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We narrowed to 588 results for: cel.2

Showing: 181 - 200 of 588 results

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

    Type
    Blog Post
    ...detect your targeting vector or gene of interest. 2. Breed your parental mice to generate F1 Once you’...strain, the promoter being used should be tested in cell lines to determine how it impacts the expression...interest. Cre may be expressed globally (in every cell of the mouse) or in a tissue-specific manner (with...

  2. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    ...then packaged into AAV’s protein shell, or capsid; 2) the pHelper plasmid which expresses adenovirus genes...takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR run and data analysis. Refer to...quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Human ...therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2. Choi, V.W., Asokan, A., Haberman, R.A., & Samulski...packaging AAV is transfecting HEK293 cells with AAV packaging plasmids. The cells need three different plasmids...precipitation. Instead of sonicating the cell pellet, I lyse the cells with this AAV lysis buffer and freeze...bath to release the virus particles from the cells. Cell lysates are then treated with benzonase immediately...

  3. Viral Production at Addgene

    Type
    Blog Post
    ...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Wang,...maps section for images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre...Digital PCR Method. Human Gene Therapy Methods, 29(2), 96–103. https://doi.org/10.1089/hgtb.2017.198   ... preparations are added to cells in culture. Three to five days later, cell cultures are inspected for...a new vial of early passage cells. Approximately two weeks post-thaw, cell culture supernatant is tested... (55634-AAVrg) alone at 1.7E6 viral genomes (vg)/cell, pAAV-CAG-FLEX-rc [Jaws-KGC-GFP-ER2] (Addgene 84445...preparations ("-LV” catalog items) are produced in 293T cells using the 2nd generation psPAX2 and pMD2.G packaging...

  4. Hot Plasmids: Spring 2025

    Type
    Blog Post
    ...imaging and loose-seal, cell-attached recordings in primary visual cortex (V1) layer 2/3 (L2/3). B) Fluorescence...protocols for viral generation and stable cell line creation (Figure 2). Looking for more? Explore our empty...a protein core, 2–4 glycosaminoglycans (GAG), and are (typically) tethered to the cell surface by a GPI-anchor... Lentiviral backbone plasmids here! Figure 2: Addgene cloned EGFP into pAG Lenti CMV N-HA Puro ...either the optogenetic activator channelrhodopsin-2 or fluorescent reporter dTomato under the control ...PV-positive cells out of all BiPVe3-dTom cells. C) Confocal stack of a biocytin-filled cell expressing ...particles in HEK293T cells with pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260). HeLa cells were infected...

  5. 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...expression in non-dividing cells because they remain in episomal form in the cell’s nucleus. But since nothing...hope are many more clinical successes for gene (and cell) therapies. One may wonder why the first AAV-based... providing long-term gene expression in retinal cells - AAV - were becoming available. At the same time...

  6. Adenovirus Guide

    Type
    Guide
    ...use of pAdEasy-2 (Addgene #16401) can increase the capacity of the rAdV vector. pAdEasy-2 does not contain...gene and cell-based therapies in the new era of personalized medicine . Genes & Diseases, 4 (2), 43–63.... incorporate additional genomic deletions. Figure 2: First-generation rAdV vectors. Created with BioRender.com...and have to be handled at biosafety level two (BSL-2). They are highly immunogenic and can trigger a strong...adenoviruses using the AdEasy system . Nature Protocols, 2 (5), 1236–1247. https://doi.org/10.1038/nprot.2007.135...Adenoviral vector vaccine platforms in the SARS-CoV-2 pandemic . NPJ Vaccines, 6 (1), 97. https://doi.org...backbone plasmid that lacks E1 and E3. pAdEasy-2 Adenoviral backbone plasmid that lacks E1, E3, and E4. Useful...

