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We narrowed to 674 results for: abo.1

Showing: 61 - 80 of 674 results

  1. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

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    Blog Post
    ...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required...from the 5’ end and generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ ...recombineering with an ssDNA oligo substrate.   Figure 1: Components of the Lambda Red Recombineering system...and tags.  The typical frequency of recombinants is 1 positive clone out of 104 to 105 colonies. Pro Tip...dsDNA with a recombination frequency between 0.1% to 1%, and can be increased to as high as 25-50% by designing...replication. There are two ways to avoid activating MMR: 1) use a strain of bacteria that has key MMR proteins...or 2) specially design ssDNA oligos to avoid MMR: 1) E. coli with inactivated MMR: Using E. coli with ...

  2. Opto-Nanobodies: Using Light to Manipulate Cell Signaling and Protein Purification

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    Blog Post
    ...loops 1 and 6 resulted in light controlled binding, but with opposite effects: insertion into loop 1 caused...experiments.  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced...favorite protein (YFP). You have two options:  Option #1: Affinity tag purification You tag YFP and use an ...and one against GFP. Insertion of AsLOV2 into loop 1 always generated OptoNBs with dark-induced binding... induced dark-induced blue-light induced Table 1: Summary of the effect of AsLOV2 insertion site on...Addgene blog Learn about using nanobodies to control protein activity Read about RANbodies, reporter ...reporter nanobody fusions Learn more about using the secondary nanobody toolbox for immunodetection Resources...

  3. Viral Vectors 101: AAV Variables That Matter

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    Blog Post
    ...promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1 Oh, J. Y., Han, J. H., ...starting the selection process. Factors to think about are (1) your experimental conditions and measurements...serotype, promoter, and titer.     Figure 1: Flow chart depicting a) steps for selecting serotypes...injection site than AAV2 (Watakabe et al., 2015) (Figure 1). You’ll need to consider if such properties could...AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two different brain regions....Image modified from Aschauer et al., 2013, Figure 1. Used under Creative Commons Attribution License. ...Applications in Gene Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org/10.3390/cells12050785 Murlidharan...

  4. PEI Calculator for Planning AAV Packaging Transfections

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    Blog Post
    ... regularly use the ratios below: 1 Transfer Plasmid : 1 pHelper: 1 RepCap OR 3 Transfer Plasmid : 5 pHelper...know what you think about the calculator in the comments section below. References 1. Choi, Vivian W., ...how many dishes or stacks you plan to transfect: 1 to 10 Fill in experimental details on the spreadsheet...

  5. X-CHIME: Context Dependent Germline Knockout in Immune Cells

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    Blog Post
    ...in the immune system. Nature Immunology, 25(1), Article 1. https://doi.org/10.1038/s41590-023-01689-6 ... strains and lentiviral expression vectors (Table 1). The four systems Name Function Plasmid ... GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME systems Proof of Concept These systems were...was lethal (LaFleur et al., 2024).   Figure 1: Schematic showing the X-CHIME approach. Figure from...genes in the immune system. Nature Communications, 10(1), 1668. https://doi.org/10.1038/s41467-019-09656-2...LaFleur is a Postdoctoral Fellow in Arlene Sharpe’s laboratory at Harvard Medical School and is interested in...K.B., Gillis, J.E., Sen, D.R., Gaudiano, E.F., Al Abosy, R., Freeman, G.J., Haining, W.N., & Sharpe, A.H...

  6. Typing CRISPR Systems

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    Blog Post
    ...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems...archaea, though Class 1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible...optimal plasmids to harness the power of Class 1 systems. Class 1 CRISPR systems can be further broken down...binding to a target. Class 1 is the most abundant of the two, comprising about 90% of identified CRISPR-...types, 33 subtypes, and 17 variants. Figure 1: Overview of the CRISPR classification hierarchy. ...incredible abundance, Class 1 CRISPR systems are less popular in the lab. This can be attributed to the multi-Cas...Type I CRISPR systems make up the majority of Class 1, and are the most common type of CRISPR overall. Type...

  7. When Fidelity Matters: A frank discussion about ligase fidelity

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    Blog Post
    ...phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably T4 DNA ligase) are ... enzymology of DNA repair enzymes.     References 1. Shuman, Stewart, and Christopher D. Lima. "The polynucleotide...thermostable DNA ligase-encoding gene." Gene 109.1 (1991): 1-11. PubMed PMID: 1756968. 7. Lauer, G., et al. "Cloning... And what is ligase fidelity anyway? Let’s talk about it. DNA ligases are enzymes that seal breaks in ...to accelerate characterization of nucleic acid metabolic enzymes." Nucleic acids research 44.2 (2015): ...

