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We narrowed to 297 results for: hal.2

Showing: 81 - 100 of 297 results

  1. Adeno Associated Virus (AAV) for Cell and Gene Therapy

    Type
    Blog Post
    ...AAVs in the clinic Thus far, AAV vectors AAV1, AAV-2, AAV1-AAV2 hybrids, AAV-6, AAV-7, AAV-8, AAV-9 and...neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are thought to be as high as 70% among the... 10.24 (2003): 1999-2004. PubMed PMID: 14566358.  2. Hirsch, M. L., et al. "Self-complementary AAV mediates... cargo (DNA or RNA) to target cells has been challenging. A variety of vector delivery technologies have...treatment appears to be both safe and effective. Challenges for AAV gene therapy Although AAV-based gene ...promising, scientists are working to overcome key challenges to their efficacy:  Pre-existing immunity: Many...

  2. Educational Planarian Tools for Teaching Developmental Biology Techniques

    Type
    Blog Post
    ...neoblast cells and the gene Prohormone convertase 2 (pc2) labels the central nervous system. Evaluating... characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching a specific mRNA sequence...223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver...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...

  3. Viral Vectors 101: An Introduction to AAV

    Type
    Blog Post
    ..., AAV2/8 has a type 2 ITR and a type 8 serotype. Almost all vectors contain type 2 ITRs, which comes from...differentiated, non-dividing cells.   Figure 2: DNA rescue and transduction of a conventional single-stranded...quantitative PCR-based method for the titration of AAV-2 vector stocks."Molecular Therapy 6.2 (2002): 272. ...strand synthesis. In contrast, a scAAV vector with half the size of the ssAAV genome has a mutation in the...

  4. An Introduction to Adenovirus

    Type
    Blog Post
    ...either SARS-CoV-2, Adenovirus type 41 (AdV-41), or Adenovirus-Associated Virus type 2 (AAV2) – or some... was either 1) a physiological effect of SARS-CoV-2 toward destabilizing one’s natural immune response...response to Adenovirus (and/or to its partner, AAV) or 2) a sociological effect of the COVID-19 lockdowns causing...… Chiu, C. Y. (2023). Adeno-associated virus type 2 in US children with acute severe hepatitis. Nature... like other viral vectors, though significant challenges remain (Wold & Toth, 2013).  While the pre-existing...

  5. 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...2015; Koike-Yusa et al., 2014; Ma et al., 2015; Shalem et al., 2014; Wang et al., 2014), knock down (Gilbert... 26190106. PubMed Central PMCID: PMC4559080. 8. Shalem, Ophir, et al. "Genome-scale CRISPR-Cas9 knockout...

  6. Fluorescent Tagging of Endogenous Genes with SapTrap

    Type
    Blog Post
    ...digestion of the destination vector with SapI yields 2 sites- the first site accepts the sgRNA target sequence...marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type will produce... a gRNA vector that targets the gene of interest, 2) a plasmid to specify the reading frame of the insertion...the tag for each insertion. Also, it remains challenging to screen for the genetically modified strains...fluorescent (EGFP, tagRFP, mCherry) and nonfluorescent (Halo, SNAP) tags, a selectable marker (floxed Cbr-unc...small epitope tags (HA, Myc, Strep tag II, AviTag, HaloTag, SpyTag). The Foerstemann lab developed a polymerase...

  7. Hot Plasmids and Viral Preps - January 2021

    Type
    Blog Post
    ... Opto-Cre-Vvd is fast and can cut in as little as 2 hours.  Sheets, MB, et al., ACS Synth Biol. 2020....they investigated three Casɸ orthologs, Casɸ-1, Casɸ-2, and Casɸ-3. This system is notable for several reasons...a minimal PAM requirement. For instance, for Casɸ-2 the PAM sequence is 5’-TBN-3’ (where B is G, T, or...deposited by Leopold Parts. This library delivers 2 randomly paired guides per construct and allows a ... is only ~70kDa, with a molecular weight roughly half of Cas9 or Cas12a. This is in part due to the fact...

  8. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    ...termed fluorescence activated cell sorting or FACS (2). By analyzing single cells instead of the population...expression. A routine flow cytometry experiment uses 2 or 3 different fluorescent colors each measuring a...distinct populations on a FSC versus SSC plot (Figure 2).  A user can then “gate” around the specific population...cell and T cell into distinct populations (Figure 2). An immunologist who studies B cells, might then ... B cells, plasma cells or others.   Figure 2: Due to differences in their size and complexity, ...then flow cytometry can help you overcome these challenges and make these types of experiments easier and...fluorophores Fluorescent antibodies get around the challenges of fluorescent protein fusions by binding to ...

  9. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

    Type
    Blog Post
    ...Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow 3.2. ...M-CFP expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate... each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for example, Brainbow...processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...First, the imaging of Brainbow mouse tissue was challenging due to low fluorescence intensity, caused partially...expression patterns. Hampel S, Chung P, McKellar CE, Hall D, Looger LL, Simpson JH. Nature Methods. 2011 Mar...

  10. 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...Adeno-Associated Viral Vector-Mediated Gene Delivery to the Hypothalamus. Human Gene Therapy, 21(6), 673–682. https:/...

  11. Synthetic promoter AAVs for cell-type specific expression in retinal cells

    Type
    Blog Post
    ...retinal cell-type specific genes from mice. Strategy #2 (ProB): Create promoters using phylogenetically conserved...would express Cre-mCherry and GFP.   Figure 2: Synthetic promoters can be paired for targeted expression...1345-1356. https://doi.org/10.1038/s41593-019-0431-2  Neff, E.P. An AAV library for retinal cell expression... at the Institute of Molecular and Clinical Ophthalmology Basel recently took a different approach to ...to test for cell type-specific expression of GFP. Half (113) expressed in retinal cells four weeks post-injection...