  7. DNA Purification Without a Kit

    Type
    Blog Post
    ...level of sensitivity. Figure 2: Steps to purify DNA using a cellulose dipstick. Image: Zou et al., 2018...alkaline lysis plasmid miniprep Starting material: 2 mL bacterial culture Product: plasmid DNA This kit-free...protocol: 1) it solubilizes both DNA and agarose, and 2) it helps DNA stick to the glass. After binding DNA...Academy of Sciences of the United States of America, 76 2, 615-9 . https://doi.org/10.1073/pnas.76.2.615  Zhou...broken out of cells with a solution of salts, detergent, and ball bearings. Then a cellulose dipstick is... kits and typically follow the same principles: cells are lysed, RNAs and proteins are removed to leave... could be digested with restriction enzymes.  Cellulose nucleic acid purification Starting material: plant...

  8. Optogenetics Guide

    Type
    Guide
    ...nonspecific cation influx into the cell when stimulated. Channelrhodopsin-2 (ChR2), the first widely adopted... (Phy) interaction 660 Cryptochromes Cryptochrome 2 and CIB1 Blue-light–mediated induction of protein ...interactions based on Arabidopsis thaliana cryptochrome 2 and CIB1 450 LARIAT Inhibits protein function by reversibly...customizable TALE DNA-binding domain with cryptochrome 2 protein and its interacting partner CIB1 to control...transcription effectors OPTOSTIM PHR domain2 of cryptochrome 2 (Cry2) from Arabidopsis thaliana is fused to truncated... 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...

  9. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    ... to DFHBI in the presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more...PMID: 25713381. PubMed Central PMCID: PMC4364232. 2. Baker, Monya. “RNA imaging in situ”. Nature Methods...emerging environmental pollutants.” Front Chem. 2014; 2: 41. PubMed PMID: 25019067. PubMed Central PMCID: ...biosensors.” TrAC Trends in Analytical Chemistry 27(2):108-117 · February 2008. 11. Svensen, Nina and Jaffrey..., peptides, amino acids, drugs, metal ions, and cells (even pathogenic bacteria). That range is possible... over half a century; the Addgene blog has an excellent primer and tips from experts on FRET techniques... combination of SELEX and fluorescence-activated cell sorting (FACS) of E. coli transformed with aptamer...

  10. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

    Type
    Blog Post
    ...mechanism of action are presented in Figure 2. Figure 2: Single-stranded donor DNA interaction with...Nature biotechnology (2016). PubMed PMID: 26789497.  2. Lin, Steven, et al. "Enhanced homology-directed human... between Cas9 (which causes the breaks) and host cell DNA repair factors (which repair those breaks) makes...example, correcting the allele that causes sickle cell anemia. However, HDR occurs much less frequently...a structure could drive sequence replacement in cells. Targeting three dCas9 molecules to a precisely ...functional Cas9 protein and the targeting guide RNA into cells by electroporation. Results from our lab and others...Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins...

  11. The Developmental Studies Hybridoma Bank: Over 25 Years of Antibody Sharing

    Type
    Blog Post
    ...human transcription factors such as PAX7, PAX6, AP-2 alpha and ISLET-1. In addition, we distribute monoclonal... example through the use of less toxic adjuvants; 2) generating complex antibody chips that allow assessment...produced by a single B cell. To create a monoclonal antibody cell line, myeloma cells are fused with activated...activated B cells from the spleen of mice injected with the antigen of interest. The immortalized cells, called...distributing over 3,000 antibodies and associated hybridoma cell lines. The DSHB was created by the National Institute... individually isolated and expanded. The clonal cells are screened to identify hybridoma lines, each producing...

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

    Type
    Blog Post
    ...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...What works well in one cell or neuron type may not work well for a neighboring cell type, even within the...options while AAV8 (82%) and AAV9 (84%) were both excellent at infecting our target neurons (Figure 1). However... conditions (3,5,8), injection titers (1,2), and cell types used for packaging viral particles (6), can...

  13. IBEX Knowledge-Base: A Data Resource for Multiplex Tissue Imaging

    Type
    Blog Post
    ...imaging of diverse tissues. Nature Protocols, 17(2), Article 2. https://doi.org/10.1038/s41596-021-00644-9 ...directly from the Zenodo dataset.     Figure 2: Screenshot showing reagent entries with associated...approach for deep phenotyping and spatial analysis of cells in complex tissues. Proceedings of the National ...

