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We narrowed to 592 results for: cel.2;

Showing: 561 - 580 of 592 results

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

    Type
    Blog Post
    ...PMCID: PMC3010411. 2. Kohli, Manu, et al. "Facile methods for generating human somatic cell gene knockouts...K562 leukemia cell line and ~8 and 9% efficiencies in T-cells and hematopoietic stem cells respectively...transient transfection of 293T cells with helpers, and used to infect recipient cells. After G418 selection, ...using AAV vectors for gene editing in mammalian cells since 1998. In that year, Russell and Hirata from...used to routinely deliver sequences of interest to cells with efficiencies of 1-40% after G418 selection ...the p53 and PTEN tumor suppressor genes in human cells (Kim et al 2008). They have also used AAV gene editing...endogenous allele of the STAG2 tumor suppressor in human cells (Kim et al, 2016). The Waldman lab has designed ...

  2. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ...or other cell spreading device that fits the plate. Incubate plates at 37 ℃ for 18 hours. Day 2 Observe...individual colonies and effective selection. 150 µL of 2 mg/mL Carbenicillin plated over-agar Plate shows less...individual colonies with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection. Selection Curve...investigator to conveniently plate and select transformed cells containing plasmids differing in their resistance...bent to create an “L” shape, and then used like a cell spreader. Several other devices may be used for.... During the incubation, transform DH5α E. coli cells by heatshock with the plasmid of interest. See our...the liquid is mostly absorbed. The spreading of cells can be done in the same way as the antibiotic, using...

  3. Plasmids 101: E. coli Strains for Protein Expression

    Type
    Blog Post
    ...gene1 [lon] ompT gal sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10--TetS) General protein expression... want to make sure your cells have first reached an appropriate density. Cells in the exponential growth...collecting dead cells, and, conversely, you cannot induce too early as there are not enough cells in the culture...host protein, using up valuable resources in the cell thus leading to slowed growth. Additionally, some...such as TB or 2XYT is optimal because of the high cell-density they support; however, minimal media supplemented...

  5. 5 Reasons to Use Reddit for Science Communication

    Type
    Blog Post
    ...community is active, engaged, and has great questions! 2. The r/Science subreddit is well moderated. One of...questions from the AMA - as you’ll see below, it pretty nicely displays her energetic, truthful and helpful style... training is abundant, helpful, and sometimes excellent. Finally, as I said in the AMA, it’s incredibly...topics to the Addgene blog The blog has many other excellent posts on science career topics as well. Instead...

  6. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    ...start selecting and testing systemic capsids! Figure 2 contains a decision tree for selecting a capsid, while... highlighted below the table.     Figure 2: This decision tree can help guide your choice on ...transfer to the adult brain. Nature Biotechnology, 34(2), 204–209. https://doi.org/10.1038/nbt.3440 Goertsen... your cell type of interest, while miRNA target sites can be used to prevent expression in cell types ...endothelial cells in rodent CNS AAV-PHP.V1 shows higher transduction in CNS endothelial cells (60%) and ...endothelial cells in the retina and spinal cord (Krolak et al., 2022). Systemic capsids for broad cell type ...mouse to primate neocortex. Cell Reports, 34(13). https://doi.org/10.1016/j.celrep.2021.108754 Ravindra Kumar...

  7. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    ... genome from every infected cell and 2. prevent reinfection of those cells. Kaminski et al. designed gRNAs...infected cells, the other using wild-type Cas9 to remove the HIV-1 genome from infected cells. Read on...1 reservoirs HIV-1 infects cells in the immune system, notably CD4+ T-cells, and eventually leads to acquired...percentage of cells with activated HIV-1, measured via HIV-1 promoter-driven GFP expression. In cell lines that...Kaminski et al. observed no negative effects on cell viability, cell cycle progression, or apoptosis with Cas9...in HIV-1 infected T-cells to see if they could be rescued by CRISPR. CD4+ T-cells were isolated from healthy...1 is limited to a subset of T-cells, but it is unclear if enough T-cells could be reached to ablate a ...

  8. Five Popular Model Organisms

    Type
    Blog Post
    ...variety of environmental conditions, and double every 2 hours. Yeast are also the first eukaryotic genome ... properties and processes with our own cells. Like human cells, yeast DNA is packaged into chromosomes...differentiation of β-cells in the the developing zebrafish pancreas. They first tagged these cells using plasmids...recapitulate the complex interactions between cancer cells, therapeutic drugs, and the rest of the body than...than studies in other model organisms or cell culture. For example, Scott Lowe’s lab utilized mice to study...to identify a set of mutations that made cancer cells more resistant to chemotherapy. You can find these...to optimally control gene expression in specific cells of the fly (Pfeiffer et al., 2010). Yeast (Saccharomyces...