  8. MXS Chaining

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    Blog Post
    ...assembly. Journal of biological engineering. 2010;4(1):1. Pubmed PMID: 20205762. PubMed Central PMCID: PMC2822740...with minimal spectral overlap between them (Table 1). In each construct, 3 copies of the fluorescent protein... enabled labeling of particular structures (Table 1). Each construct was flanked with a CMV promoter (...construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling, with...fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave researchers the ability ... all four constructs into one 15 kb insert. Table 1: MXS Constructs for Labeling Cellular Structures ...maxima Tethering partner Subcellular localization 1 TagBFP 399nm/ 456nm histone 2B (H2B) Chromatin 2...

  9. The Effect of COVID-19 on Liu Lab Plasmid Requests From Addgene Data

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    Blog Post
    ...was around 33% of that observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation...paper. Green and red lines depict the sum of mean +1 and -1 std for each paper, respectively. The fitted...complication in the China data – there is a period of 1 month, from 1/22/20–2/23/20, where there were zero plasmid... from 5/22/14 to 5/19/20). Each paper has between 1–32 plasmids and a total of 2–2,590 requests. We received...correspond to publications of popular papers. Figure 1: Plasmid requests by date. Parentheses depict the ...China. However, it is evident that if we believe that 1/22/20–2/23/20 are the more accurate dates of lockdown...in his laboratory. Learn more here. Additional resources on the Addgene blog Learn more about plasmid...

  10. Recombinase-based State Machines Enable Order-dependent Logic in vivo

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    Blog Post
    ...recombinase-based state machines in living cells” (1), into a more straight-forward, concise explanation...an RSM is to sequence the register. In our paper (1), we allowed individual bacterial cells to form colonies...in the Materials and Methods section of our paper (1). Lastly, it is possible to build gene regulatory ... not. How might one implement RSMs? In our study (1), we implement RSMs in E. coli with two plasmids (...inputs to endogenous gene expression (2). Our paper (1) explains how to arrange attB-attP pairs on a register...performance data from the indicated figures in our paper (1)) Input Plasmid Output Plasmid Fig. 3A pNR64 pNR160...in the Material and methods section of our paper (1). How might one troubleshoot GSRMs that don't work...

  11. Deep Mutational Scanning with One Pot Saturation Mutagenesis

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    Blog Post
    ...introduce all desired codon changes (step 1 in the image above). A second universal primer is used to synthesize...strand. Both produced about the same number of positive clones. See Supplementary Note 1 in Wrenbeck et al...strand of DNA degraded in the first digest (step 1 in above diagram). Remember that the BbvCI enzymes nick...think about one pot saturation mutagenesis? How would you use it in your research? References 1. Natarajan...making some or all of the following modifications: 1) increased concentration of Taq polymerase, 2) increased...introduced, and scalability (see Supplementary Table 1 of Wrenbeck et al). It uses a uracil labeled wild-...strand is nicked by the BbvCI variant not used in step 1. Then this strand is degraded with ExoIII and ExoI...

  12. The Breast Cancer Microenvironment: A Tumor’s Backstage Team

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    Blog Post
    ...enzyme (among others): caveolin 1, also known as CAV 1. Usually, caveolin 1 promotes the cell life cycle ...caveolin 1, there’s less malignancy because there’s less proliferation. However, the lack of caveolin 1 in ...believes that miRNA may decrease the expression of TAK 1, an enzyme that controls apoptosis and regulates transcription...Agnieszka K., et al. "An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor... related blog posts Learn about our cancer collection pages Read more about the tumor microenvironment...the tumor. How do microenvironment enzymes and metabolic reprograming influence tumors development? Being... vice versa, let’s take a look at cancer cell metabolism. Cancer cells promote oxidative stress leading...

  13. Important Considerations When Using AAVs

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    Blog Post
    ...in movement. Follow her on twitter @katrinaboards.   References 1. Russell, David W. "AAV vectors, insertional...vectors without specific facilities (Biosafety Level-1). Other possible tools, lentiviruses for example, ...suspected cases of insertional mutagenesis in humans (1) and AAVs are non-enveloped viruses, so they are resistant...Subscribe to Viral Vector Blog Posts from Addgene 1. Location, location, location! What do buying real...important factors related to location. Location (1): What tissue? Location (2): Where in the tissue/which...the right serotype? Keep asking questions!  Table 1: AAV Serotypes and Tropism Experiment  Detection...the vicinity of the injection. Examining serotypes 1, 8 and 9, not only do they undergo retrograde and ...

  14. Live and Let Dye: Self-Labeling Protein Tags

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    Blog Post
    ...intracellular targets. Nature Communications, 7(1), Article 1. https://doi.org/10.1038/ncomms10778 Erdmann...molecules in vivo. Nature Biotechnology, 21(1), Article 1. https://doi.org/10.1038/nbt765 Lavis, L. (2016... envelope design. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023-40788-8 ... capping protein. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023-39655-3 ...up linked to whatever molecule you choose (Figure 1). Just express the fusion protein and add a specific...proteins, opening up new applications.   Figure 1: Live-cell labeling strategies. The green structure...minimally-disruptive genetic tag on the protein of interest. Table 1: Summary of popular self-labeling tag systems ...

