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We narrowed to 845 results for: ELL

Showing: 261 - 280 of 845 results

  1. Choosing the B(right)est Fluorescent Protein: Photostability

    Type
    Blog Post
    Published
    June 8, 2017, 2:30 p.m.
    ...intensity evolves over time in living cells at a power used for live-cell imaging. To conclude, the most photostable...molecule will photobleach at some point. For live-cell imaging, it is desirable to have fluorescent proteins...experiment. The best fluorescent proteins for live cell imaging can be excited many times, thereby producing...these factors, environmental conditions such as cellular redox state and oxygen concentration may affect...protein is a key requirement for quantitative live-cell imaging, it is important to be able to quantify ...it is advisable to perform time-lapse imaging of cells producing the fluorescent protein of interest under... a substrate. These in vitro methods allow for a well-controlled environment, but they do not mimic the...

  2. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    Published
    Sept. 28, 2020, noon
    ...Leonard SP, Perutka J, Powell JE, Geng P, Richhart DD, Byrom M, Kar S, Davies BW, Ellington AD, Moran NA, Barrick...Control of Gene Expression. Cell 152:1173–1183 . https://doi.org/10.1016/j.cell.2013.02.022 Ronda C, Pedersen... is primarily a means of selection that targets cells in which homologous recombination has not occurred...ensures high editing efficiency; the only non-edited cells to survive have inactivating mutations in the Cas9... E. coli, the system was used successfully in Tatumella citrea, another species of Enterobacteriaceae,...one or more genes in E. coli. and in a mammalian cell line (Qi et al., 2013). Two important features of...other Cas9 orthologs to identify a more efficient, well tolerated, Cas enzyme. They identified the Cas9 ...

  3. Split Fluorescent Proteins for Studying Protein-Protein Interactions

    Type
    Blog Post
    Published
    May 3, 2022, 1:15 p.m.
    ...measuring protein expression or localization in cells, or even single-particle tracking of the bound complex...) for use with GFP(11) Multiplexed labeling of cellular proteins with split fluorescent protein tags. ...(1-10) and GFP(11) Versatile protein tagging in cells with split fluorescent protein. Kamiyama et al. ... and mNG3A(1-10) for use with mNG2(11) Improved yellow-green split fluorescent proteins for protein labeling... S, Brown D, Huang B. PLoS One. 2020 Bo Huang Yellow Venus pBiFC-VN173, pBiFC-VC155 Identification of...pairs with sfCherry2(11) Multiplexed labeling of cellular proteins with split fluorescent protein tags. ...

  4. What's New in CRISPR - March 2020

    Type
    Blog Post
    Published
    March 10, 2020, 1:15 p.m.
    ...for studying the bacterial cell cycle, asymmetric cell division, and cellular differentiation, but it takes...crescentus Multiplexed gRNA for human pluripotent stem cells CRISPR-based DNA demethylation Optogenetic repressors...complex remains in the cytoplasm. Read the paper in Cellular and Molecular Bioengineering Find the optogenetic...Figure 1: Serial dilutions of CRISPRi in Caulobacter cells for ctrA gene expression using various dCas9. Image...popular Streptococcus pyogenes system does not work well in Caulobacter). To test their CRISPRi system, the...lab repressed ctrA, a gene encoding an essential cell cycle regulator. The lab expressed both dCas9 and...piggyBac transposon system for human pluripotent stem cells Lindy Barrett’s lab developed new fluorescently ...

  5. The AAVantages of AAV in CRISPR Screens

    Type
    Blog Post
    Published
    Feb. 4, 2025, 2:15 p.m.
    ...same: Deliver CRISPR library to cells of interest. Select for cells of interest (positive or negative...infect specific cell or tissue types. Using serotypes that have a known tropism for your cells of interest... a host cell’s genome. In this state, AAV cargo can be stably expressed in non-dividing cells for long...mice are involved. Ex vivo starts with transducing cells with the CRISPR library in vitro before transplanting...interest — for example, AAV8 for kidney cells — can help improve infection rates in your experiment. ...AAV vectors, enhancing their affinity for specific cell and tissue types. The combination of serotypes and...integration sites seem to only occur in the target cells or tissues. AAV also shows improved transduction...

  6. Pooled Library Amplifications

    Type
    Blog Post
    Published
    May 30, 2024, 1:30 p.m.
    ...Genome-wide Inhibition Library. DNA and cell amounts After deciding on a cell type, you will need to determine...DNA and how many cells to use for your transformation(s). The amount of competent cells you need total ... depending on the particular type of cells you are using, as well as other factors, such as your lab’s...generalized amplification protocols tend not to work as well.   Defining your library First, you’ll need to collect... generally electroporated into electrocompetent cells to ensure the highest possible transformation efficiency...efficiency. Using Stbl4 or other ultra-high efficiency cells ensures that plasmid diversity in the library is...transformations, such as splitting 400 ng of DNA in 100 μL of cells over four individual electroporations. The specifics...

