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Showing: 181 - 210 of 1076 results

  1. Addgene: The First Twenty Years

    Type
    Blog Post
    ...process, which was a major barrier for depositors and requestors alike. We transformed this process by ...ready-to-use viral vectors from some of our most popular lentiviral and AAV vectors, as well as developing...privilege to find and share what stories we can, and to hear the stories others have to tell.  "Also thanks...amazing years in front of us. So in our last celebratory moment, we wanted to walk through some of our...service would allow people to add a gene to a repository others could request from — Addgene!  Addgene...meant we could grow accessibility alongside our repository.  “I’m sure you hear it all the time, but Addgene...took almost six years to reach our first major repository milestone — 10,000 plasmids deposited and available...

  2. CRISPR Activation: A Practical Guide

    Type
    Blog Post
    ...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...plastic tool and has taken the scientific world by storm. While Cas9 has been most widely used to create ...significant work on constructing Cas9 transcriptional activators. These constructs allow for the upregulation ...no longer activated. However, there are several factors that may prevent your gene from being up-regulated... we would recommend using a cDNA overexpression vector rather than going through all the troubleshooting...genome-wide studies One of the best uses for Cas9 activators is in genetic screening. gRNAs targeting every...

  3. The Challenges of Cell Culture

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    Blog Post
    ...validation carried out by the commercial repository. Repositories generally ensure cell line identity and...which can be stored in liquid nitrogen long term, preferably some of these vials will be stored in a separate...in a cryostore log. Keeping good passage records allows you to track a cell line’s age and monitor any ...and money but cells lose provenance (this is the history of the cell line consisting of records of the origin...invariably much older than those available from biorepositories leading to genotypic and phenotypic artefacts...easier to conduct high quality cell culture. Biorepositories around the world ensure that the cell lines...The oldest, largest, and most diverse of these repositories is the American Type Culture Collection aka ...

  4. Adeno Associated Virus (AAV) for Cell and Gene Therapy

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    Blog Post
    ...makes AAV vectors especially amenable to correcting disease-associated mutations. AAV vectors maintain...gene delivery by affinity-purified receptor-targeted viral vectors." Nature communications 6 (2015): ncomms7246... Check out our Viral Vectors Featured Topic Page Learn about CRISPR-AAV vectors Learn about Addgene AAV... is caused by mutations in the common cytokine receptor gamma chain (c) and is characterized by a lack...thereby correct the mutations using retroviral vectors. However, many diseases require techniques that...target cells has been challenging. A variety of vector delivery technologies have been investigated including...usability in clinical settings. On the other hand, vectors derived from AAV have recently gained popularity...

  5. Starter Guide to induced Pluripotent Stem Cells (iPSCs) Part 2:  Reprogramming and Transdifferentiation

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    Blog Post
    ...are similar except that the vectors carrying the transdifferentiation factors are directly injected into... Kotton Four Factors that Differentiate the Stem Cell Field Learn how lentiviral vectors can be used for... involves overexpression of four reprogramming factors- OCT4, SOX2, KLF4, and C-MYC - that induce a differentiated...another cell type of choice. The four reprogramming factors can be delivered and expressed in multiple somatic...supplements, bioactive small molecules, and growth factors are used to control the cell fate of iPSCs and ...         Adipocytes [28] Keratinocytes [29] Photoreceptors [30] Otic hair cells [31] Neurons [32, 33... intermediate pluripotent state is not always obligatory in cell conversion processes [35]. Rather than...

  6. Early Career Researcher Toolbox: Free Online Molecular Biology Tools

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    Blog Post
    ...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...data repository for validation data on Addgene/CLOVER viral vectors.  Caspedia: A data repository for ....)  DailyCalcs Science Calculator app for iPhone Reagent and Data Repositories These links can help you... for plasmid design and mapping. ApE (A plasmid Editor): A free, donation-based plasmid analysis tool ...enzyme cut sites Open reading frame finder  Addgene vector database: Find sequences for plasmids assembled...Golden Gate Reactions Cloning ligation reaction calculators These links can help you calculate how much insert...

