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We narrowed to 736 results for: RAN-1

Showing: 121 - 140 of 736 results

  1. Deep Mutational Scanning with One Pot Saturation Mutagenesis

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    Blog Post
    ...used in step 1. Then this strand is degraded with ExoIII and ExoI. Synthesize 2nd mutant strand: The second...are the same sense as the strand of DNA degraded in the first digest (step 1 in above diagram). Remember...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-...primers to introduce all desired codon changes (step 1 in the image above). A second universal primer is ...number of positive clones. See Supplementary Note 1 in Wrenbeck et al for details on how nicking was optimized...time no, but there are 2 instances where it does. 1) When the backbone vector has multiple BbvCI sites...

  2. Adenoviral Vector Production and Troubleshooting

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    ...references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid is identified, re-transform into a bacterial...rAdV-S). HEK293 cells are transfected with the AdV plasmid construct from step #1 and allowed to stay in ... (ITR) present at both ends.   Figure 1: A) The structure of an adenovirus: the virion is ~ 100nm... 5 key steps: Construction of the rAdV plasmid (~1 week): The AdEasyTM system is the most popular method... it will result in very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more HEK293...-4 rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification should result...CsCl are commercially available. Titer calculation (1 or 10 days) Physical titer (particle count, rAdV ...

  3. Harnessing TnpB for Genome Editing: A Compact and Versatile Tool for Your Research

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    Blog Post
    ... Oligos Oligo 1: Add 'tcaa' to the 5′ end of your forward guide sequence (see Figure 1). Oligo 2: Add... Figure 1). Perform standard phosphorylation and annealing of the two oligos. Figure 1: Components... promoter for ωRNA (reRNA) expression (see Figure 1). Both plasmids contain a hygromycin resistance gene...provide a step-by-step guide for using a Dra2TnpB. Step 1: Design the 20-nt Guide Sequence Identify a TAM site...protocols (pp. 245–260). https://doi.org/10.1007/978-1-0716-4358-7_20  Karmakar, S., Panda, D., Panda, S... induction of meristems. Nature Biotechnology, 38(1), 84–89. https://doi.org/10.1038/s41587-019-0337-2...1166–1187. https://doi.org/10.1038/s41477-021-00991-1  Qiao, J., Zang, Y., Gao, Q., Liu, S., Zhang, X.,...

  4. Important Considerations When Using AAVs

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    Blog Post
    .... "Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and...Examining serotypes 1, 8 and 9, not only do they undergo retrograde and anterograde transport, but they also...serotypes 1, 8, and 9 share conserved mechanisms for anterograde and retrograde axonal transport." Human...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...

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

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    ...intracellular targets. Nature Communications, 7(1), Article 1. https://doi.org/10.1038/ncomms10778  Erdmann...small molecules in vivo. Nature Biotechnology, 21(1), 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 ...

  6. Viral Vectors 101: The Retroviral Lifecycle

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    ...can use Figure 1 to compare the genomes of these two family members.   Figure 1: Gamma-retrovirus...Retroviruses” is an umbrella term which encompasses 1.) the gamma-retroviruses — that have been developed...of retroviruses called the lentiviruses (like HIV-1) — that have been developed into lentiviral vectors...Academy of Sciences of the United States of America, 54(1), 137–144. https://doi.org/10.1073/PNAS.54.1.137. ...1997). Functional RT and IN incorporated into HIV-1 particles independently of the Gag/Pol precursor protein...RNA as template. Journal of Molecular Biology, 79(1), 163–183. https://doi.org/10.1016/0022-2836(73)90277...Aids Full-Length Human Immunodeficiency Virus Type 1 DNA Synthesis. Journal of Virology, 82(5), 2376. https...

  7. 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...infect. Finally, the transfer plasmid encoded the desired transgene flanked by HIV-1 long terminal repeats...packaging, envelope, and transfer plasmids. The packaging plasmid carried a mutated HIV-1 provirus that couldn...section below! References 1. In vivo gene delivery and stable transduction of nondividing cells by a ...Trono and collaborators turned to the lentivirus HIV-1, well-known to infect non-dividing cells. The first... 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...

