We narrowed to 614 results for: cat.1

...treatment and improving patients lives. References 1. Ong-Abdullah, Meilina, et al. "Loss of Karma transposon...Mechanisms of epigenetic modifications The main mechanism of epigenetic modification is chromatin remodeling...literally means ‘on top of the genome’). Epigenetic modifications can result in phenotypic changes without any...any change in genotype. Epigenetic modifications play a large role in turning genes ‘off’ and ‘on’, signaling...two main mechanisms, either through histone modifications, which can influence the transcription of genes...hypomethylation, on the other hand, has also been implicated in the development and progression of cancer ...enzyme digestion, DNA adenine methyltransferase identification, and bisulfite sequencing. However none of ...

...If you already have an end product in mind, steps 1 and 2 will generally be straightforward. Likewise ...well as suitable applications. Expression System Components of System Application Mammalian expression...CRISPR mammalian expression systems, the typical applications for each, and potential delivery methods.   ...Select the appropriate CRISPR tools for your application: Wildtype Cas9 or Cas9 nickase are appropriate... or introducing mutations and tags, while a catalytically dead dCas9 can be used in conjunction with activator...or more sgRNAs and Cas9 are required for any application. If you want to include a homology directed repair...Cas9-gRNA complex and delivered to cells using cationic lipids. Transient expression of CRISPR components...

... – a mechanical device that could be used to bend 1/8th inch steel wire into a backbone structure of a...development programs for bioscience educators. Using these hands-on tools, educators introduce their students to...biologists have always struggled to effectively communicate the nuances of protein structure in a flat 2-...dangerous. Subscribe to CRISPR Blog Posts How do educators and students use molecular models?   We created...something tangible. Under the guidance of a trained educator, these instances of focused attention become magical... their labs. Soon after we began working with educators in our Center for BioMolecular Modeling, we spun...through flexibility It is common knowledge in the education community that All models are wrong….Some models...

...viruses - 2 cas systems regulate at the DNA level and 1 at the RNA level. Udi Qimron from Tel Aviv University...chemical replication of RNA in the absence of evolved enzymatic machinery (i.e. RNA replication with no...Guangzhou and the Science City The conference was located in the modern, relatively new “Science City” area...and technology companies and start-ups choose to locate there. I made time to go into the city of Guangzhou...inheritance that might be less error prone than DNA replication. He too mentioned how “surprising” small RNAs...Steitz quipped that RNA in the ribosome is in a complicated conformation “not like DNA which has a boring...

...which you’ve already fluorescently labeled. Figure 1: Using Split-BioID to Identify Protein Protein Interactions...methods for identifying PPIs, such as affinity purification mass spectrometry (AP-MS), lack the ability ...the validation of a binary PPI as well as the identification of additional interacting factors. Problems...detect weak and/or transient PPIs. Many PPI identification techniques rely on protein interactors to stay... AP-MS. This requirement often prevents the identification of weak and/or transient interacting proteins...spatiotemporally defined protein complexes. Nature communications. PubMed PMID: 28585547. PubMed Central PMCID...

...is best for larger genes such as Cas9. See figure 1 for an example of how changing transfection ratios...prevent the formation of replication competent lentivirus (RCL). Replication competence can arise from...end-users, size of the transgene, and downstream applications should all be considered when choosing the correct... as resources available, cost and downstream applications. For example, while the PEG precipitation method...

... inhibitory synapses in the brain.  Figure 1:Schematic diagram of different FingR constructs and... labeling proteins with different subcellular locations. Image from Goudeau et al., 2020. Goudeau... show a high proportion of blue fluorescence, indicating cell proliferation. BaF3 cells maintained in ...in high IL-3 showed mostly blue fluorescence, indicating a quick cell cycle. Cells that had IL-3 withdrawn...Hybrid for Multiplexed Editing and screening Applications (CHyMErA) Knockout Libraries express hybrid ...

