtesy of Ron Mader, via Flickr, under the Creative Commons license...education that is provided through a structured environment such as the classroom or a defined learning program...

...miRNA regulation have yet to be fully elucidated. Ron Weiss’s lab at MIT pushed the field further with ...halves to enable VAMP2 cleavage, and suppresses neuronal transmission within minutes. Constructs using ...split BoTN/B plasmids at Addgene. Liu Q et al., Neuron. 2019. PubMed PMID: 30704911. Acoustic reporter...

...hTLS.pBSKS(+) FUS T7 ALS David Ron 21828 TLS 2: hTLS.pCDNA1 FUS CMV ALS David Ron 21829 TLS 3: TLS.pRSET.C ...TLS.pRSET.C FUS His T7 ALS David Ron 21830 TLS 4: hTLS-CHOP.pBSKS(+) FUS T7 ALS David Ron 21831 TLS 5:hTLS-CHOP....Friedreich ataxia Michael Ristow 15239 pWBC KIF5A T7 ALS Ron Vale 15402 pBlue-hFX(AA88)-HA FXN HA, HIS, AU1 T7...hTLS-CHOP.pCDNA1 FUS CMV ALS David Ron 22106 pWJPrP5 PRNP Dementia Susan Lindquist 22109 pWJPrP1 PRNP Dementia...Friedreich ataxia Ronald Kahn 11443 pBS human Frataxin (myc tag) FXN Myc T7 Friedreich ataxia Ronald Kahn 11487...Friedreich ataxia Ronald Kahn 11504 pBabe hygro human Frataxin FXN Friedreich ataxia Ronald Kahn 11505 pBabe...telangiectasia Didier Trono 14581 pSUPER.ATMi ATM H1 Ataxia telangiectasia Didier Trono 14666 pQE9-His-p97...

...23086203. PubMed Central PMCID: PMC3699407. Milo, Ron. "What is the total number of protein molecules per..., that strongly binds the glycoprotein, avidin or a similar protein, streptavidin. This strong interaction...are approximately 2-4 million proteins per cubic micron in bacteria, yeast, and mammalian cells (Milo, ...ascorbate peroxidase (APEX), used as a genetic tag for electron microscopy, could be used for efficient proximity...of hydrogen peroxide. This radical reacts with electron-rich amino acids such as tyrosine on neighboring...Stephanie S., et al. "Directed evolution of APEX2 for electron microscopy and proximity labeling." Nature methods...peroxidase as a genetically encoded reporter for electron microscopy." Nature biotechnology 30.11 (2012)...

...°C. 0.1% Pluronic-F68 in PBS (A) Add 500 µL of 10% Pluronic-F68 to 49.5 mL PBS 0.01% Pluronic-F68 in PBS...Stocks of Pluronic F68 Pluronic F68, 10% solution A: 0.1% in PBS: 49.5 mL PBS + 500 µl Pluronic F68 B: 0.01%..., Hamilton 1X PBS pH 7.4 1X PBS-MK buffer 100X Pluronic-F68 NaCl MgCl 2 KCl Centrifugal filter units (...PBS (B) Add 5 mL of Buffer A to 45 mL PBS 0.001% Pluronic-F68 in PBS + 200 mM NaCl (C) (formulation buffer...Procedure Cover the filter membrane with 15 mL of 0.1% Pluronic F68 PBS and incubate for 10 min at RT Spin at ...and discard the flow through. Add 15 mL of 0.01% Pluronic F68 PBS and incubate for 10 min at RT. Spin at... discard the flow through. Add 15 mL of 0.001% Pluronic F68 + 200mM NaCl PBS. Spin at 3000 rpm for 5 min...

...Supermix for Probes no dUTP, Bio-Rad,1863023 10% Pluronic F-68, Thermo Fisher, 24040032 Droplet generation...Reagent Preparation 1X PCR Buffer containing 0.05% Pluronic F-68: Prepare immediately before use and vortex...using. 500 µL of GeneAmp 10X Buffer 25 µL of 10% Pluronic F-68 4475 µL Molecular Biology Grade Water Procedure...

...expensive to ensure adequate quality, but this upfront cost will save headaches and expense later. At ...that are suitable for unstable or recombination-prone DNA. The use of electrocompetent cells is essential...commercially prepared electrocompetent cells is strongly recommended. 800 ng pooled library DNA (100 ng...

...A or T between position 15-19. Avoid targeting introns. Avoid stretches of 4 or more nucleotide repeats...must follow safety procedures and work in an environment (e.g. BL2+) suitable for handling HIV-derivative...responds differently to puromycin selection. Addgene strongly recommends that you determine the optimal puromycin...

... do not sequence the entire plasmid. Addgene strongly recommends that you sequence any portion of the...position. Sometimes an “N” is the result of an erroneous insertion by the sequencing reaction....

... to be centrifuged Water Background Information Front view of an open tabletop centrifuge with labels ... same components. The photo to the left shows a front view of an open tabletop microcentrifuge with the...

...chemicals that present a danger to people and the environment in biohazard containers. Biohazardous materials...protocols require bloodborne pathogens training. It is strongly recommended that anyone participating in BSL-2...

... Linear, Transfection Grade, VWR 75800-188 10% Pluronic F-18, Thermo Fisher 24040032 Glutagro, Corning...BCD TFX 1000 mL BalanCD HEK293 Media 10 mL 10% Pluronic-F68 40 mL 200 mM Glutagro Do not add selective...