  8. Degrading DNA with Cascade-Cas3

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    Blog Post
    ...Cascade's complexity. Figure 1: Schematic of Class 1 CRISPR types. Class 1 includes type I with a Cas3 ...collection! A "complex" system Cas3 is part of the Class 1 CRISPR family, which means it requires multiple Cas...the brunt of the mechanical work for editing. Class 1 CRISPR systems make up the majority of CRISPR-based...CRISPR-based immune systems in bacteria and archaea. Class 1 is further broken down into different types, with ...making up type I, the most abundant type in the Class 1 category. These type Is can then be further broken...editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-13226-x Cameron...non-target strand. The helicase domain is then activated, and Cas3 begins unraveling the double-stranded DNA ...

  9. 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... dimerization.   1. Transmembrane fluorescent protein fusions Transmembrane proteins, i.e. receptors... in nature are prone to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [...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...contacts that bring apposing membranes together and can wrap the membranes into stacked structures including...

  10. Antibody Validation for Flow Cytometry

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    Blog Post
    ...their HLDA workshops. Let’s use TIM-1 (Single pass type-1 membrane protein), which has been designated...dye, respectively. WT and KO cells were mixed in a 1:1 ratio and fixed in 4% PFA and permeabilized in 0.1%...antibodies were diluted to 1 µg/mL except for 14558, which was used at 0.35 µg/mL (1/100). * = monoclonal antibody...specific antibody:target combination of interest (Figure 1). It can therefore be helpful to try all three when...to label an intracellular target.   Figure 1: HCT 116 WT and SYT1 KO cells were labelled with a...cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb CoraLite® Plus...between the wild-type and the KO population (Figure 1), further optimization can be performed to both maximize...

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

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    ...approximately 1 in 16 mice that harbor homozygous alleles at both of your genes of interest. About 1 in 4 mice...ve learned in Part 1, there are many types of genetically engineered mice: transgenic mice, knockin and...In Part 1 of our mouse modeling blog series, we covered techniques that can be used to introduce genetic..., 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... integration of a novel gene randomly into their genome, some transgenic strains are difficult to breed...

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

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    ...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 ... expression of TAK 1, an enzyme that controls apoptosis and regulates transcription. Without TKA1, cells...Agnieszka K., et al. "An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor...microenvironment. This results in increased expression of transforming growth factor-β signaling, possibly inducing...let’s take a look at exosomes. Proficient in transportation, exosomes are tools of communication and provide... (micro RNA) and other molecules that can be transferred to other cells, inducing epigenetic changes in...

  13. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
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    ...and no viral progeny are produced (Figure 1).  Figure 1: Comparison of the life cycle of a lentiviral...2004). HIV-1 gene expression: lessons from provirus and non-integrated DNA. Retrovirology, 1, 13. https... cannot integrate it into the host genome (Figure 1). IDLVs were first discovered by researchers investigating...you might choose an IDLV over a lentiviral vector: 1. No risk of insertional mutagenesis Lentiviral vectors..., H. E. (1996). Human immunodeficiency virus type 1 integrase mutants retain in vitro integrase activity...Berry, C., Ecker, J. R., & Bushman, F. (2002). HIV-1 integration in the human genome favors active genes...https://doi.org/10.1186/1742-4690-1-13. PubMed PMID: 15219234 PubMed Central PMCID: PMC449739 Yew, C.T., ...

  14. Four Ways to Package Transgenes That Exceed the Size Limit of Adeno-associated Virus

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    ...and drawbacks of four split AAV vector approaches. 1. Overlapping  Overview: The overlapping strategy has...Adeno-Associated Virus Capsids. Human Gene Therapy Methods 27:1–12 . https://doi.org/10.1089/hgtb.2015.140 Frischmeyer... successfully mediated transduction and expression of the full-length transgene in vitro and in vivo (...this, larger functional transgene products were produced following transduction.  So what’s going on here...between part A and B of the transgene to reconstitute the full-length transgene (Chamberlain et al., 2016...cells in mice. This vector transduced ~10% of cells, similar to the transduction rate of a single AAV. Odom...muscle physiological performance.  2. Trans-splicing Overview: For trans-plicing, splice site donor and acceptor...

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

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    ...nucleotides 1-10 of the guide RNA, prearranged for base pairing with a complementary DNA strand. These 10...4CMP) with domains colored as in Figure 1. HNH, red; RuvC, orange; CTD, yellow; REC lobe, light blue; bridge...10.1146/annurev-biophys-062215-010822 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I.... basic parts of the Cas9 enzyme.   Figure 1: A cartoon depiction of Cas9’s two major lobes, REC...the CRISPR system (Nishimasu et al., 2014). Table 1. Summary of Cas9 domains, the lobe each domain belongs... on itself at the bottom of the image. Stem loops 1 and 2 extend horizontally across both lobes. The guide...outside of the protein, spanning both lobes. Stem loop 1, nestled between the REC and CTD domains, is required...