...significant progress in clinical trials.     Figure 1: Types of delivery and editing strategies for CRISPR... therapeutic potential in mice with a dominant cataract disorder caused by a single copy of a gene known...known as Crygc (Wu et al). They found that the cataracts phenotype could be rescued by zygote co-injection...substantial relief from both disorders. As of publication, submissions to the UK, EU, and USA federal drug...10.1186/s12943-022-01559-z Zarghamian P, Klermund J, Cathomen T. Clinical genome editing to treat sickle cell...

...). References 1. Li, Chaokun, et al. "FastCloning: a highly simplified, purification-free, sequence-...methodologies, with a special emphasis on these five categories. Please note that many of the following cloning...relies on site-specific recombination reactions catalyzed by BP Clonase and LR Clonase enzyme mixes. After...there is no need for restriction digests, gel purifications, or ligations. The general Gateway cloning protocol...

...article, they investigated three Casɸ orthologs, Casɸ-1, Casɸ-2, and Casɸ-3. This system is notable for several...including excision or insertion, inversion, translocation or cassette exchange. By utilizing recombinases...BioRender.com In their recent ACS Synthetic Biology publication, Mary Dunlop’s lab generated a photosensitive...regions with Vivid (Vvd) photodimers. Upon the application of blue light, Vvd dimerizes bringing the C-terminal...

...SpCas9 base editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated...Lower on-target activity than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very ... plasmid or group of plasmids' functions and applications. We hope that these articles make it easier ...FLIM (Fluorescence Lifetime Imaging Microscopy) applications. To demonstrate the power of mCyFP1, the Lin... Coselection efficiency is so high that some applications may permit working with edited “pools” of cells...showed high editing efficiencies with AsCpf1, indicating that ouabain coselection is a robust and flexible...be found at Addgene and in Figure S12 of the publication. Agudelo D, et al. Nature Methods. 2017. PubMed...

...technologies (Bradbury & Plückthun, 2015a).   Figure 1: (Top) Polyclonal antibodies are produced in animals...cells, for antibody production and subsequent purification.  Academic laboratories and companies have already...Edition. 2104. Cold Spring Harbor Laboratory Publications, New York. Helma J, Cardoso MC, Muyldermans ...

...most comprehensive for searching. Timesaving Tip #1: If you know the species that your sequencing result...search to check for expected ORFs, mutations or truncations. A primary advantage of blastx is that you do...other labs speed up their plasmid and cloning verification steps and free up more time for using your plasmids...

...genome engineering and microbiology.     References 1. Chen, Sean, et al. "Highly efficient mouse genome...valuable to scientists, there are some potential complications that must be considered when using this method...alternative approach, which avoids many of these complications, is to directly deliver a ribonucleoprotein ...used for most of the current genome engineering applications of CRISPR including: generating single or multi-gene...pathways. This makes Cas9 RNPs useful for CRISPR applications where limited expression of Cas9 is required...development. David Liu’s Lab has demonstrated the use of cationic lipid-mediated methods to deliver Cas9-gRNA RNPs... of eventually using this method for in vivo applications. This technique utilizes Cas9 proteins harboring...

... questions of how the brain operates. References 1. Callaway EM. Transneuronal Circuit Tracing with Neurotropic...able to replicate, and for a virus to cross a synapse from one cell to another, it must replicate. Modified...like how our stem cells are housed in specific locations and give rise to progenitors during growth and...infects one cell (the “starter cell”). It then replicates within that cell, and nascent RABV cross synapses...infection occurs, subsequent cycles of viral replication and retrograde transmission occur, spreading ...limitations. First, these high levels of viral replication are toxic. Second, infecting subsequently higher...spread monosynaptically from these cells. These modifications take advantage of the fact that RABV is an enveloped...

...in restraint. The very first subheading in Chapter 1 asks “What is science?” That’s a very big question...drives and their ramifications. Readers unfamiliar with academia’s challenges with publication and funding ...Connecticut College who has been teaching and communicating science for over two decades, attempts to answer... primarily on fluorescent proteins and their applications, tackles much broader issues in this brief volume...sexism in science, problems with peer-reviewed publications and funding, bioethics in the age of CRISPR,... and her essential-but-unlauded role in the identification of DNA’s double helix. Perhaps to the dismay...dismay of middle-aged scientists, the identification of Green Fluorescent Protein (GFP) and its development...

