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We narrowed to 250 results for: hal.2

Showing: 21 - 40 of 250 results

  1. Typing CRISPR Systems

    Type
    Blog Post
    Published
    March 18, 2025, 1:15 p.m.
    ...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems use...systems to carry out various functions. Class 2 Class 2 CRISPR systems rely on a single protein Cas effector...explore Class 2 CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...complex for antiviral defense) complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade...1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class...components present in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits) with...

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

    Type
    Blog Post
    Published
    Sept. 3, 2020, 12:15 p.m.
    ...N-terminal (residues 2-713, N713) and C-terminal (residues 714-1368, C714) halves, rendering the Cas9 ...the light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 ...inactive, dead Cas9 protein (dCas9) fused to CIB1; and 2) the activator - the CRY2 photolyase homology region... fused to both the N- and C-termini of dCas9; and 2) CRY2FL-VP64, a fusion of full-length CRY2 and the...researchers saw an 11-fold increase in mRNA levels after 2 hr and a 400-fold increase after 30 hr. The system...nMagC714 instead of nMagHigh1-C714, generating paCas9-2. This change did not significantly alter the system...prior work, the Sato lab also showed that the paCas9-2 system could be spatially controlled and reversibly...

  3. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    Published
    Jan. 6, 2015, 5:21 p.m.
    ...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...

  4. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    Published
    June 27, 2023, 1:15 p.m.
    ...for controlling neuronal activity, channelrhodopsin-2 (ChR2), a blue light-activated cation channel, from...used in tandem with the opsin.      Figure 2: Diagram of excitatory (ChR2) activation by light ...) domains, phytochrome B (PhyB), and cryptochrome 2 (CRY2) (Zhang & Cui, 2015). When stimulated with light...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 References...comparative analysis of microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 Nagel...Hegemann, P., & Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane ...intracellular signaling pathways. Trends in Biotechnology, 33(2), 92–100. https://doi.org/10.1016/j.tibtech.2014.11.007...

  5. CRISPR Antimicrobials

    Type
    Blog Post
    Published
    May 3, 2016, 2:30 p.m.
    ...Citorik Plasmids here). In both cases, they used 1-2 CRISPR crRNAs targeting sequences found only in certain...resensitize antibiotic-resistant bacteria (Figure 2). The first phage introduced is lysogenic, integrating...machinery targeting 1) the given resistance gene and 2) a second lytic phage. Bacteria that receive this ...bacteria have previously flourished.   Figure 2: Two-phage strategies to resensitize bacteria to antibiotics...237163. Find plasmids from this paper at Addgene. 2. Bikard, David, et al. “Exploiting CRISPR-Cas nucleases...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...

  6. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    Published
    April 17, 2018, 1:37 p.m.
    ...transfection where 2 of 11 clones tested had plasmid integration. Another challenge is that multiple transfection...Recent progress and future challenges." Journal of Cellular Physiology 228, no. 2 (2012): 267-75. PubMed ...plasmid-based expression requires serial transfection of 1 or 2 plasmids that express the reprogramming factors of...understanding the mechanisms of reprogramming, and 2) studies with clinical endpoints. In the first scenario...PubMed PMID: 27764162. PubMed Central PMCID: 5072705. 2. Sommer, Cesar A., and Gustavo Mostoslavsky. "The ...PubMed PMID: 23546745. PubMed Central PMCID: 4176696. 2. Yusa, Kosuke, Roland Rad, Junji Takeda, and Allan...generation. Expert opinion on biological therapy, 10 2, 231-42. PubMed PMID: 20088717. PubMed Central PMCID...

  7. The Materials Science of Optogenetics Experiments

    Type
    Blog Post
    Published
    Sept. 17, 2015, 2:30 p.m.
    ...different steps and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail,...stimulation         In the video above, Channelrhodopsin 2 (Chr2) has been expressed in the motor cortex of a...lasers are relatively cheap and will run you about $2-3,000/laser. However, an important caveat is that ... the light-gated cation channel, Channelrhodopsin 2 (ChR2), exists in several states: closed/light receptive...PMID: 21628464; PubMed Central PMCID: PMC3138296. 2. Sparta DR, et al. Construction of implantable optical...board, Jacqueline N Crawley  [et al]. 2012;Chapter 2:Unit2 16. doi: 10.1002/0471142301.ns0216s58. PubMed...validation experiments we discussed last week are only half of the battle when using optogentics to answer a...