  7. Plasmids 101: Common Lab E. coli Strains

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    Blog Post
    ...Stable   For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences...derived. For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences... HB101. For cloning into and storage of lentiviral and retroviral vectors or cloning or repeated sequences...cells for cloning and storage of unstable DNA and lentiviral/retroviral vectors. Used by Addgene for pooled... your plasmid or carrying out your experiment. History of E. coli strains E. coli are gram-negative, rod... DH5alpha and DH10b (also known as TOP10). The history of B strain is a bit more convoluted due to researchers...researchers sharing and renaming samples throughout history. It was likely isolated in 1918 but was first referred...

  8. Fluorescent Tagging of Endogenous Genes with SapTrap

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    Blog Post
    ...reaction to generate high-throughput targeting vectors. These vectors can be used to tag endogenous genes and...the final targeting vector (Fig. 1, Step 4), and co-injection of the targeting vector and a Cas9 expression...site destination vectors for inserting a tag at multiple target sites. SapTrap vectors can be easily modified...cells. To use CRISPaint, users need 3 vectors:  1) a gRNA vector that targets the gene of interest, 2)...PCR or cloning, as digestion of the destination vector with SapI yields 2 sites- the first site accepts...screening of the insertion event, and a variety of connector modules (linker sequences between the tag and ...with SapI allows the tag, selectable marker, and connector to be released (Fig. 1, Step 2-3). Since donor...

  9. CRISPR 101: Which Cas9 Do I Choose for My CRISPR Experiment?

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    Blog Post
    ...has been exploited to localize factors such as transcriptional activators and repressors to target genes...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 ...promoter region. Cas9 activators include Cas9 fused to VP64, VP64 along with other SAM factors, and the Suntag...with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 2020 387 38, ...array of Cas9s (many available through Addgene’s repository) and make it easy to find your perfect match....precise edit without even making a break.  Base editors are dead Cas9 (dCas9) or nCas9 enzymes fused to...

  10. Advanced Uses of Cre-lox and Flp-FRT - A Neuroscientist’s View

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    Blog Post
    ...are transfected with both vectors. Flex vectors are the solution. Flex vectors expressing fluorescent cytoplasmic.... It became possible to design FLEx vectors (Flip-excision vectors) that only allow one inversion event...concentrations of vectors expressing the required recombinase. The high concentration of FLEx vectors guarantees...experiments. Mutated FRT and loxP sites in Flex Vectors – inversion only once All recombination events ...between target sites of the same sequence in FLEx vectors, the DNA sequence that should be inverted is flanked... target sites are cleaved out, leaving behind a vector with the inverted DNA flanked by non-compatible...non-compatible target sites (Schnütgen et al. 2003). Flex vectors revolutionize neuroscience Manipulation of specific...

  11. Tips for Using FRET in Your Experiments

    Type
    Blog Post
    ...imaging microscopy (FLIM) method. Acceptor photobleaching method The acceptor photobleaching method is simple...don’t measure acceptor fluorescence, this method is also less sensitive to direct acceptor excitation artifacts...interactions in living cells using a microscope. Historically, molecular interactions were detected by indirect...fluorophore (the donor) to another fluorophore (the acceptor) by means of intermolecular long-range dipole-...dipole-dipole coupling." The emission of the acceptor fluorophore can be measured using microscopy techniques...photons) and the extinction coefficient of the acceptor (linking the quantity of absorbed light, at a ...highest quantum yield donor, the highest absorbing acceptor and fluorophores with significant overlap in their...

  12. Data Freedom: The Expansion of Data Sharing in Research Publications

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    Blog Post
    ...responsible for storage.  If datasets are too large to transfer to a journal repository, they are likely...publishers. Nonetheless, the initial release caused a storm as the organization left open the question of how...for advice about the scope of the policy. End of story? Not quite. The PLOS policy on data access is a...statistical methods, omission of some data, etc.  The firestorm that followed implementation of the policy comprised... with limited acknowledgement of the initial contributors. This is probable to some extent - but that ...tools and assumptions to reveal findings that the originator was oblivious to, no matter how much time they...they had. It could be argued that data generators may, in light of the policy, hold onto data longer before...

