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We narrowed to 594 results for: cel.2

Showing: 101 - 120 of 594 results

  1. Viral Vectors 101: Pseudotyping

    Type
    Blog Post
    ...safely. Recently, many SARS-CoV-2 researchers began pseudotyping the SARS-CoV-2 spike protein into a lower ... et a., 2018).   Figure 2: To pseudotype with the SARS-CoV-2 spike protein, scientists encode...of vesicular stomatitis virus, HIV-2, and human foamy virus in cell lines expressing galactosyl(alpha1...spike protein’s role in infecting cell lines without using intact SARS-CoV-2 virus. Scientists have been using...Particles Produced in Human Cells Are Inactivated by Human Serum. Molecular Therapy 2:218–222 . https://doi....production uses three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the packaging plasmid... pseudotyping methods with other viruses in a BSL-2 lab instead of a BSL-3 or BSL-4 lab. For more details...

  2. University of Florida Serotype Testing Panel for the Eye and Brain

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    Collection
    ...AAV2 vectors in the mouse retina. Mol Ther . Feb;19(2):293-301. PMID: 21045809 Other citations include: ...adipose tissue-derived mesenchymal stem cells into pluripotent stem cells by a mutant adeno-associated viral...serotype also demonstrates enhanced transduction of cells in culture for ex-vivo conditioning. The AAV2(trpYF...displays enhanced transduction of hematopoietic stem cells and microglia. The AAV6(dbY-F+T-V) serotype was ...AAV-mediated transduction and targeting of retinal bipolar cells with improved mGluR6 promoters in rodents and primates...human gene therapy of glaucoma and their reversed cell entry characteristics. Gene Ther . 2020 Apr;27(3...delivery of a novel AAV vector targets ON bipolar cells and restores visual function in a mouse model of...

  3. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

    Type
    Blog Post
    ... State University. Since its appearance, SARS-CoV-2 has spread to almost every part of the world manifesting...Rapid detection of 2019 novel coronavirus SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-...primers that target the E and the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences... method has been optimized to detect the SARS-CoV-2 virus directly using saliva without having to perform...and visual detection of novel coronavirus SARS-CoV-2 Sensitivity: 4.6-11 copies/microliter of RNA targets...single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification was...easy-to-deploy, protocol for Cas13-based detection of SARS-CoV-2 genetic material (CREST) Sensitivity: 10 copies of...

  4. Viral Vectors 101: Virus Safety

    Type
    Blog Post
    ...cancer. These viruses need to be handled in a BSL-2 (or BSL-2+) lab and require additional safety measures.... BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL-2 materials, such...respiratory diseases in humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) ...recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus, many organizations...sprayed into the air. For lentivirus and other BSL-2 materials, you’ll need to use aerosol-tight caps, ...percentage of the decontaminate to be used for BSL-2 waste, and of course, if you are working with a substance...producer cell line, such as HEK-293T, production of viral particles, and transduction of a target cell line...

  5. 10 Basic tips for mammalian cell culture

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    Blog Post
    ...: 24901218. PubMed Central PMCID: PMC4047077. 2. Pagliara, Patrizia, et al. "Ostreopsis cf. ovata induces...times), depending on the cell line so old cells should be retired! Conversely, cells younger than the 3rd ...Make sure cells don't stay out too long While you’re preparing things, make sure your cells don’t stay...contaminants. Set up a routine to split cells Don’t be lazy about splitting cells, instead try to form a routine...’s best to split cells before the plates are more than 80-85% confluent because cells like their space...lactic acid (a by-product of cellular metabolism that is toxic to the cells), and need to be split very...numbers! When starting off with a new cell line, grow the cells, aliquot them into a number of vials, ...

  6. The Challenges of Cell Culture

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    Blog Post
    ... York: Crown Publishers, 2010. 2. Gold, Michael. "A conspiracy of cells." State University of New York...quality work in cell culture. The problems with cell lines Simply put, the majority of cell line problems...HeLa cells, the first immortalized human cell line. He proved this by examining whether the cell lines...Cancer Cells, Marker-independent identification of glioma-initiating cells. The International Cell Line ...working cell bank has been made by a research institution (an excellent practice), or maybe cells have been...Animal Cell Culture Guide Thermofisher Guidelines for Maintaining Cultured Cells Invitrogen Cell Culture... Outreach Scientist Nick Amiss. Cell culture in the present day Cell culture is a widespread tool used...