  12. Optogenetics Guide

    Type
    Guide
    ...Cryptochrome 2 and CIB1 Blue-light–mediated induction of protein interactions based on Arabidopsis thaliana cryptochrome...OPTOSTIM PHR domain2 of cryptochrome 2 (Cry2) from Arabidopsis thaliana is fused to truncated forms of cytosolic...influx into the cell when stimulated. Channelrhodopsin-2 (ChR2), the first widely adopted optogenetic tool,...cryptochrome 2 and CIB1 450 LARIAT Inhibits protein function by reversibly sequestering targets into large...customizable TALE DNA-binding domain with cryptochrome 2 protein and its interacting partner CIB1 to control... with single-spike temporal resolution. PLoS One. 2(3):e299. PMID 17375185 Kim B, Lin MZ. 2013. Optobiology...comparative analysis of microbial opsins. Nat Methods. 9(2):159-72. PMID 22179551 Saunders A, Johnson CA, Sabatini...

  13. Twenty Years of Addgene Sharing: CRISPR

    Type
    Blog Post
    ... were depositing and requesting each year (Figure 2). We did a similar analysis with CRISPR pooled libraries...different categories (Figure 2C). Note that in Figure 2, the numbers on the X axis refer to distribution and...rather than a cumulative total.    Figure 2: Addgene CRISPR deposit and distribution by function...rapid adoption of new tools, illustrated in Figure 2, and the longevity of tools seen in Figure 3, indicates... the easy part; using them can be a bit more challenging. Addgene’s free educational resources help scientists...

  14. No Llamas Required - Synthetic Nanobodies Against Membrane Proteins

    Type
    Blog Post
    ...select for optimal sybody candidates. Figure 2: Outline of the process for selection of sybodies ...identify and isolate sybodies targeting the SARS-CoV-2 Receptor Binding Domain found on the membrane-bound...Generation Toolbox kit. Find the Seeger lab's SARS-CoV-2 sybodies! References Geertsma ER (2013) FX Cloning...2020) Synthetic nanobodies targeting the SARS-CoV-2 receptor-binding domain. BioRxiv.  https://doi.org...access to routinely, and it can be especially challenging when trying to develop antibodies against unstable...

  15. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    ...the absence of genome editing (Fig. 2 and 3) (5, 3). Figure 2: Gene activation with nuclease-competent...shown to exhibit more limited activity than SpCas9 (2). In many cases this is due both to lower relative...PMID: 24076762. PubMed Central PMCID: PMC3844869. 2. Chari, Raj, et al. "Unraveling CRISPR-Cas9 genome...different perturbations. Consider, for instance, the challenge of attempting to simultaneously and selectively...

  16. Tetbow: Bright Multicolor Labeling for Neuronal Tracing

    Type
    Blog Post
    ...Figure 2). We’ve also created constructs with containing chemical tags such as the SNAP-tag, Halo-tag, ...possible when each of the XFP genes is introduced at ~2 copies per cell per color, following a Poisson distribution...AAV titer - The XFP genes have to be introduced at ~2 copies per cell per color to achieve the highest color...

  17. What's Your Organism? Expanding Genomic Tools via the NSF EDGE Program

    Type
    Blog Post
    ...presented in a series of quick (10 minute) or very quick (2 minute) talks so we got to hear from everyone. Addgene...evolutionary tree at least 6-7 times and on at least 2 separate continents. Mimulus (Andrea Sweigart) Monkeyflowers...Organisms, Part 1 Five Popular Model Organisms, Part 2 ...share all of these new organisms continues to be challenging. Organism specific viral vector systems would...thus making progress on the NSF “G to P grand challenge.” Creating a productive, new scientific community...proteins, and DNA. Euprymna scolopes (Joshua Rosenthal) The Hawaiian bobtail squid has an extreme number... a different protein. Did you know that most cephalopods die after they lay their 1 clutch of eggs? Propagation...

  18. Cloning Mammalian Cells with the Agarose Method

    Type
    Blog Post
    ...3 plates at different densities for selection). A 2% agarose solution is then melted (and thereby sterilized...96-well assay plate. We generally freeze at least 2 ampules each of 4 clones with different levels of ...of agarose, which is then overlaid with the firmer 2% agarose gel solution discussed above for picking....incubator, additional growth medium is added to about halfway up each well. Tips for using the agarose cloning...Time CRISPR User (by a 1st Time CRISPR User) The Challenges of Cell Culture Plasmids 101: Mammalian Vectors...

  19. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    ...procedure to be performed using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As...Primer design Using specific primer design (Figure 2), IVA cloning can be used to perform any plasmid modification... for each type of modification is shown in Figure 2, and is discussed in more detail below. The main requirement...Biotechniques 10.1 (1991): 62-66. PubMed PMID: 2003926. 2. Bubeck, Peter, Monica Winkler, and Wilfried Bautsch... to amplify away from the insertion site. Encode half the new sequence at the 5’ end of each primer, with...

  20. AAVs in Retinal Gene Therapy

    Type
    Blog Post
    ...therapy had fallen - plummeted really -  from grace 2 years prior with the death of Jesse Gelsinger in an... the whole field to a grinding stop and only now, 2 decades later, is it finally back in the limelight...the other hand, have broad tropism in the eye. AAV-2, the first identified serotype and the one used in...reading 1. FDA approves gene therapy for blindness 2. Gene Therapy emerges from disgrace to be the next... completing her PhD and medical training in Ophthalmology. Gene therapy was in its infancy, but the eye...
Showing: 81 - 100 of 297 results