  16. Plasmids 101: Expressing shRNAs for RNA Interference

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    ...vectors. BMC Biotechnology, 6(1). https://doi.org/10.1186/1472-6750-6-1 O’Brien, J., Hayder, H., Zayed...machinery for processing (Figure 1). The shRNA sequence is transcribed to produce hairpin shRNA structures... recruits other silencing proteins. Figure 1: Comparison of shRNA and miRNA processing and silencing...,"rgb":"rgb(0, 82, 169)","rgba":"rgba(0, 82, 169, 1)"},"button_font":{"size":19,"styles":{"font-family...:"rgb(255, 255, 255)","rgba":"rgba(255, 255, 255, 1)"}}{% endraw %}{% end_module_attribute %}{% module_attribute...RNA-induced silencing complex. Molecular Cell, 82(1), 30–43. https://doi.org/10.1016/j.molcel.2021.11.026...progress in miRNA biogenesis and decay. RNA Biology, 21(1), 36–43. https://doi.org/10.1080/15476286.2023.2288741...

  17. CRISPR-mediated Plant Base Editors

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    Blog Post
    ...of the desired T-A base pair. Figure 1: Transition mutation (C>T, G>A, A>G, and T>C) installation...hAPOBEC3 deaminase can edit in a stretched window ranging from 1-17 bp relative to the PAM sequence (Zong et...excision repair mediated by uracil glycosylase (Figure 1). The presence of a uracil glycosylase inhibitor (... 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...which recognize PAM sequences other than NGG (Table-1). Type Nickase Cas9 (D10A) Deaminase PAM requirements...locus/loci, choose a base editor variant from Table 1 depending on the PAM sequence. Avoid targeting a ‘...

  18. New Neuroscience Tool: The iGluSnFR3 Glutamate Sensor

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    Blog Post
    ...sensing glutamate using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation and ...hSynap: Human Synapsin-1 promoter. Good for neuronal expression. hSynap-FLEX: Synapsin-1 promoter, FLEXed for... v857. Image adapted from Aggarwal et al., Figure 1   Increased versatility through two different...expression in Cre-expressing cells. hSynap-FLP: Synapsin-1 promoter, FLP-dependent expression GFAP: Glial fibrillary...three different membrane-targeting flavors – PDGFR, GPI, and SGZ, to improve neuronal membrane trafficking...properties such as kinetics, ∆F/F0, SNR, dynamic range, photostability, quantum yield, and expression. ...segment (PDGFR TMD) that anchors the sensor to the membrane and displays it on the extracellular side. In ...

  19. CRISPR-based Genome Editing Systems For Editing in E. coli

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    ... Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from Li et...coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella citrea, with a total of 70 sites...the pCas/pTargetF system.   Figure 1: The editing process begins with the addition of the sgRNA sequence...updates involved the following changes. In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2)...biosynthesis, we constructed a strain library with 1-11 copies of integrated genes. We obtained a stable...in E. coli BL21(DE3) as transformants failed to grow after the transformation of pTargetF into the BL21...pTnsABC expresses the transposases TnsA, TnsB, and TnsC pQCascade expresses the transposase TniQ, the Cas proteins...

  20. History of CRISPR Cas - A tale of survival and evolution

    Type
    Blog Post
    ...Davidson AR.  Nature. 2013 Jan; 493, 429–432. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...sequences were initially discovered in E. coli in 1987 (1), the concept that these clustered repeat sequences...neutralize CRISPR/Cas. Earlier this year, expression of 1 of 5 anti-CRISPR genes were found to inactivate the...and are not translatable to the other CRISPR subtypes. Still, the large and dynamic range of CRISPR systems...well-suited for various innovative biological and translative technologies. Addgene has empowered researchers...http://en.wikipedia.org/wiki/File:Crispr.png Barrangou R, Horvath P. Science. 315, 2007 Jan; 1709–1712...CRISPRdb. Date: 2013-09-18. Makarova K, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, Moineu...
Showing: 121 - 140 of 736 results