  7. TALEN Plasmids and Kits

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    Collection
    ...Golden Gate TALEN Kit and are used in place of pTAL1, 2, 3, or 4. For both plasmids sequence positions 1214...effectors to visualize subcellular positioning of target DNA sequences in living cells (TALE-mediated Genome...serial ligation. Validated in zebrafish somatic cells. Keith Joung Zhang Lab TALE Toolbox PCR/Golden Gate...ligation-independent cloning (LIC). Validated in human cells. Veit Hornung Musunuru/Cowan Lab TALEN Kit Kit comprised..., both in microinjected embryos and transfected cells. TALEN expression is driven by the strong CAG promoter...destination vectors and a reporter vector for mammalian cell-based validation assay that are designed for use... the evaluation of TALEN activities in cultured cells. Golden Gate TALEN 2.0 47388 pcDNA3.1-GoldenGate...

  8. 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 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...). Class 2 CRISPR/Cas: an expanding biotechnology toolbox for and beyond genome editing. Cell & Bioscience...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...

  9. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    ...the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed to...construct. For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required, but...Deliver your construct to your target cells. After recombination, exon 2 of the targeted gene will be removed... through an experiment designed to knock out exon 2 of a given gene.   Figure 1: A knockout targeting...the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted gene is disrupted (knocked out...recombination event will remove NeoR only and leave exon 2 floxed, as seen in line 4 of Figure 4. Since the loxP...recombined cells. Recombination is a rare event, so you must select as opposed to screen for cells where recombination...

  10. Addgene's Top Blog Posts from 2020

    Type
    Blog Post
    ...CRISPR to detect SARS-CoV-2, including many with plasmids deposited at Addgene! 2. DNA Purification Without...detection methods, synthetic nanobodies, SARS-CoV-2 protein expression, and more. We also highlighted ...empty backbones, fluorescent protein tags, SARS-CoV-2 plasmids, etc. It is our hope that by providing cloning...RNA and extracting RNA without a kit. 3. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas by ...multiple plasmids in one cell, it might be that you can't stably maintain them in the cell. This blog post features...

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

    Type
    Blog Post
    ...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the...but these cells are prone to mutations and will have more unintended changes to their genomes. 2) Designing...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required for recombineering...generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering system...pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically ~70 nucleotides...bacteria that has key MMR proteins knocked out or 2) specially design ssDNA oligos to avoid MMR: 1) E....

  12. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

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    Blog Post
    ...   Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with ... a range of reasonable MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially if you’re doing...(Figure 2) and I was surprised at how variable Cas9-expression was across the different cells (Figure ...Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but I didn’t know what...knockouts and knockins in human somatic cells. Nature Protocols 2007; 2(11): 2734-46. PubMed PMID: 18007609...despite having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity using this assay, you really...Western blotting against GFP expression, but I didn’t. 2. In the original publication they see GFP expression...

  13. Antibodies 101: Fab Fragments

    Type
    Blog Post
    ...fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced by proteolytic... multiplexed super-resolution microscopy. Cell Rep Methods 2(10):100301. https://doi.org/10.1016/j.crmeth...fragments are produced by mild reduction of F(ab')2 fragments, which reduces the hinge region disulfide...Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common...advantages, though with some caveats.  First off, F(ab’)2 fragments are bivalent, so may have higher avidity...or Fab fragments. Higher avidity means that F(ab’)2 fragments may have overall stronger interaction with...Clinical Prospects of Antibody Fragments. Antibodies 8(2):28. https://doi.org/10.3390/antib8020028    Wessel...

  14. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    ...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Lock,... and keep the cells happy. Harvesting virus Once you have transfected your cells, wait 2–5 days and then...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human ..., R., & Kleinschmidt, J. (1999). Titration of AAV-2 particles via a novel capsid ELISA: Packaging of genomes... genomes can limit production of recombinant AAV-2. Gene Therapy, 6(7), Article 7. https://doi.org/10.1038...Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material. Human Gene Therapy, 21...however, you’ve collected the producer cells, you’ll need to lyse the cells to let the virions out. We recommend...