... NGT PAM GAA PAM GAT PAM xCas9 3.7 41 (1) 32 (1.6) 13 (2.1) 38 (4.5) 7.2 (5.2) ...targeting anywhere in an exon. But things get more complicated if you’d like to use homology directed repair...They created a selection phage (SP) to carry catalytically dead SpCas9 (dCas9) fused to the bacterial polymerase...test xCas9 3.0-3.13, Hu et al. first restored catalytic residues D10 and H840. To ensure that the clones...loci displayed a broad range of PAM sequences, indicating they may be less predictable than SpCas9 off-... that we have only scratched the surface of modifications that can be made to Cas9. It is a pleasant surprise...editing rates. This PAM flexibility should expand applications that require very specific targeting, like homology...

...Addgene with interests in education and scientific writing.     References 1. Martz, Colin A., et al. ... millions of people die from cancer-related complications each year. Despite these grim statistics, new...any of these genes through several different applications such as fluorescent protein fusion studies and.... "Systematic identification of signaling pathways with potential to confer anticancer drug resistance...

... digging deeply into their data.     Fig. 1: Genomic fraction for each of five major ancestry ...Dependency Map cancer cell lines (left). Heatmap indicating the breakdown of ancestry associated dependencies...machinery where to cut.      Fig. 2: Heatmap indicating loci where SNPs reside on sgRNA target sequences...Understanding the impact Fig. 3: Histograms indicating the frequency (x-axis) which SNPs map to thetargeting...same reference genomes and libraries. Further complicating the issue is the long and ugly history of reporting...

...for your first journal club to begin! Figure 1: Discussing next-generation sequencing at the Addgene...emerging work by collaboratively discussing recent publications in a specific field(s). Most anyone who has ...the company at large, by polling and informal communications we made a decision to try out a Bioinformatics...major findings and then focusing more on broader implications rather than diving deep into the individual ....  These variations are suited for different applications and should be carefully chosen for each group...

...cereblon, leading to rapid target degradation (Figure 1). Plasmids: We generated a series of plasmids that... and we will provide an update with vendor and catalog numbers. As we are happy to share aliquots of dTAG...

...effects (Zibitt et al. 2021).    Figure 1: A schematic illustration of CRISPR/Cas9-based approaches...it is important to carefully select the target locations of knock-in studies to avoid off-target effects...it comes to targeting precision and practical application. If the CRISPR/Cas9 system is not effective for...lncRNAs with high specificity, particularly in applications requiring smaller delivery systems (Zibitt et...

...editing machinery such as CRISPR-Cas. Figure 1: Schematic of targeted gene knock-out (left) and targeted...and quiescent, post-mitotic mammalian cells. Applications of CRISPR delivery by adenoviral vectors Since...Strategies for Tissue-Specific Targeting. In: Applications of viruses for cancer therapy. Elsevier, pp ...Holkers M, Maggio I, Henriques SFD, Janssen JM, Cathomen T, Gonçalves MAFV (2014) Adenoviral vector DNA...

...dollar contracts behind closed doors. Fig. 1: The mythical land of negotiations.    You negotiate...fingers or Ethiopian?) to where you are going on vacation (New York City or Hawaii?). So how can you approach...been: A job offer conflicts with a pre-planned vacation I’m looking forward to. How can I negotiate my... I’m excited while still being able to take my vacation? I want to learn more about a different area of...

...impacts of neural circuits on behavior. Figure 1: An overview of the chemogenetics system. Why use...combined with. Combining a PSAM with the IPD of a cation channel, such as the serotonin 3 receptor, will...chemogenetics for research and potential clinical applications. Science 6436: eaav5282. https://science.sciencemag.org...