  9. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    ... more from the authors in this blog post. Figure 2: iGluSnFR3 displays enhanced brightness, expression...two-photon microscopy. Cell. 2022 Aug 16. pii: S0092-8674(22)00916-3. doi: 10.1016/j.cell.2022.07.013 pYtags...Calcium Indicator. Front Cell Dev Biol. 2022 Jun 29;10:880107. doi: 10.3389/fcell.2022.880107. KRaION potassium...fluorescence unit fused with TRansmembrane region-to-IntrAcellular loop linkers” (MTRIA for short), where a fluorescent...inserting a circularly-permuted GFP into an intracellular loop of various G-protein-coupled receptors....fluorescent sensor for real-time measurement of extracellular oxytocin dynamics in the brain. Nat Methods....sensor relies on a change in localization to the cell membrane and not a direct change in the fluorescent...

  10. Quick Guide to Near-Infrared Fluorescent Proteins

    Type
    Blog Post
    ...Oligomeric state Photo-stabilityc, t1/2, s pKa Brightness in HeLa cells vs. iRFP713, % d Ref BphP-based...mammalian cells. dDetermined as effective NIR fluorescence in transiently transfected live HeLa cells with ...Near-Infrared Fluorescent Proteins in Mammalian Cells. Cell Chem Biol 24, 758-766 e753 (2017). Baloban, M...developers and users, BV is present in eukaryotic cells as an intermediate of heme metabolism (Shemetov ...to GFP-like probes by supplying a single gene to cells. Still in bacteria, which do not produce BV, an ... to consider effective brightness of NIR FPs in cells, in addition to their molecular brightness (a product...the concentration of BV-containing molecules in a cell and depends on the efficiency of BV incorporation...

  11. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

    Type
    Blog Post
    ..., whereas the mVenus-APT1 fusion does not (figure 2). In the mVenus-APT1, a lipidation motif, which is...proteins enable the observation of proteins in living cells and organisms. Both components of the chimera are...However, these may not reflect the situation in cells. Therefore, the localization of critical fusions...currently the standard for assessing oligomerization in cells. In addition to measuring stickiness, several researchers...fluorescent proteins that behave as monomers in mammalian cells include: mTurquoise2, mEGFP, mNeonGreen, mScarlet...protein to the existing pool of protein within a cell and may lead to over-expression artifacts. This ...heterotrimeric G protein Gq activation in living cells." BMC biology 9.1 (2011): 32. PubMed PMID: 21619590...

  12. Plasmids 101: Biotinylation

    Type
    Blog Post
    ...Nutr, 129 (2009a): 477–484. PubMed PMID: 10064313. 2. Chapman-Smith, A., & Cronan, J. E. “The enzymatic...carboxyl groups to organic acids to produce important cellular metabolites. Biotin acts as a carboxyl carrier...chemically. Enzymatic biotinylation occurs naturally in cells and is the process by which biotin is covalently... Immunosorbent Assay) Southern/Northern blotting Cell surface labelling Attaching polypeptides to a substrate...substrate Flow cytometry/fluorescence-activated cell sorting (FACS) In situ hybridization Methods for...proteins in bacteria, yeast, insect and mammalian cells. Check out Kay et al., 2009 for methods on biotinylating...Application of avidin-biotin technology to improve cell adhesion on nanofibrous matrices.” Journal of Nanobiotechnology...

  13. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ... glacial acetic acid 57 mL of dH 2 O Store Solution III at 4°C Grow 2 mL overnight cultures from single...Resuspension buffer Denaturing solution Renaturing solution 2 mg/mL RNase A TE or water-saturated phenol-chloroform...100 μL of cold Solution I. Vortex the solution for 2 min or until all bacteria are fully resuspended. Add...pipetting or carefully pouring. Optional: Add 5 μL of 2 mg/mL RNase A to the supernatant in the new tube and... to the recovered aqueous DNA layer. Repeat steps 2-4. Note: Phenol-chloroform is a hazardous waste - ...: Ethanol Precipitation To your DNA solution, add 2-2.5 volumes 95% or 100% ethanol and 1/10 volume of...min at 12,000 g. Notes: Pellet contains proteins, cell fragments, salt and other extra particles from solutions...