  15. Antibodies 101: Normalization and Loading Controls for Western Blots

    Type
    Blog Post
    ...the resulting fraction is reported. Figure 2: Normalization with loading controls. Under each lane...control band is weaker in condition 1 than condition 2, the normalized values for the two conditions are ...opposite change in your loading control. Assumption #2: The loading control can be accurately quantified ...significantly lighter in “condition 1” than “condition 2.” By eye, the loading control appears about the same...) are smaller than the analogous green “condition 2” points by the exact same value. For the two solid...consider a different loading control. Assumption #2: The loading control can be accurately quantified ...factors we’ve covered in our previous blog posts (1, 2) on western blotting. If the abundance of your POI...

  16. Plasmid Preps: Different Purity, Different Quantities, Different Uses

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    Blog Post
    ...electrocompetent cells is minimal (picogram range). It can be extracted from small bacterial cultures (i.e. 2-3 ml...ratios measured at 260 and 280 nm should fall ~1.8 - 2 and give scientists an indication of purity versus...contaminants. The 260 and 230 nm ratios should fall ~2 - 2.2, and provide an indication of purity versus ...hydrochloride used during plasmid extraction. Figure 2: Use cloning grade DNA for direct cloning of your ...lipopolysaccharide of the cell wall of Gram negative bacteria like E. coli that affects tissue culture cell line viability...introduces transformation grade plasmids into a bacterial cell. Transformation grade DNA  Transformation is ...Figure 3: Need to introduce plasmids into mammalian cells? Use transfection grade DNA. Transfection is ...

  17. Hot Plasmids - October 2022

    Type
    Blog Post
    ...GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...challenges in 2-photon in-vivo imaging of voltage fluctuations. Image credit: Liu Z, et al. Cell. 2022.  ...suitable for in vivo optogenetic use.   Fig. 2: A) HcKCR1 is highly selective for K+ over other Na...slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies...two-photon microscopy. Cell, 18, 3408 - 3425.e29. (2022). https://doi.org/10.1016/j.cell.2022.07.013.   ...human c-myc proto-oncogene product. Molecular and Cellular Biology, 5(12), 3610–3616. doi: https://doi.org...

  18. Fluorescent Proteins: FRET

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    Collection
    ...referred to as intermolecular or bimolecular FRET) or (2) conformational changes within a protein where the...influenced by many factors, such as the organism or cell type, relative position and orientation of the fluorophores... (Unpublished) FRET Indicators - Live and Fixed Cells Additional Resources at Addgene Learn more on the...

  19. Viral Vectors 101: AAV Serotypes and Tissue Tropism

    Type
    Blog Post
    ...α2,3-/α2,6 N-linked sialic acids1 AAV receptor (AAVR)2   AAV2 Heparan sulfate proteoglycan (HSPG)3 ...3 AAV receptor (AAVR; not strictly required)2 Human fibroblast growth factor receptor 1 (FGFR1)3 αVβ5...AAV5 2,3-N-linked sialic acid4 AAV receptor (AAVR)2 Platelet-derived growth factor receptor (PDGFR...Heparan sulfate proteoglycan (HSPG)3 AAV receptor (AAVR)2 Epidermal growth factor receptor (EGFR)3 AAV8... AAV8 Laminin receptor (LR)3 AAV receptor (AAVR)2 Carboxypeptidase D (CPD), also called AAVR25   ... Terminal N-linked galactose3 AAV receptor (AAVR)2 Laminin receptor (LR)3 Putative integrin3 ... AAV2, so they are formally called rAAV2/1, rAAV2/2, and so on, although we often use abbreviated nomenclature...

  20. Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

    Type
    Blog Post
    ...Table 2 for more details on the effect of pH on the fluorescent properties of Rosella.   Table 2: Effect...in yeast. Autophagy,4(2), 205-213. doi:10.4161/auto.5331. PubMed PMID: 18094608 2. Sargsyan, A., Cai, J...environment like the vacuole or lysosome. See Table 2 for a summary of what color fluorescence Rosella emits...fluorescence accumulates in the vacuole. See Figure 2 for an example of what fluorescence looks like for... Mammalian Autophagy Research. Cell,140(3), 313-326. doi:10.1016/j.cell.2010.01.028. PubMed PMID: 20144757...analyzing autophagy of cytosol and organelles in yeast cells. Autophagy (Greek for “self-eating”) is induced ...radio-labeled long-lived proteins as a proxy for autophagy. Cells are cultured with isotope-labeled amino acids for...
Showing: 101 - 120 of 594 results