...necessary.   Tips for barcoding and tracking samples 1. Start with the tube - Barcoded tubes come in many.... Tracking samples - it's all about location, location, location There are many different types of samples...but we also have a dedicated and detail-oriented lab team that works carefully to catch mistakes. Each sample...assigning a physical location to your samples, include as much detail about that location as possible. Are...sample a unique location in a specific freezer will help ensure that samples are easy to locate in freezers...also help us keep track of the tubes’ physical location in the lab. These barcodes additionally allow ...Addgene. Each sample at Addgene has four layers of location which can all be extrapolated from the 2D barcode...

...plasmids are passed down through generations (Figure 1). These so called “plasmid addiction systems” require...activity of CcdB. Applications of ccdB in plasmid cloning Perhaps the most widely used application of CcdB is...selection markers. Restriction modification systems Because restriction modification systems consist of two enzymes...used as biological tools for a wide range of applications in molecular biology or in vivo studies. Let...types of plasmid addiction systems and their applications. The discovery of toxin-antitoxin systems Toxin-antitoxin...CcdA (antitoxin) where CcdB inferes with DNA replication by binding the GyrA subunit of DNA gyrase and...construct. Other toxin-antitoxin systems and their applications MazE/MazF Aside from CcdB/CcdA, there are many...

...Figure 1: Flowchart showing the process for using pooled lentiviral libraries. A) Amplification, validation...you.  Starting from plasmids requires amplification, verification, and lentiviral production steps before...the same proportion after amplification as they were prior to amplification. And as anyone who has worked...pooled library is transformed into E. coli for amplification. Transformed E. coli are allowed to grow on ...into HEK cells allows production of lentivirus. Purification of this lentivirus results in the pooled library...//pubmed.ncbi.nlm.nih.gov/27366891/   Amplification When you request a DNA pooled library from Addgene...might receive a similarly-sized aliquot from an amplification done by Addgene.) This small aliquot is not ...

...designed to shuttle between species. Figure 1: Shuttle vectors are vectors that can function in ...are used across species: replication, selection, and expression. Replication It’s usually not enough to...chromosome has a single origin of replication (ori), where DNA replication begins, so a bacterial plasmid...Eukaryotes are a bit more complicated. Yeast vectors may contain an autonomously replicating sequence (ARS), a...fast-growing populations of cells, the vector needs to replicate alongside its host, or it will be lost from the...compatible ori that will be recognized by its host’s replication machinery. A shuttle vector designed for unrelated...experiment. Yeast shuttle vectors have these replication sequences for their yeast targets as well as ...

...sample’s absorption at 260 nm (A260). Figure 1: Characteristic absorption spectra for pure DNA and...present.  Purification reagent contaminants The reagents used during nucleic acid purification can leave...Selecting Your Plasmid Purification Kit Resources on Addgene.org DNA Quantification Protocol Recovering... and are ready to move on to your downstream application — but wait! Do you know how much plasmid DNA ...(Figure 1B). The A260/230 ratio can be a good indicator of protein contamination, but a number of other...researchers traditionally use the A260/280 ratio as an indicator of protein contamination. However, this ratio ...230 ratio of a sample. Ethanol carry-over from purification protocols is also a common concern, and while...

...successful and meaningful career in the grants profession: 1. Build on your existing skills & passion  Skills such...development, certification, degrees, foundations, coaching and even Twitter Chats dedicated to the Grant...the candidate application process, the candidate’s ability to write a grant application through a written... much more than writing. In fact, many grant applications and grant makers have moved away from long written...Now they require grant seekers to use online applications with significant character limits for the written...written portion of the grant application. They have increased their demand for stronger budgets with clear...duties can range from coordinating the grant application and submitting it, to evaluating the capacity...

