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We narrowed to 224 results for: ENDO 2

Showing: 81 - 100 of 224 results

  1. R Bodies: Membrane-Rupturing Microscopic Tools

    Type
    Blog Post
    ...be decorated with proteins and remain functional, 2) break membranes outside of their natural context ...PubMed PMID: 3759909. PubMed Central PMCID: PMC213417. 2. Schrallhammer, M. et al. Tracing the role of R-bodies...Silver.  Most types of biological motion (whether endocytosis, vesicle trafficking, or muscle contractions)...video. Most types of biological motion (whether endocytosis, vesicle trafficking, or muscle contractions)... conditions. More speculatively, R bodies might endow divergent organisms with new functions. For inspiration...bodies assemble and function, we might be able to endow cells with new ways to interact mechanically with...

  2. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ...Feb 2;33(2):139-42. doi: 10.1038/nbt.3149. Light-inducible CRISPR-Cas9 system for endogenous gene activation...and on your favourite signalling pathways (Figure 2). They are all available now at Addgene. 1 Lungu...20;19(4):507-17. 10.1016/j.chembiol.2012.02.006. 2 Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; ...Nihongaki et al., Chemistry and Biology. 2015 Feb 19; 22(2):169-74. High efficiency cloning for enzyme screening...inducible, gene knockdown with shRNA or shRNA-miR30, (2) tamoxifen-inducible CreERT2 gene deletion, (3) simultaneous...different fluorescent proteins, 8 constitutive promoters, 2 includible promoters, 3 polyA terminators and various...of plasmids required for each input/output system; 2) removing all chemically inducible promoters; 3) improving...

  3. Plasmids 101: TOPO Cloning

    Type
    Blog Post
    ... the two DNA strands and releasing topoisomerase (2).  These days commercially available TOPO kits provide...Taq polymerase using your favorite PCR protocol.  2. Set Up TOPO Cloning Reaction: Mix together the PCR...PubMed PMID: 1658796. PubMed Central PMCID: PMC52876. 2. Novel approach to molecular cloning and polynucleotide...green cloud) functions both as a restriction endonuclease and as a ligase by cleaving and rejoining supercoiled...

  4. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    ...testing your AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two different ...) your experimental conditions and measurements; (2) the cells you’re targeting; (3) how much expression... Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain. PLOS ONE, 8(9),... Comparison of four promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1...analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral...Astrocyte-selective AAV gene therapy through the endogenous GFAP promoter results in robust transduction ...

  5. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    ...satisfactorily accepts DNA as its substrate (see figure 2).          The artificially evolved adenosine deaminase...editing platforms for plant systems.  Figure 2: Plant base editor platforms and their molecular components...available for plant systems. Target-AID based (CBE-2)-  and rAPOBEC based (CBE-1)-  CBEs are successful...based CBEs are known to deaminate cytosine placed in 2-6 bp and 4-8 bp, respectively, of the protospacer....PMID: 27096365. PubMed Central PMCID: PMC4873371.  2. Nishida, Keiji, et al. "Targeted nucleotide editing...CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system." Proceedings of the National...

  6. CRISPR 101: Making a Knock-In Cell Line

    Type
    Blog Post
    ... However, if you have a larger knock-in (200 bp – 2 kb), a dsDNA donor will need to be used due to synthesis...accidentally change your protein of interest.   Fig. 2: Stop re-cutting with PAM and guide RNA disrupting... the other repair mechanisms Mammalian cells have 2 primary repair mechanisms besides HR - Non-homologous.... Com., 8(2024), (2017). 10.1038/s41467-017-01836-2 Charpentier, M., Khedher, A. H. Y., Menerot, S., Brion...from a template molecule, which is usually an endogenously available sister chromatid, though exogenous...

  7. A Needle in a Base-Stack: Cas9 Structural Biology

    Type
    Blog Post
    ...Cas9 from Streptococcus pyogenes.   Figure 2:  Crystal structure of S. pyogenes Cas9 in the apo...NUC and REC lobes laying open like a book (Figure 2). The portion of the CTD that recognizes the PAM is...it doesn’t appear in the crystal structure (Figure 2) at all. When this crucial recognition region is disordered...regions, so crystal structures like those in Figures 2 and 3 don’t show these regions, even if they are present...itself at the bottom of the image. Stem loops 1 and 2 extend horizontally across both lobes. The guide RNA...is required for Cas9 nuclease function. Stem loops 2 and 3 are not strictly necessary for in vitro cleavage...open conformation, but highly flexible, as in Figure 2. The guide RNA is bound primarily by the REC lobe,...

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

    Type
    Blog Post
    ...non-specific proteins, and then elute YFP.  Option #2: Opto-Nanobodies (OptoNBs) purification You skip adding...blue lights. Which option do you choose?  Option #2 may sound like fiction, but light-controlled protein...experiments.  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced...purification as well as reversibly regulation of endogenous signaling activity in cells.  Let’s shed some...

  9. The PAM Requirement and Expanding CRISPR Beyond SpCas9

    Type
    Blog Post
    ... F (2015) Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . ...is unique compared to the rest of the genome and (2) the sequence is located just upstream of a Protospacer...pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from species other...Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 34:863–868 . ...

  10. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    ... than control, 3; same as control (Cetn1/sgRNA1), 2; darker than control, 1; very dark). Brighter fluorescence...you can hold off on genotyping until the pups are 2-4 weeks old and use the ear tag method (creating different...PMID: 24253446. PubMed Central PMCID: PMC3875854. 2. Mashiko, Daisuke, et al. "Generation of mutant mice...target sequence was digested by sgRNA guided Cas9 endonuclease, homology dependent repair (HR, homologous recombination...off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases." Genome research 24.1 (2014): ...