  14. CRISPR 101: Any Base Transversion Editors

    Type
    Blog Post
    ...of concept for further development. Figure 2: Mechanisms of deaminase-based adenine transversion...base editing. Molecular Cell, 84(7), 1257-1270.e6. https://doi.org/10.1016/j.molcel.2024.01.021 Tong, H.,... edit A → Y (Y = T or C) in 67.17% of screening cells (Tong, Wang, et al., 2023). Based on this promising...the base editor. Black arrows indicate natural cellular processes. Created with BioRender.com. The...Cas9 nicks the non-edited strand to encourage the cell to repair the DNA using the edited strand as a template... efficient A•T-to-C•G base editing in mammalian cells and embryos. Nature Biotechnology, 42(4), 638–650...efficient T-to-G and C-to-G editing in mammalian cells. Nature Biotechnology, 42(10), 1538–1547. https:...

  15. Harnessing Bacterial Toxins for Allelic Exchange

    Type
    Blog Post
    ...two visible blue or magenta chromoproteins (Figure 2) (Liljeruhm et al., 2018). This system is a nice confirmation... that converts sucrose into a toxic metabolite. Cells that have undergone a double crossover will not ...contain sacB, allowing them to grow on sucrose. Cells that have only gone through the single cross over...Giacalone et al., 2006). The Chen lab demonstrated excellent counter-selection which enabled very efficient...desired deletion directly into cut vector with excellent efficiency using a commercial isothermal assembly... from Harvard. Jake received his MD and a PhD in Cell and Molecular Biology from the University of Pennsylvania...

  16. A Control for All Seasons

    Type
    Blog Post
    ...as the protocol remains the same.    Figure 2: Microscopy-based antibody experiments should include...to detect dead or dying cells so that you can base your analysis on living cells only (for example, propidium...This tends to be most problematic when staining cells or tissues that are the same species as your primary...propidium iodide only stains dead cells). Compensation controls address the spectral overlap that occurs...Quantitative Fluorescence Microscopy.” Journal of Cell Biology, vol. 185, no. 7, Rockefeller University...

  17. Thoughts on the Future of Research 2015 Boston Symposium

    Type
    Blog Post
    ...Postdoctoral Associations around Boston - From Panel Session 2: Defining the Postdoc: An Institutional Perspective... can treat the underrepresented individuals too nicely. When the work of the underrepresented individual...

  18. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    ... Methods 2009; 6, 343-345. PubMed PMID: 19363495. 2. Wang JW, Wang A, Li K, Wang B, Jin S, Reiser M, Lockey...is immediately ready to transform into competent cells. The master mix of enzymes can be purchased from...mix for 1 hour, and then transform into competent cells. The success rate of this reaction is typically ...

  19. Hot Plasmids: Summer 2024

    Type
    Blog Post
    ... from damage (Abe & Lim, 2024).    Figure 2: A) When proteins (blue) interact with the air-water...epigenome editing. Cell, 184(9), 2503–2519.e17. doi: https://doi.org/10.1016/j.cell.2021.03.025.    Recombinant...tissue is identifying the cells you are studying. Antibodies can help reveal cellular identities by targeting...particles (eVLPs) offers an option to transduce cells directly with ribonucleoprotein cargo. eVLPs contain... will only be transiently present in transduced cells, limiting off-target editing. Cargo size limits ...01133-w Pandey, S., Gao, X. D., Krasnow, N. A., McElroy, A., Tao, Y. A., Duby, J. E., Steinbeck, B. J.,...site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime ...

  20. Four Ways to Package Transgenes That Exceed the Size Limit of Adeno-associated Virus

    Type
    Blog Post
    ...improvement in muscle physiological performance.  2. Trans-splicing Overview: For trans-plicing, splice...human T cells and hematopoietic stem and progenitor cells (HSPC)s to ~40% expression in the K562 cell line...gene to airway epithelial cells in mice. This vector transduced ~10% of cells, similar to the transduction...Using AAV Donor Vectors. Cell Reports 20:750–756 . https://doi.org/10.1016/j.celrep.2017.06.064  Carvalho... then independently packaged into an AAV. When a cell is co-infected with both part A and part B AAVs,... always.  The combination of serotype and tissue/cell type could have a big impact on the rate of transgene...since both of these variables influence whether cells will be co-transduced with multiple copies of both...
Showing: 561 - 580 of 592 results