...can make this fun activity relate to STEM.” Figure 1 shows the activities we had for my school’s Math &...my opinion, it was more important to communicate the applications of CRISPR over the details of how it...m a father of three, a scientist and science communicator, and I strive to be a volunteer and contribute...recent experiences. STEM in elementary schools - catching them early I’m fortunate to live in a town with...unlike elementary schools, middle schools have dedicated science teachers. I tried contacting our school...simplistic and I would insult the students. Too complicated and I would quickly lose their interest. I won...great feedback from teachers and other science communicators.  What can CRISPR do? I spent more time on...

...two (Knutson et al., 2010a,b).      Fig. 1: Undergraduates in Provost’s CURE prepare to analyze... put it best when he stated, “the value of an education . . . is not the learning of many facts but the...possible to bring the kinds of transformative educational experiences afforded to those fortunate enough...which can lead to greater success in meeting educational goals (Kowalski et al., 2016; Scott, 2021). In...CUREs are beginning to transform undergraduate education in the sciences by making a greater number of ...scientific research more inclusive. CBE Life Sciences Education. 13(4):602-606. Brian, D. 1996, Einstein: A Life...positive STEM attitudes in CUREs. CBE Life Sciences Education. 21(4):ar74. Duboue, E., Kowalko, J., Keene, A...

... such a popular model organism? Zebrafish (Figure 1) were initially recognized as a useful vertebrate ... are not ideal for every application. Disadvantages include their duplicated (i.e., redundant) genome,...mutant, which regulates a developmental pathway implicated in congenital heart defects and cancer. These...DNA into the genome is useful for a variety of applications. For example, specific tissues such as blood...pathways control this process. Additional zebrafish applications and conclusions Zebrafish are also popular outside...—have proven useful in a variety of research applications. Lineage tracing studies highlight how these...Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments." Methods...

...determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV ...Addgene has a growing catalogue of viral preps available to request. In this post, we’ll walk you through...minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic properties...posted on the material page for the corresponding catalog item (see maps section for images).     ...Lentivirus Production Lentiviral preparations ("-LV” catalog items) are produced in 293T cells using the 2nd.... Concentrated lentiviral preparations (“-LVC” catalog items) are generated from lentiviral preparations...typically prepared using endotoxin-free plasmid purification kits.     Figure 4: Schematic of the...

...antibody bonding strengths.    . Steps of an IP: 1) Protein extracts from cells or tissues contain a ...a protein’s stability or post-translational modifications, or study how a target protein interacts with...Protein A and Protein G are not suitable for all applications, such as protein isolation from serum. In this...capture antibody interaction and/or the downstream application. Typically, if the target protein will be detected...and an isotype control antibody. Alternative applications Oftentimes, an IP-appropriate antibody simply...identify DNA binding proteins involved in histone modification. Similarly, RNA IP (RIP) can be used to isolate...resources References:  Björck L, Kronvall G (1984) Purification and some properties of streptococcal protein...

...template including the desired edit. Figure 1: Prime Editing schematic. Created with BioRender.com...BioRender.com. The pegRNA specifies both the genomic location to be edited and the sequence of the edit, so ...its design changes for every new prime editing application. And optimizing its sequence is important, as...left), while pegRNAs that use other bases in this location bind to Cas9 with canonical sgRNA structure (right...structured motifs like mpknot, use the pegRNA Linker Identification Tool (pegLIT) to create linkers that minimize...

... reached labs in over 75 countries. Figure 1: SARS-CoV-2 proteins and other components. Image from...a method that uses loop-mediated isothermal amplification after reverse transcription (RT-LAMP) and a ...receptor for SARS-CoV. The lab also found that cathepsins play a role in SARS-CoV infection and deposited...deposited plasmids for the expression of cathepsin S, L, and B. This became a great starting point for SARS-CoV..., Zengel JR, Sathyan V, Nguyenla X, Van Dis E, Catamura C, Yamashiro LH,  Begeman A, Stark JC, Shon DJ...

... end sequences (R and L ends).  Figure 1: (A) Components involved in each type of CRISPR-Transposon... and have been adapted for a wide variety of applications - as mutagenesis tools for genetic studies (... The process also creates a 5-bp target-site duplication (TSD), a hallmark characteristic of the Tn7-family...the new streamlined VchINT system was shown to catalyze insertion of mini-transposons up to 10 kb in size...at one or multiple desired genomic loci. Many applications of these systems have already been demonstrated...

