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We narrowed to 328 results for: hal.1

Showing: 61 - 80 of 328 results

  1. Droplet Digital PCR for AAV Quantitation

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    Blog Post
    ...range of titers. We usually dilute our samples 1:6 million to 1:25 million.  After making the dilutions, they...evaluation in challenged monkeys and TB diagnosis." Emerging microbes & infections 7.1 (2018): 1-9. PubMed...note that the dynamic range of the ddPCR is between 1 and 100,000 genome copies (GC) per reaction. Since...In the image to the right, there is approximately 1 positive copy per microliter in the no template control...elongation time to 2 minutes and the denaturation time to 1 minute has been shown to increase droplet separation...generating the standard curve for qPCR can be challenging to get right. Here at Addgene, we use a linearized...

  2. CRISPR Antimicrobials

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    ..., they used 1-2 CRISPR crRNAs targeting sequences found only in certain bacteria (Figure 1). Citorik et...from 50% to 11% in just 24 hours.   Figure 1: Single-phage strategies for bacterial CRISPR targeting...genome, and it carries CRISPR machinery targeting 1) the given resistance gene and 2) a second lytic phage...the fight against bacterial superbugs. References 1. Citorik, Robert J., Mimee, Mark, and Timothy K. Lu...Targeted Bacterial Population Editing.” Cell Syst. 1(3):187-196. (2015). PubMed: PMID 26973885. Additional...may provide a method for doing just that. While challenges remain in the delivery of these agents, CRISPR...CRISPR-induced double-stranded break (DSB) in the genome is lethal. If this DSB occurs on a plasmid, the plasmid will...

  3. Optogenetics + CRISPR, Using Light to Control Genome Editing

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    ...2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins. The goal of both groups was to create... the Sata lab is composed of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein...combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal fragment.... crassa) (Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and...fold-induction of Cas9 activity (16.4-fold). This paCas9-1 light-inducible system was able to recognize the same...flCas9). When triggered by blue light (470nm), paCas9-1 induced indel mutations via NHEJ (frequency of 20.5%...background activity of the system by modifying paCas9-1 using nMagC714 instead of nMagHigh1-C714, generating...

  4. Viral Vectors 101: AAV Variables That Matter

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    ...promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1 Oh, J. Y., Han, J. H., ...the selection process. Factors to think about are (1) your experimental conditions and measurements; (2...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...

  5. Adeno-associated Viruses (AAVs) for Genome Editing

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    ...infection, gene targeting vectors with relatively short (1 kb) homology arms underwent homologous recombination...sequences of interest to cells with efficiencies of 1-40% after G418 selection (Kohli et al 2004, Kim et...with polylinkers for the addition of gene-specific 1 kb homology arms flanking a FLOXed neoR gene. pAAV-SEPT-Acceptor...since it routinely generates knockin efficiencies of 1-40% with G418-selected clones. However, there are ...efficiency of gene editing is occasionally less than 1%, and virtually never exceeds 40%. While this is comparable...gene inactivation in human cancer.     References 1. Russell, David W., and Roll K. Hirata. "Human gene...double strand break repair, it has proven more challenging to develop high efficiency CRISPR-based approaches...

  6. Measuring Kinase Activity at the Single-Cell Level with Kinase Translocation Reporters (KTRs)

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    ...Kinases: they regulate many proteins, with ~1/3 of human proteins predicted to be phosphorylated on at...which have previously been characterized. See Box 1 in Kudo et al. for more details on the design of the...using translocation reporters. Nature protocols, 13 1, 155-169. PMID: 29266096. Find the KTR plasmids here...kinase activity is FRET, but FRET reporters are challenging to design and difficult to multiplex. The Covert...which fluorophores can be multiplexed together. Challenging  to Design: FRET reporter activity depends on...

  7. PiggyBac-ing Through the Genome Editing Field

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    ...Correct Path.,” J. stem cell Regen. Biol., vol. 1, no. 1, Jan. PubMed PMID: 26702451. PubMed Central PMCID...genetic manipulation in mammalian cells until 2005 [1]. Like other transposons, piggyBac™ has two components...The predicted frequency of ‘TTAA’ in the genome is 1 in every 256 base-pairs of DNA sequence, making it...has been demonstrated) with no known upper limit [1]. The piggyBac™ technology can be used for numerous...genetic loci of integration [8].   Figure 1: Mechanism of the piggyBac™  transposase/transposon...technologies and strategies for their projects. References 1. L. E. Woodard and M. H. Wilson, “piggyBac-ing models...CRISPR/Cas9, TALENs, and ZFNs). However, several challenges still remain, such as making gene edits completely...