  15. Lentiviral Vector Uses and Overview

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    Blog Post
    ...HIV-1 were transduced with a lentiviral vector containing an antisense sequence against the HIV-1 envelope...Addgene depositor Didier Trono and collaborators turned to the lentivirus HIV-1, well-known to infect non-dividing...plasmids. The packaging plasmid carried a mutated HIV-1 provirus that couldn’t package itself due to a few...plasmid encoded the desired transgene flanked by HIV-1 long terminal repeats (LTRs) that facilitate viral... targeting and tracing cells in the brain (Figure 1). 2nd generation lentiviral packaging system The graphic...infectivity, is often used as a substitute for HIV-1 Env, which can only infect CD4+ cells. 3rd generation...lentivirus in the comments section below! References 1. In vivo gene delivery and stable transduction of ...

  16. Viral Vectors 101: Producing Your rAAV

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    Blog Post
    ...Inactivation of AAV Serotype 1 Vectors. Human Gene Therapy Methods, 28(1), Article 1. https://doi.org/10.1089...called “triple plasmid transfection” (Figure 1).    Figure 1: Overview of key steps of AAV production, ...repeat sequences. Human Gene Therapy Methods, 23(1), Article 1. https://doi.org/10.1089/hgtb.2011.034 Benyamini...in the CNS. Molecular Neurodegeneration, 15(1), Article 1. https://doi.org/10.1186/s13024-020-00361-z ...dehydration (Howard & Harvey, 2017). You can learn more about common AAV applications by reading our Viral Vectors...

  17. Mouse Modeling, Part 2: Breeding and Crossing Mice

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    Blog Post
    ...approximately 1 in 16 mice that harbor homozygous alleles at both of your genes of interest. About 1 in 4 mice...In Part 1 of our mouse modeling blog series, we covered techniques that can be used to introduce genetic...mice in your experiment. As you’ve learned in Part 1, there are many types of genetically engineered mice..., and assuming Mendalian patterns of inheritance: 1. Genotype your parental mice In order to ensure that...example of mice that are derived from crosses. In Part 1, we explained how genetic recombination in mice expressing...expressed, or inducible (this is explained more in Part 1 of this series). In any case, Cre is a dominant phenotype...Find our Plasmids 101 blog posts Read blog posts about using Cre-lox Read our genome engineering blog posts...

  18. When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

    Type
    Blog Post
    ...Figure 1).   Figure 1: Each problem with dimerizing fluorescent proteins is illustrated. 1) Membrane... in nature are prone to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [...active state due to closure by FP dimerization.   1. Transmembrane fluorescent protein fusions Transmembrane...bright, non-physiological, and difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several... For a more detailed discussion see [5]. Problems 1 and 2 depend on protein concentrations. An imperfect... distance runner, gardner, and cook.   References 1. Snapp, E.L., et al., Formation of stacked ER cisternae...sold by Clontech and is in a freezer box in just about every lab in the world, is not inert. In all seriousness...

  19. Cancer, Inflammation and Immunity - Harnessing the Body’s Defenses to Fight Cancer

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    Blog Post
    ...aggregation, and potentiates the effect of PD-1 blockade.“ (Ref 4). PD-1 helps tumor cells evade the immune system...addgene.org to find out more about our free deposit and distribution service! References: 1. Pietrocola, Federico...fasting has a positive effect on cancer treatment (Ref 1). As described in the presentation, starvation or ...of the immunosuppressive cell surface receptor PD-1. This immunosuppressive environment in turn may represent...  (Chimeric Antigen Receptors) CAR T cells and PD-1 modulators. An exciting development in immunotherapy...Cancer, Inflammation and Immunity and learn more about “... recent findings in basic immunology, cancer-immune...curing cancer in cross-disciplinary projects and collaborations. Summarized by Professor Michael Karin from...

  20. CRISPR-mediated Plant Base Editors

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    Blog Post
    ...rice crop improvement in the laboratory of Yinong Yang at PSU.   References 1. Komor, Alexis C., et al. ...excision repair mediated by uracil glycosylase (Figure 1). The presence of a uracil glycosylase inhibitor (...generation of the desired T-A base pair. Figure 1: Transition mutation (C>T, G>A, A>G, and T>C) installation... A-T base pair is converted to a G-C pair (Figure 1). As the cellular inosine excision repair is not as...Target-AID based (CBE-2)-  and rAPOBEC based (CBE-1)-  CBEs are successful base editors in rice, tomato...deaminase can edit in a stretched window ranging from 1-17 bp relative to the PAM sequence (Zong et al., 2018...which recognize PAM sequences other than NGG (Table-1). Type Nickase Cas9 (D10A) Deaminase PAM requirements...
Showing: 61 - 80 of 674 results