  13. Plasmids 101: CcdB - The Toxic Key to Efficient Cloning

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    Blog Post
    ... years ago and developed cloning vectors to harness it. These vectors, called pKIL18 and pKIL19, contained.... If, however, an investigator were to successfully clone an insert into the vector, the ccdB reading ...cells that contained non-recombinant vectors (re-ligated empty vectors, for instance) would still express...ccdB makes cloning easier by selecting against vectors that did not take up your insert. But exactly how...ccdB accomplish this? Let’s start with a brief history of the gene and how molecular biologists have harnessed...potent toxin... The ccdB gene, located on the F sex factor plasmid of E. coli, is part of a toxin-antitoxin...from Bernard, P., et al. showing the pKIL 18/19 vectors and demonstrating the concept of restriction enzyme-mediated...

  14. 5 Tips for Troubleshooting Viral Transductions

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    Blog Post
    .... Viral vectors undergo DNA rearrangements. Depending on the composition of the viral vector and the ... researchers prefer to store freshly harvested virus at 4 °C instead of storing it briefly at -80 °C. ...of a recombinant adenoviral vector: optimization of conditions for storage, transport and delivery." Japanese...about Lentiviral Vectors Additional Resources on the Addgene.org Check out Our Viral Vector Guide Pages ...perform complex biological tasks, both of which are factors in the technical challenges they can present. Many... one point or another. After amplifying a viral vector, collecting virus, and transducing target cells...the repeat sequences present in the LTR, the vector may undergo genomic rearrangements during replication...

  15. Experimenting in an Open Source lab: from CRISPR to Cats

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    Blog Post
    ... space is the Victoria Makerspace in Canada, which I belong to. The team at the Victoria Makerspace is..., we intend to focus on motor neuron knockouts so that we can perturb factors related to worm motion. ...student and then instructor at Singularity University at NASA Ames, and an instructor at Biocurious.org...was contributed by Derek Jacoby from Makerspace Victoria, CA Over the decades science has become increasingly... of a bigger movement in a variety of research sectors to provide open source technologies and spaces ... are constantly moving so we need good ways to monitor and track them. We have a number of microscopes...instance, we use a mutant known as CX51 that exhibits motor defects above 25 degrees. Computer scientists and...

  16. Harnessing Bacterial Toxins for Allelic Exchange

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    Blog Post
    ...counterselection vector is the pDS132 sacB-based allelic exchange vector. This vector contains sacB, an...This post was contributed by Jacob Lazarus, a postdoctoral researcher at Harvard. There’s an astounding...selecting for an antibiotic resistance marker from the vector (abR, Figure 1). The resolution step (“double crossover...leads to the desired mutation and excision of the vector backbone. This step requires “counter-selection...colonies isolated are those where the allelic exchange vector has undergone single crossover and integrated into...recombineering (Khetrapal et al., 2015). They created vectors for Lambda Red that contained one of eight bacterial...aeruginosa. These toxins were a mix of Type VI secreted effector toxins, toxin-antitoxin type toxins, and phage...

  17. Thoughts on the Future of Research 2015 Boston Symposium

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    Blog Post
    ... to be nice, the mentor fails to effectively mentor the underrepresented - the mentor over praised and...received a doctoral degree (or equivalent) and is engaged in a temporary and defined period of mentored advanced...to your peers and other mentors who can help you. You Can Talk to Your Editors I wish I had been more...well-meaning mentors often fail to judge the work of underrepresented groups under their mentorship with the...research community to develop a plan to make postdoctoral pay/benefits commensurate with work training...image below). Slide of information gathered by Postdoctoral Associations around Boston - From Panel Session... staff scientists. This results in a glut of postdoctoral trainees who, even though they are aware that...

  18. Illuminating Choices: A Guide to Selecting Fluorescent Dyes and Ligands

    Type
    Blog Post
    ...Interaction with receptors: Understanding how fluorescent dyes interact with specific receptors is key, especially...especially for G-protein-coupled receptors (GPCRs). Mutations in receptor sites can affect the fluorescence...Throughout their history, fluorescent dyes have enabled the visualization of both organic tissues and... intensity during exposure to light, is another factor to consider. If your application requires longer...fluorescent protein (Mahmoudian et al., n.d.). Factors such as exciting the fluorophore at peak wavelength...It's important to consider how dyes can influence receptor conformation and signaling activity​​ (Sridharan...varying degrees of β-arrestin-2 recruitment to receptors, indicating their agonistic nature. The potency...