  8. Viral Vectors 101: Systemic Capsids

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    ...total dose between 1 x 1011 and 5 x 1011 vg per animal (i.e., 10 and 50 µL of vector at 1 x 1013 vg/mL titer... and 1 x 1012 vg*. Most systemic capsids are easy to produce in high titer, but higher doses (>1 x 1012...before using it in an application.     Figure 1: AAV toolkit for targeted gene delivery to the brain...decision tree for selecting a capsid, while Table 1 gives a brief overview of published descriptions of...Capsids page to learn more about that collection. Table 1: Systemic capsid plasmids available from Addgene ...AAV9. At lower viral titers than AAV-PHP.B (e.g., 1 x 1011 vg/mouse), AAV-PHP.eB can transduce a greater...Diverse Brain Cells. Annual Review of Neuroscience, 45(1), 447–469. https://doi.org/10.1146/annurev-neuro-111020...

  9. Which Fluorescence Microscopy Technique is Best for Me?

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    ... microscopy contrast technique in modern biology (1). When performed correctly, fluorescence microscopy...relatively thicker three dimensional sample (see Figure 1); similar to an MRI or CT scanner. This is accomplished... the resolution of a light microscope (see Figure 1). What microscopy technique should I use for my experiment...new sample I always start by asking two questions: 1) Is the sample dynamic or static? 2) Is the sample...of modern day fluorescence microscopes (see Table 1). Thin dynamic samples Ex: monolayers of live cells...excitation light can push imaging depths closer to 1 mm. Traditionally, 2-photon has been a very slow imaging...manufacturers have similar information References 1. Lichtman JW, Conchello JA. Fluorescence microscopy...

  10. Adenoviral Vector Production and Troubleshooting

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    ...repeats (ITR) present at both ends.   Figure 1: A) The structure of an adenovirus: the virion is ... 5 key steps: Construction of the rAdV plasmid (~1 week): The AdEasyTM system is the most popular method...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...transfected with the AdV plasmid construct from step #1 and allowed to stay in culture for up to 20 additional... 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 ...

  11. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

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    ...preventing your primary antibody from binding (Fig.1). The level of cross-linking that occurs depends on...proteins, which could disrupt your target epitope (Fig. 1). So, while organic solvents may improve antibody ...thing depending on your experiment.   Figure 1: Choice of fixation method impacts antibody binding...New York, pp 299–311. https://doi.org/10.1007/978-1-4939-8935-5_26  Rolls G (2022) Process of Tissue Fixation...www.leicabiosystems.com/us/knowledge-pathway/fixation-and-fixatives-1-the-process-of-fixation-and-the-nature-of-fixatives...samples in a given state or point in time. You want to halt degradative processes while maintaining the structure...fixation-artifacts-and-how-to-minimize-them/  Goldenthal KL, Hedman K, Chen JW, et al (1985) Postfixation...

  12. Educational Planarian Tools for Teaching Developmental Biology Techniques

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    ... hybridization experiments. A recent publication [1] from the Sánchez lab describes the plasmids and protocols...deposit) are described in the Sánchez lab publication [1] so that the students performing these experiments...can easily investigate in planarians. References 1. Alice Accorsi, Monique M. Williams, Eric J. Ross,...Proceedings of the National Academy of Sciences 100.suppl 1 (2003): 11861-11865. PubMed PMID: 12917490. PubMed...experiments are either not accessible or too challenging for amateur scientists. Planarians, wormy creatures...available planarians: Girardia sp., Dugesia dorotocephala, Phagocata morgani, and Phagocata gracillis....differ. For example, Girardia sp. and Dugesia dorotocephalacan can be fed dsRNA-expressing E. coli that are...

  13. Typing CRISPR Systems

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    ...   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...types, 33 subtypes, and 17 variants. Figure 1: Overview of the CRISPR classification hierarchy. ...effector complex after binding to a target. Class 1 is the most abundant of the two, comprising about ...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...