...blood test for HMGB1, the high mobility group box 1 protein. Normally localized to the nucleus, if the...cancer. These later stages of a cancer diagnosis indicate, for Stage 3, that the cancer is large in size...size and may have spread while Stage 4 indicates that the cancer has spread from where it originated to ... dire prognosis for mesothelioma patients is to catch the disease earlier. New techniques are currently... working to fight the cancer.  Beyond early modifications to standard treatments and early detection, ...

...testing. You can find Nick on LinkedIn. References 1. Skloot, Rebecca, and Bahni Turpin. The immortal life...Marker-independent identification of glioma-initiating cells. The International Cell Line Authentication Committee...propagation of the line, and can include assays for authentication and contamination) and cell lines can become...let alone the human cost. Here are just a few publications which have since been retracted due to cell ...

...mammalian expression from human elongation factor 1 alpha Constitutive  Tends to give consistent expression...resistance gene and the origin of replication are necessary to replicate plasmids and make sure the cells...length version is strong with high expression. Truncated promoters are constitutive with lower expression...metabolic operon Inducible by arabinose and repressed catabolite repression in the presence of glucose or by competitive...

...such as model systems and primary cells. References 1. Doench, John G., et al. "Rational design of highly...is only effective when sgRNAs target a specific location on the gene. For CRISPRa, sgRNAs were targeted...Calabrese pooled library and lentiviral prep here! Applications for the Root and Doench CRISPR libraries Overall...et al. "A protein-tagging system for signal amplification in gene expression and fluorescence imaging....

...antibiotic selection. Essentially, this gene is a 2-in-1 providing both bacterial and eukaryotic selection,...common antibiotic resistance genes and which applications are best (or worst) for each antibiotic. The...is expensive (relative to amp), and for some applications, the benefit it offers is marginal. Fortunately...antibiotic resistance ultimately comes down to your applications and needs. The more niche and specific your ...

...fun together! Laura’s Top 10 Event Planning Tips! 1. Take suggestions Some of Addgene’s best events have...be an obvious one, but don’t make things too complicated for yourself (especially if you are the sole ...sole event planner)! If you have a very intricate itinerary, there is a higher likelihood that something ...Delegate- it will save you time and sanity!    10. Communicate Make sure people know that there is something...

...drives our bioinformatics needs       Fig. 1: QC'ing data at Addgene is like standing under a Niagara... from depositing laboratories and associated publications, but our quality control (QC) standards have...such as 96-well microplates, and thanks to our dedicated product and development teams, frequent updates...can be easily shared with others without any complicated setup. We created, tested, and debugged each ...

... of low-toxicity RNA editing tools.   Fig. 1 Schematic of luciferase RNA knockdown assays using...experimental design and its potential for clinical applications. Specifically, efficacy rates of Cas7-11 were...place, while increasing the number of potential applications. But they weren’t done engineering quite yet... at the bench and potentially in therapeutic applications. The potential of CRISPR has yet to fully taken...

... done and we’re onto the next story. Figure 1: Addgene’s Agile Approach Sharing is caring There...diverse group of ethnicities, cultures, genders, educational, and professional backgrounds. Sometimes this... information. It is quite possible that the clarifications lead to the story being scrapped or broken ...While writing the code, we may need additional clarification and again we collaborate with the Product Team...

...inclusion bodies and is folding properly. References 1. Angov, Evelina, et al. "Heterologous protein expression...ribosome may therefore stall at inappropriate locations or fail to make it through the entire transcript...generally segregate with the cell pellet during purification procedures. Even if you produce a large amount...

...commandments for making it all work – most of the time. #1 Thou shalt find enjoyment in your work If you are ...other activities. Take care of yourself. Take a vacation alone with your partner – no kids. Get a massage...