  19. Typing CRISPR Systems

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    Blog Post
    ...This also means that between two types, the Cas effector may have the same name but be completely different... understand. Classes are based on the main Cas effector; types are based on main function and composition...Class 2. Class 1 CRISPR systems use multi-Cas effectors, referred to as Cascade (CRISPR-associated complex...complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade complex doesn’t usually...specific CRISPR type but often recruits the main effector complex after binding to a target. Class 1 is ...the lab. This can be attributed to the multi-Cas effector, Cascade. Developing tools using a Cascade complex...the CRISPR types and is thought to be the common ancestor of all other CRISPR systems. Type III systems ...

  20. Addgene’s Viral Service - Why Virus? Why Now?

    Type
    Blog Post
    ... to all of our generous, sharing depositors. Over 3,000 laboratories are sharing plasmids (with 16 labs...sponsored the capital construction of our new BL2+ laboratory and the infrastructure capabilities to produce...currently sharing viral preps) through the Addgene repository. Without these scientists and the support of ...blessed to have an incredibly supportive Board of Directors at Addgene. Our Board helps keep our focus on ...helping thousands of sharing scientists from laboratories all over the world. With this reach and exposure...new team to push this project forward. The viral vector project team develops methods, performs extensive...generates  educational materials, and provides viral vector technical service (details about virus production...

  21. Protein Tagging with CRISPR/Cas9: A Conversation with Mendenhall and Myers

    Type
    Blog Post
    ...different assays, not just transcription factors. The second vector is a homology directed repair donor plasmid...system is so flexible. Other investigators can easily adapt these vectors for use with their favorite ...in Huntsville explains it, a major goal in his laboratory is to understand the function of the non-coding...focused considerable attention on transcription factors, starting with an established method called ChIP-seq...antibodies to recognize and bind transcription factors directly, but that’s a slow-going process and it...effort to track down every last one of those gene regulatory proteins and the places in the genome where they...is to look at all 1,500 or 1,600 transcription factors and figure out where they bind to the genome. We...

  22. Hot Plasmids April 2018 - Protein Degradation, Nanoscopy, FIRE-Cas9, and Yeast Expression Tools

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    Blog Post
    ...controlled transcription factor and a ZIF268/progesterone controlled transcription factor as well as reporters...transcription factors in series and independently changes inducer levels. That is, if transcription factor 1 controls...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles... to drug inhibition, with TRIM-away targeting of mTOR producing similar results to rapamycin inhibition... super-resolution microscopy.” Endosomes are historically difficult to study in living cells using conventional...the Hell lab designed their super-resolution pH indicators (SRpHi) biosensors to meet all of them. These...Ford Listen to the Synthetic Yeast Transcription Factors Podcast Segment Researchers from the El-Samad lab...

  23. New Tool for Lineage Tracing: The ClonTracer Library

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    Blog Post
    ...in epidermal growth factor receptor (EGFR) with the ClonTracer library and monitored population dynamics...paths to become resistant to epidermal growth factor receptor inhibition." Nature medicine (2016). PubMed...Library, deposited by Carrie Bhang, a research investigator in the In Vivo Pharmacology group at Novartis...mammalian cells through lentiviral infection and to monitor their abundance and clonal dynamics over time using...drug resistance, Bhang says “the [drug] resistance story panned out quickly,” and was summarized in a recent.... The library consists of a pool of lentiviral vectors with roughly 73 million semi-random, 30 bp DNA ... abundance of each barcoded clone can then be monitored over time by sequencing the barcodes in the population...

  24. Genetically-encoded Sparse Cell Labeling - A SPARC of Innovation

    Type
    Blog Post
    ...recombination step, the effector is expressed. In cells where the stop cassette remains, the effector is not expressed...inactivate any read-through transcripts of the effector gene. Effector genes, any gene that you want to control...organisms by replacing the transcription factor binding site and effector in the construct. In Drosophila, the...T5 neurons by placing the calcium indicator GCaMP6f in the effector position, as well as to optogenetically...CsChrimson) attached to a fluorescent indicator (tdTomato), as the effector. They also expanded the SPARC system...A predictable number of cells will express the effector or retain the stop cassette following recombination...Drosophila (Fig. 1):  GAL4-UAS, a transcription factor binding site. GAL4 expressed in the fly binds to...