  11. Plasmids 101: Degron Tags

    Type
    Blog Post
    ...required components are 1) a SMASh-tagged protein and 2) asunaprevir (small molecule). dTAG The degradation...gone through an additional round of evolution (AID-2) to reduce some of the leakiness associated with the...become increasingly popular for the modulation of endogenously and exogenously expressed proteins. Here we ...Secondly, these tags can be used to manipulate endogenously and exogenously expressed proteins. Thirdly,...dimerization of the FKPB12F36V fusion protein with the endogenous CRBN E3 ligase complex which will polyubiquitinate...the dTAG uses a small molecule, a tag, and the endogenous cellular degradation machinery to function. The...The TIR1 F-box protein will associate with the endogenously expressed SCF complex members, an E3 ubiquitin...

  12. Hot Plasmids: Spring 2025

    Type
    Blog Post
    ...cell-attached recordings in primary visual cortex (V1) layer 2/3 (L2/3). B) Fluorescence from axonal boutons of neurons... generation and stable cell line creation (Figure 2). Looking for more? Explore our empty and EGFP retroviral... Lentiviral backbone plasmids here! Figure 2: Addgene cloned EGFP into pAG Lenti CMV N-HA Puro ...Glypicans are proteoglycans composed of a protein core, 2–4 glycosaminoglycans (GAG), and are (typically) tethered...either the optogenetic activator channelrhodopsin-2 or fluorescent reporter dTomato under the control ...2025). In retrotransposon insertion, a combined endonuclease-reverse transcriptase uses homology arms flanking...

  13. Quick Guide to Working with Drosophila Part 3: Genome Engineering in Flies

    Type
    Blog Post
    ...are many docking sites available for integration. 2. How soon would you like your flies ready for use?...beautiful immunofluorescent images like in this paper (2). Metabolic assays such as Seahorse can be employed...PMID: 16547094. PubMed Central PMCID: PMC1526508. 2. H. Jiang,  et al. Cytokine/Jak/Stat signaling mediates...which restores eye color to fly lines where the endogenous white gene is knocked out. Flies with your integration...

  14. Antibodies 101: Conjugation

    Type
    Blog Post
    ...conjugation. 1) Endogenous lysine residues, which can be found throughout the antibody. 2) Endogenous cysteines...Detecting antigens by quantitative immuno-PCR. Nat Protoc 2:1918–1930. https://doi.org/10.1038/nprot.2007.267 ...the disulfide bonds between antibody chains. 3) Endogenous carbohydrates added to antibodies post-translationally...

  15. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...Currently available from the Kim lab are 2 presynaptic and 2 postsynaptic targeting mGRASP plasmids. Additionally... cells were pulsed with 2µg/ml of Doxycycline for 2 hours, and imaged on the Keyence BZ-9000 fluorescence...work! Robinson et al., Dev Cell 2010 Feb 16;18(2):324-31. Cheeseman et al., J Cell Sci 2013 May 1...promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated... of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly...1 modules can be similarly assembled into a Level 2 vector, thus forming a functional genetic circuit....circuit. Flexibility has been built into the Level 2 vectors to allow for additional iterations of Level 1 assembly...

  16. Viral Vectors 101: AAV Serotypes and Tissue Tropism

    Type
    Blog Post
    ...proteoglycan (HSPG)3 AAV receptor (not strictly required)2 Human fibroblast growth factor receptor 1 (FGFR1... AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated nomenclature...suggest these serotypes as a good place to start. Table 2: AAV tissue tropism for mice in vivo. To target... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PloS One, 8(9),...Retrograde Access to Projection Neurons. Neuron, 92(2), 372–382. https://doi.org/10.1016/j.neuron.2016.09.021...reported to have tropism for lung, liver, vascular endothelial, and smooth muscle cells (Issa et al., 2023)....

  17. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

    Type
    Blog Post
    ...CombiGEM lentiviral vector backbone pAWp12 (Figure 2). Cloning from the pAWp28 storage vector to the pAWp12... before beginning your experiments. Figure 2: Strategy for assembling barcoded combinatorial gRNA...PMID: 25114216. PubMed Central PMCID: PMC4151723. 2. Doench, John G., et al. "Optimized sgRNA design to...targeting your genes of interest. Thanks to the tremendous efforts made by various research teams to build...

  18. Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9

    Type
    Blog Post
    ... (2,3). Stable strand separation is maintained by 2 sets of interactions : 1. Interactions between the...formed by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand and the gRNA... Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal structure of ...PAM-dependent target DNA recognition by the Cas9 endonuclease." Nature 513.7519 (2014): 569-573. PubMed PMID...

  19. Viral Vectors 101: Calcium Sensors

    Type
    Blog Post
    ...calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) ...user-controlled illumination with violet light and (2) exposure to increased intracellular calcium. It has...dynamics in vivo. Neuroscience Research. 2021 Aug 1;169:2-8. Suzuki J, Kanemaru K, Iino M. Genetically encoded... prevent aberrant binding of the biosensor to endogenous calmodulin. Multiplexing Are you combining your...

  20. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    ...Nature Cell Biology, 26(2), 294–304. https://doi.org/10.1038/s41556-023-01342-2 Koblan, L. W., Arbab, M...order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent...region of separate strands called an R-loop (Figure 2). This allows the guide RNA to base pair with the ...accessible on the outside of the enzyme. Figure 2: CRISPR R-loop. Created with BioRender.com. Base...), 35–40. https://doi.org/10.1038/s41587-020-0592-2 Additional Resources on the Addgene blog CRISPR 101.... They conducted a CRISPRi screen to identify endogenous genes that impact CGBE efficiency. In addition...
Showing: 81 - 100 of 224 results