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

    Type
    Blog Post
    Published
    April 16, 2024, 1:15 p.m.
    ...super-resolution or single-molecule techniques.   Figure 2: Two-color super-resolution imaging of ER and mitochondria...stimulated emission depletion (STED) nanoscopy. Scale bar, 2 μm. Inset shows comparison with standard confocal ...enables applications like multi-color imaging (Figure 2). If you’re working with purified proteins in vitro...Labeling in Living Cells. Chemistry & Biology, 15(2), 128–136. https://doi.org/10.1016/j.chembiol.2008.01.007...conjugates: A flexible chemical tag. Nature Methods, 2(4), Article 4. https://doi.org/10.1038/nmeth749 Strebinger...known as HaloTag is actually HaloTag7, and recent specialized variants run up to HaloTag11. HaloTag is now... (Gallagher et al., 2009). HaloTag: Scientists at Promega developed HaloTag in 2008 (Los et al., 2008)...

  9. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    Published
    April 5, 2018, 12:42 p.m.
    ...of conventional gene targeting is lowered to about 2-3 months. But certain models remain more difficult...included in the final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair template... floxed conditional allele requires you to target 2 loxP sites to intronic regions 0.5 - 0.8 kb apart....Typically, Easi-CRISPR works best with insertions of 2 kb or less. Luckily, most of the commonly used cassettes... induction systems (ttA or rtTA) are between 1 to 2 kb in length. There are plenty of Addgene plasmid ...create the ssDNA construct as described in Figure 2.  You can also use Addgene plasmids to purify Cas9...Cre-dependent conditional alleles, has remained a challenge. Enter Easi-CRISPR: a method that harnesses the...

  10. Editor's Choice, October 2016

    Type
    Blog Post
    Published
    Nov. 4, 2016, 2:30 p.m.
    ...Part 2: Building Community  My editor’s choice pick for this month is “Grad School Advice Part 2: Building...Options for CRISPR Genotyping Grad School Advice Part 2: Building Community Engineering the Plant Genome Using...Superhero 5k and, of course, we had our yearly Halloween party (see all of our wonderful costumes in the...

  11. Negative Can Be Positive: Open AAV Data with Addgene

    Type
    Blog Post
    Published
    Sept. 6, 2022, 1:15 p.m.
    ...indicated they wanted to see more negative data (Figure 2.) At the same time, when scientists who support open...is not only housed, but encouraged.    Fig. 2: Results from a survey conducted by Addgene in 2021...detailed methods and image descriptions, a minimum n = 2, and well labeled high- and low-magnification images...gene therapy trials. Human Gene Therapy Methods, 26(2), 71–76. https://doi.org/10.1089/hgtb.2015.040 Initiative... peripheral nervous system presents a special challenge to scientists. What works well in one cell or ...vectors in neuroscience Let’s consider my first challenge when I started working with AAV vectors. I wanted...Adeno-Associated Viral Vector-Mediated Gene Delivery to the Hypothalamus. Human Gene Therapy, 21(6), 673–682. https:/...

  12. Lentiviral Vector Uses and Overview

    Type
    Blog Post
    Published
    May 19, 2016, 2:30 p.m.
    ...create the 2nd-generation lentiviral system (Figure 2). The HIV genes that do remain are very important ...1996 Apr 12;272(5259):263-7. PubMed PMID: 8602510. 2. A third-generation lentivirus vector with a conditional...CH, Humeau LM, Rebello T. J Gene Med. 2013 Feb;15(2):78-82. PubMed PMID: 23322669. 6. Genetic treatment... Banasik MB, McCray PB Jr. Gene Ther. 2010 Feb;17(2):150-7. PubMed PMID: 19847206. Additional Resources...hematopoietic disorders sickle cell anemia and beta-thalassemia. Lentiviral vectors are promising agents for ...genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014 Aug;11(8):783-4. ...HMGA2 activation after gene therapy of human β-thalassaemia. Cavazzana-Calvo M, Payen E, Negre O, Wang G...

  13. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    Published
    June 28, 2018, 3:01 p.m.
    ...purify IVT sgRNA in the lab. Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively...into stem cells. Cells are allowed to recover for 2-5 days and then analyzed for tagging efficiency using...IVT sgRNA) approach (Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly efficient...cell suspension, transfect and let cells recover for 2-5 days.Note: We use the Nucleofection method for delivery...success was ~%50. If you get low knock-in efficiency (<2% positive by ICC for the tag) or do not see any cells...PMID: 28096221. PubMed Central PMCID: PMC5312033. 2. Dewari PS, Southgate B, Mccarten K, Monogarov G, ...Creating epitope-tagged clonal cell lines remains a challenge as it relies on inefficient homology-directed ...

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

    Type
    Blog Post
    Published
    Aug. 6, 2019, 12:55 p.m.
    ...detect your targeting vector or gene of interest. 2. Breed your parental mice to generate F1 Once you’...that can impact reproduction include embryonic lethality, a short lifespan, or infertility and can arise... number or expression level effects.  Another challenge that may arise when crossing mice with multiple...