  25. Multiple Plasmids at a Low Price: Kits from Addgene

    Type
    Blog Post
    ...of pExpression vectors) plasmids. The type IIS restriction sites used in these vectors are of low abundance...of the many useful kits you can order from the repository: FX Cloning Kit BIOFAB Plasmid Set pCri System...to produce the constructs you need. Expression vectors within this kit contain a variety of promoters ...insects, and even Xenopus oocytes. The expression vectors additionally contain various combinations of protein...blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen." Scientific...Series Confronted with the problem of creating vectors for predictable protein expression in bacteria,...this so-called bicistronic design (BCD). The 49 vectors in this kit can be used to drive production of ...

  26. The AAVantages of AAV in CRISPR Screens

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    Blog Post
    ...genes with minimal effort. The versatility of Cas effectors allows for a wide variety of screens, including...packaging capacity and stable expression of lentiviral vectors make them an attractive option. However, safety... particularly in vivo ones. In those cases, AAV vector screens may be AAVantageous!       In vivo, in ...delivery method for in vivo CRISPR screens. AAV vectors can be made at high titers equal to those achieved... customization that is unmatched by other viral vector delivery methods. Lentivirus has limited packaging...packaging options, with most lentiviral vectors being packaged using VSV-G. In contrast, AAV can be packaged...psuedotypes to further improve the tropism of AAV vectors, enhancing their affinity for specific cell and...

  27. Plasmids 101: The Promoter Region – Let's Go!

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    Blog Post
    ...controlled by still other regulatory factors: enhancers, boundary elements, insulators, and silencers; however...tetracycline operators. Transcription can be turned on or off depending on what tet transactivator is used.... controlled by lac operator. Can be induced by IPTG. Commonly found in pET vectors. Very tightly regulated...the promoter (RNA polymerase binding site) and operators (response elements). Promoters are about 100 to...binding of the RNA polymerase and transcription factors, therefore promoters play a large role in determining...we will discuss these in a future post on viral vector parts. Promoter specificity Aside from choosing...Strong mammalian expression from human elongation factor 1 alpha Constitutive  Tends to give consistent ...

  28. A Guide to Getting Started in Undergrad Research

    Type
    Blog Post
    ...I’ve learned the value in finding mentors wherever I go. This story by Dr. Erika Moore in Science Careers....351.6276.994 Moore E (2021) One mentor isn’t enough. Here’s how I built a network of mentors. Science. https://doi.org...undergraduate years. We each have a unique “origin story,” but taking the first step into the world of academic...lot of help from my peers, professors, and other mentors, and four years after I started college, I will...postdoc” is, and that “PI” stood for principal investigator. In basic science (as opposed to translational...in a lab. Position Common tasks Principle investigator (PI) Runs the lab Writes a lot of grants Varying...teaches graduate and/or undergraduate classes Postdoctoral researcher (postdoc) Getting more scientific...

  29. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

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    Blog Post
    ...homology to the vector or neighboring fragment. Then you would amplify the fragments and vector by PCR, verify...linearized vector, the Cas9 enzyme along with a specific gRNA was used to cleave the 22kb vector. When followed...and then assembled into a vector using Gibson assembly. In this case the vector was PCR amplified to contain...be used in basic cloning of an insert into your vector of choice as shown in the figure below. To start...you want to insert your gene of interest into a vector with a large tag at the N-terminus, but you don...don’t have the tag already included in the vector you want to use. If you inserted these two pieces of ...gene of interest and the tag sequences into the vector in one step without scars as depicted below. First...

  30. Three Tips for Preventing Viral Plasmid Recombination in Your Samples

    Type
    Blog Post
    ...plasmids are formed. Viral vectors are prone to recombination In lentiviral vectors, recombination often occurs...problems that I help troubleshoot involves viral vectors and plasmids with repeating sequences. Repeating.... Over time, bacteria containing the recombined vector backbone will also outgrow bacteria that contain...into two smaller plasmids. The ~3.7 kb recombined vector backbone contains the antibiotic resistance gene...samples? Plasmid recombination can affect viral vectors (LTRs and ITRs), plasmids with multiple polyA sites...recommendations from the depositing lab. If your vector is large (>10 kb) or has a low copy number, try...that in Stbl3 bacterial cultures with lentiviral vectors, the flat white colonies contained the full plasmid...
Showing: 181 - 210 of 1076 results