  14. Who Gives a Tweet? 9 Facts About Scientists on Twitter

    Type
    Blog Post
    ...Occupational Classification System  and Wikipedia. 2. Social and computer scientists are overrepresented...international Twitter users were included in the dataset; 2) users not part of a scientific Twitter list were ...scientists on Twitter are male and 40% are female Figure 2. Gender Ratio of Scientists on Twitter. 60% of scientist...tweeted by biologists were: Nature, Sciencemag, Wiley, Cell and Scientific American Figure 4: Top Scientific...

  15. Plasmids 101: Gateway Cloning

    Type
    Blog Post
    ...is excised from the donor vector.   Figure 2: The Gateway system adopts phage integration into ...our website to search for your favorite gene! STEP 2: Generate an Expression Clone When making the expression...expression construct in only 1 day, as opposed to 2+ days with traditional restriction and ligation cloning...Clon Transgen 4:138. doi:10.4172/2168-9849.1000138 2. Hartley JL. Use of the Gateway System for Protein...the next step is to transform competent E. coli cells and select the positive clones. The entry clone ... expression of the human KRAS gene in mammalian cells. STEP 1: Generate an Entry Clone There are a few...ccdB gene. Since the ccdB product is toxic to the cell, Gateway cloning efficiency can reach >99%. ...

  16. The Importance of High Titer for AAV Transductions

    Type
    Blog Post
    ...drastically with lower titers. As you can see in figure 2, the expression from the 5x1011 titer was essentially...roughly 6-fold increase in expression [3, Figure 2]. Note how expression increases exponentially with an increase...international 61.1 (2002): S9-S15. PubMed PMID: 11841606. 2. Schultz BR, Chamberlain JS. Recombinant Adeno-associated...Making contact with a cell Although an individual AAV particle’s ability to infect a cell is dependent on its...construction and the cell it’s infecting, getting these particles to infect cells is also very titer-dependent... particle to infect a cell, it has to come into physical contact with that cell. Since there is a limited...of surface area on each cell, the way to get more AAV in contact with the cell is to increase the AAV ...

  17. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    ...ultracompetent (109 CFU/μg) cells. Primer design Using specific primer design (Figure 2), IVA cloning can be...procedure to be performed using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As... 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...transformation efficiency of the strain: when asking the cell to perform multiple recombination events, all linear...linear fragments need to get into the same cell. For simple cloning requiring just one or two recombination...recombination events, homemade competent cells of 107 CFU/μg are perfectly fine, yet performing multiple modifications...

  18. Predicting Adverse Reactions to Monoclonal Antibody Drugs

    Type
    Blog Post
    ...various components of the immune system. Figure 2: In-vitro models for immunogenicity prediction of ...drug reaction. This system would somewhat act like a 2-step verification. All approaches have their own limitations...Future Enhancements. Frontiers in drug discovery, 2, 952326. https://doi.org/10.3389/fddsv.2022.952326Joubert...epitopes likely to be presented by T cells to elicit an immune response. T cell epitopes are found in either ...that take place within an antigen-presenting cell like a T cell. It is also not yet possible for these algorithms...traditional 2D assays with immune cell lines or peripheral blood mononuclear cells (PBMC) in 3D models of the...CD4+ or CD8+ T-cells or monocytes. PBMCs can be further differentiated into dentritic cells or macrophages...

  19. 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...As we celebrate twenty years at Addgene, we are sharing stories of our repository’s positive impact on... Sharing this story is a truly special way to celebrate our twentieth anniversary in 2024, and it’s all...

  20. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    ...translocation. Nature Cell Biology, 26(2), 294–304. https://doi.org/10.1038/s41556-023-01342-2 Koblan, L. W.,...order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent...region of separate strands called an R-loop (Figure 2). This allows the guide RNA to base pair with the ...accessible on the outside of the enzyme. Figure 2: CRISPR R-loop. Created with BioRender.com. Base...), 35–40. https://doi.org/10.1038/s41587-020-0592-2 Additional Resources on the Addgene blog CRISPR 101...By taking advantage of repair pathways within the cell — in some cases, the same repair pathways that were...adenosine (A) to inosine (I, treated as G by the cell). Surprisingly, the team noticed that this editor...
Showing: 181 - 200 of 588 results