  15. 5 Steps to Writing Grant Proposals for Grad Students

    Type
    Blog Post
    Published
    Feb. 11, 2014, 12:13 p.m.
    ...require application submission in-house approximately 2 weeks before the RFA due date in order for the administrative...deliverables to the aims requested in the call. Step 2: Develop a research plan Most grant applications ...proposal typically lists the deliverables, explains 1-2 potential pitfalls of the intended protocol, and offers...while writing each section. Research challenge The Research Challenge motivates the request for application...how you, the expert, will address the research challenge. Now is the time to make bold statements. While...solution is the best approach to the research challenge. Demonstrating expertise in the field and past...how your innovation will address a variety of challenges and impact the field - its encouraged to dream...

  16. Plasmids 101: How to Verify Your Plasmid Using a Restriction Digest Analysis

    Type
    Blog Post
    Published
    Aug. 28, 2014, 3:34 p.m.
    .... Diagnostic restriction digests are comprised of 2 separate steps: 1) incubating your DNA with restriction...which cleave the DNA molecules at specific sites and 2) running the reaction on an agarose gel to determine...including a 1.2 kb insert. The plasmid was digested with 2 unique enzymes (HindIII and BamHI) and run on an agarose...to serve as a reference band. The uncut DNA (lane 2) shows 3 possible plasmid conformations, with relaxed...yourself or obtained it from a colleague down the hall, it is always a good idea to take some time to confirm...

  17. Using AAV for Neuronal Tracing

    Type
    Blog Post
    Published
    Aug. 9, 2018, 1:04 p.m.
    ...for retrograde transport has been found for AAV1, -2, -5, -7, -8 (Taymans et al., 2007), AAV1 (Hollis et... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One 8:e76310...reveal novel hippocampal CA2 circuits. Nat Neurosci 17(2), 269–279. PubMed PMID: 24336151. PubMed Central PMCID... Mechanisms of Motility and Regulation. Neuron 84(2), 292–309. PubMed PMID: 25374356. PubMed Central PMCID...transduction efficiency of recombinant AAV serotypes 1, 2, 5 and 8 in the rat nigrostriatal system. J Neurochem...analysis of adeno-associated viral vector serotypes 1, 2, 5, 7 and 8 in mouse brain. Hum Gene Ther 18, 195–...retrograde access to projection neurons. Neuron 92(2), 372–382. PubMed PMID: 27720486. PubMed Central PMCID...

  18. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    Published
    Sept. 5, 2017, 1:58 p.m.
    ...improving editing accuracy by preventing re-editing and (2) optimizing “cut-to-mutation distance”. Furthermore...also takes into account that HDR is rare, usually 2-5% of the edited cells in our hands, these combined...screen a few hundred clones to find the right one Step 2 – Edit the cells a second time using a repair template...Additional CRISPR Resources on Addgene's CRISPR Guide Page 2. Optimizing "cut-to-mutation distance"  Even though... wisely (and let them cut close).   Figure 2: Relationship between cut site to mutation distance...PMID: 23287718. PubMed Central PMCID: PMC3795411. 2. Mali, Prashant, et al. "RNA-guided human genome engineering...distance is only 10 bp, the efficiency already drops by half and after only about 30 bp, it is no longer feasible...

  19. New Neuroscience Tool: The iGluSnFR3 Glutamate Sensor

    Type
    Blog Post
    Published
    April 7, 2022, 1:15 p.m.
    ...kinetics which are illustrated in figures 2 and 3.  Figure 2: Glutamate titration curves for the different...signals upon sensing glutamate using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation...adapted from Aggarwal et al…, Supplementary Figure 2.   Improved trafficking and localization through...iGluSnFR, the Looger lab wedged GFP between two halves of a bacterially derived periplasmic glutamate ...

  20. A Primer on Optogenetics: Introduction and Opsin Delivery

    Type
    Blog Post
    Published
    Sept. 10, 2015, 2:30 p.m.
    ...commonly used in optogenetics: 1) electrophysiology and 2) behavioral experiments. I’m not an electrophysiologist...choice in the brain region you wish to stimulate 2) Fabrication and implantation of the optical fiber...thoroughly elsewhere (here and here for instance) [2], [3]. Karl Deisseroth of Stanford University , one...reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn about the...monitor expression at various time points (e.g. every 1-2 weeks over 8 weeks) in order to determine the optimal...(4):251-66. doi: 10.1038/nrn3171. PMID: 22430017. 2. Fenno L, et al. The development and application of...analysis of microbial opsins. Nature methods. 2012;9(2):159-72. doi: 10.1038/nmeth.1808. PMID: 22179551; ...
Showing: 21 - 40 of 250 results