  14. New Neuroscience Tool: The iGluSnFR3 Glutamate Sensor

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    ...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...iGluSnFR, the Looger lab wedged GFP between two halves of a bacterially derived periplasmic glutamate ...

  15. Viral Vectors 101: AAV Serotypes and Tissue Tropism

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    ...them to target particular cell types. Figure 1: Diagram of AAV components. Created with BioRender.com...depends on your choice of promoter/enhancer. Table 1: AAV capsids and receptors. Capsid Primary receptor...required)2 Human fibroblast growth factor receptor 1 (FGFR1)3 αVβ5 and α5β1 integrins3 Hepatocyte growth...structure of AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated...utilizing various AAV serotypes. Scientific Reports, 15(1), 20927. https://doi.org/10.1038/s41598-025-05518-...-directed transduction. Nature Communications, 14(1), 3792. https://doi.org/10.1038/s41467-023-39554-7... and Transduction by Adeno-Associated Virus Types 1 and 6. Journal of Virology, 80(18), 9093–9103. https...

  16. Fluorescent Proteins 101: Fluorescent Protein Timers

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    ...few properties that have now become hallmarks of ideal FP timers: 1. Ratiometric determination of expression...the FT series matures from blue to red (see Table 1 for emission and absorption spectra). Subach et al...times and varied blue-red maturation times (Table 1). These separate proteins should be useful for monitoring...cellular events occurring at varied time scales.  Table 1: Monomeric FP Timers and Associated Plasmids from ... know in the comments section below! References 1. Terskikh, Alexey, et al. ""Fluorescent timer": protein...understand how protein localization changes over time.Hallmarks of fluorescent protein timers When FP timers...

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

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    ...aggregation, and potentiates the effect of PD-1 blockade.“ (Ref 4). PD-1 helps tumor cells evade the immune system...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...free deposit and distribution service! References: 1. Pietrocola, Federico, et al. "Caloric restriction...Immunologists are coming together to tackle the challenge of curing cancer in cross-disciplinary projects...

  18. Hot Plasmids - February 2022

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    ... Figure 1: (a) Anti-FLAG frankenbody (FB) construction from the complementarity determining region of...Comm. 2019. https://doi.org/10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a... to Cas9’s larger expression requirements (Figure 1), but Cas12a has generally been underutilized in genetic...SNAP-tag, and HaloTag fusions, while the anti-HA frankenbodies include mEGFP, mCherry, and HaloTag. ...screening technique, researchers identified a synthetic lethal interaction between MARCH5 and WSB2 genes using...fusion proteins, such as Cas9 and Cas12a, creates challenges for in vivo delivery and cell engineering applications..., is composed of only 529 amino acids, less than half the size of either Cas9 or Cas12a. Despite its small...

  19. Using AAV for Neuronal Tracing

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    ...but also others including pseudorabies virus type 1, vesicular stomatitis virus (VSV; Beier et al., 2011...Skeletal Muscle AAV1, AAV6, AAV7, AAV8, AAV9 Table 1: Common AAV Serotypes and their target tissues (adapted... serotypes capable of entering neurons (see table 1)(Choi et al., 2005; Taymans et al., 2007; Howard et...efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310...Wolfe, J.H. (2014). Adeno-associated virus serotypes 1, 8 and 9 share conserved mechanisms for anterograde...,9 in rat neurons and glia in vitro. Virology 372(1), 24–34. PubMed PMID: 18035387. PubMed Central PMCID...transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem...

  20. An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

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    ...pha-1(e2123) mutant worms are perfectly viable at 15 ºC, yet exhibit complete embryonic lethality at 25...effective recovery of knock-ins, but did result in 1-2 kilobases of additional sequence being introduced...facilitating recovery of edited animals. Figure 1: In the background are 504 restriction digests of ...selection for repair of a temperature-sensitive pha-1 mutation significantly enriches for knock-in of 2x... repair pathway. Reagents required to perform pha-1 co-conversion are available through Addgene. In 2019...the power of lethal mutation co-selection. Only animals rescued for the conditional lethal mutation survive...selection. Knock-in events tend to be rare, and the challenge with any experiment is in efficient recovery of...
Showing: 61 - 80 of 328 results