... 2 : Provides an optimal and stable chemical environment for the DNA polymerase to work adequately. Divalent...solvent and buffer capabilities provide a suitable environment for the DNA amplification reaction. Reference...

... strain grows, it's possible that: You used the wrong antibiotic. Your antibiotic is at too high a concentration... strain grows, it’s likely that you’ve used the wrong antibiotic, or confused your strains....

...redox. J Cell Biol. 2013 Apr 15;201(2):337-49. David Ron Redox/ROS Thiol redox-sensitive ratiometric sensor...Dopamine Ultrafast neuronal imaging of dopamine dynamics (dLight1.1-1.5) Ultrafast neuronal imaging of dopamine...electrical events imaged in neurons with a fluorescent protein voltage probe. Neuron. 2012 Sep 6;75(5):779-...and cellular processes, or other factors like environmental conditions. With proper experimental design,... fluorescence properties can be affected by environmental factors and the sensor behavior may differ from...High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nat Methods...Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature. 2013 Jul 18;499(7458):295-300...

...any growth after overnight incubation. What went wrong? Try growing the culture for more time. Some bacterial...

...instructions for each. I got no transformants. What went wrong? Check that you are plating on an LB Agar plate ...

...and 30 bp annealing to the target sequence. Avoid strong secondary structures in the homology region. Hairpins...

...CARTPT pEMS1492 intron-lacZ/NLS Ple22 CCKBR pEMS1493 intron-lacZ/NLS Ple23 CCKBR pEMS1494 intron-lacZ/NLS Ple24...CCKBR pEMS1495 intron-lacZ/NLS Ple25 CCKBR pEMS1496 intron-lacZ/NLS Ple26 CCL27 pEMS1497 intron-lacZ/NLS Ple27...CCL27 pEMS1498 intron-lacZ/NLS Ple28 CCL27 pEMS1499 intron-lacZ/NLS Ple29 CCL27 pEMS1500 intron-lacZ/NLS Ple30...CLDN5 pEMS1504 intron-lacZ/NLS Ple34 CLDN5 pEMS1505 intron-lacZ/NLS Ple35 CLDN5 pEMS1506 intron-lacZ/NLS Ple36...ICMT pEMS1593 intron-lacZ/NLS Ple123 ICMT pEMS1594 intron-lacZ/NLS Ple124 ICMT pEMS1595 intron-lacZ/NLS Ple125... pEMS1597 intron-lacZ/NLS Ple129 MKI67 pEMS1600 intron-lacZ/NLS Ple131 MKI67 pEMS1602 intron-lacZ/NLS ...pEMS1650 intron-lacZ/NLS Ple179 RLBP1L2 pEMS1651 intron-lacZ/NLS Ple180 RLBP1L2 pEMS1652 intron-lacZ/NLS...

.... AAV vector for strong & rapid SYFP2 expression in forebrain GABAergic interneurons. The enhancer core...recombinase-dependent dTomato expression in forebrain GABA-ergic interneurons under the control of the hDlx enhancer element...recombinase-dependent GFP expression in forebrain GABA-ergic interneurons under the control of the hDlx enhancer element...

...-specific labeling. Neuron. 2021 May 5;109(9):1449-1464.e13. doi: 10.1016/j.neuron.2021.03.011. PMID: ...WPRE3-BGHpA AiP11390 DLX2.0 SYFP2 Pan GABAergic interneurons Isocortex 222939 pAAV-DLX2.0-minBG-FlpO-WPRE-HGHpA...minBG-FlpO-WPRE-HGHpA AiP14532 DLX2.0 FlpO Pan GABAergic interneurons Isocortex 214518 pAAV-AiE2366m-minBG-SYFP2-WPRE3...WPRE3-BGHpA AiP1691 AiE2366m SYFP2 Lamp5 interneurons Isocortex 220737 pAAV-AiE2366m-minBG-iCre(R297T)...R297T)-BGHpA AiP20068 AiE2366m Cre Lamp5 interneurons Isocortex 224015 pAAV-AiE0724m-minBG-SYFP2-WPRE3-BGHpA...BGHpA AiP12863 AiE0724m SYFP2 Lamp5 interneurons Isocortex 224051 pAAV-AiE0724m-minBG-iCre(R297T)-BGHpA...BGHpA AiP14668 AiE0724m Cre Lamp5 interneurons Isocortex 214480 pAAV-AiE2103m-minBG-SYFP2-WPRE3-BGHpA AiP1287...

...Single polycistronic lentiviral vector for the expression of human Oct4, Sox2, and Klf4 Polycistronic lentiviral...Minicircle Human Non-integrating mini-intronic plasmid (MIP) polycistronic expression of human Oct4, Klf4, ...functional midbrain dopaminergic neurons using a single polycistronic vector. PLoS One. 2011;6(12):e28719...Fibroblasts Neurons Lentiviral Mouse Direct conversion of fibroblasts to functional neurons by defined ...Dopaminergic Neurons Lentiviral Human Direct conversion of human fibroblasts to dopaminergic neurons. Proc Natl...Fibroblasts Neurons Lentiviral Human MicroRNA-mediated conversion of human fibroblasts to neurons. Nature....cholinergic neurons. Nat Commun. 2013;4:2183. Zhang Fetal Lung Fibroblasts Cholinergic Neurons Lentiviral...