  7. Three Key Considerations for Precise Neuronal Targeting Using AAV Technologies

    Type
    Blog Post
    Published
    Nov. 27, 2019, 2:03 p.m.
    ...Table 1 from the paper. 2. Manipulate your cell of interest: Cell-type specific expression of molecular tools...in their cell bodies. Optical stimulation in a different brain area can then reveal the cells bodies of...with AAV: 1. Getting your viral vector into your cell of interest: AAV tropism and route of administration...transduction, the viral capsid proteins bind to the target cell’s surface proteins and this interaction determines...efficiency of the AAV serotype used for a specific cell type. Find some transduction characteristics of ...by restricting expression to genetically defined cell types. Specific transgene expression can be also...different approaches can increase the specificity of cell targeting even further. For example, injecting AAVs...

  8. Google Forums Round Up: First Impressions of NgAgo

    Type
    Blog Post
    Published
    Aug. 4, 2016, 2:30 p.m.
    ...degraded by the cellular machinery, leaving a small amount of active NgAgo/ssDNA complex in the cell. As proposed...results of Gao et al 2016 in a letter to Protein & Cell. Update (August 3rd, 2017) THE ORIGINAL NgAgo ARTICLE... guide, it cannot be produced from a plasmid in cells. Additionally, in vitro assembly of NgAgo/ssDNA ...dangerous, non-physiological temperature for mammalian cells and one which diminishes the endonuclease activity...guide. Therefore, researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression plasmid.... Researchers can conveniently transfect/inject cells with in vitro preassembled Cas9/gRNA complexes or...lipofectamine-based method to deliver NgAgo ingredients into the cell, the same method used by Gao et al, 2016 (1). View...

  9. Plasmids 101: Optimizing Plasmid Yields

    Type
    Blog Post
    Published
    May 26, 2016, 2:30 p.m.
    ...large amounts of carbohydrates that can inhibit the cell lysis process. To ensure that lysis is complete,...strains adapted for protein purification use the cell’s resources to produce high levels of protein, not...difference? In the simplest of terms, more volume = more cells = more plasmid. Plasmids can vary in copy number...of plasmid copies supported within each bacterial cell in your culture. Copy number is partially specified...richer media or adding supplements can improve your cell density, leading to an improved plasmid DNA yield... supplements to the media You can achieve higher cell density using a nutrient-rich broth such as Terrific...glucose (in moderation) may also serve to increase cell density and plasmid yield. Start with a single, ...

  10. Celebrating Outstanding CRISPR/Cas9 Achievements at the Dr. Paul Janssen Award Dinner

    Type
    Blog Post
    Published
    Sept. 15, 2014, 6:05 p.m.
    ...already available today to cure sickle cell anemia in stem cells but this can’t be used in people until...and how much we have yet to learn. As Dr. Mello said, “Cells have been in the information age for billions...meeting movie stars, but when I met the astronaut Jim Lovell I was speechless (the club of people who have been...genome engineering, Dr. Jennifer Doudna and Dr. Emmanuelle Charpentier. I was joined by Addgene Scientist...from them during a panel discussion with Dr. Craig Mello and Dr. Paul Stoffels from J&J. The panel was moderated...the mechanisms of RNAi, the field for which Craig Mello and Andrew Fire won the Nobel Prize. What did Dr... audience discussed how J&J is already modifying cell and animal genomes to test hypotheses of disease...

  11. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    Published
    Sept. 9, 2014, 8:20 p.m.
    ...them. Why Use multicistronic vectors? Detecting cells that are expressing your gene, especially if you...reporters allow you to easily screen or select for cells that are expressing your gene of interest at high...promoter, multicistronic plasmids ensure that any cells that are positive for your marker should also be...anytime you want to express multiple genes in the same cell. Although it is possible to drive co-expression ...promotes translation of the viral genome in eukaryotic cells.1,2 Since then, a variety of IRES sequences have...discovered - many from viruses, but also some from cellular mRNAs. What they all have in common is the ability... cistron due to factors such as the experimental cell type and the specific genes cloned into the vector...

  12. Isolating Genomic Regions of Interest with the CRISPR System

    Type
    Blog Post
    Published
    May 30, 2019, 12:44 p.m.
    ...used. The Fujii lab demonstrated this in mammalian cells by expressing dCas9 with an N-terminal 3xFLAG tag...studies (Fujita et al., 2018). In addition to the "in cell" form of this method, in which an engineered DNA-binding...molecule such as the CRISPR complex is expressed in cells to be analyzed, the Fujii lab also developed another...Protein A, 1xFLAG, 2xAM, and HA have been reported, as well as affinity purification with an anti-Cas9 antibody... disease for which there is currently no cure (Campbell et al., 2018). Find plasmids from the Fujii lab...see our interview with Dr. Fujii. References Campbell, Amy E., et al. "NuRD and CAF-1-mediated silencing...recombinant CRISPR ribonucleoproteins." Genes to Cells 21.4 (2016): 370-377. PubMed PMID: 26848818. Fujita...

  13. Summer SciComm Series: Modes of Communication

    Type
    Blog Post
    Published
    July 1, 2022, 1:15 p.m.
    ...make for beautifully compelling photos and videos, while molecular, micro, and cell biology are often more...documentaries. Can you convey enough information to tell a compelling story in an image or do you need a full exhibit...best to. And that's really natural - if it works so well for you, of course you want to share it with others...others! After all, won't it work just as well for everyone else?  In truth, people are incredibly varied...account aimed at octogenarians is going to be a hard sell!) Digital platforms often have demographic data ...should be fun and engaging for the communicator as well as the audience, so pick a mode of communication...Photography Medium (high for microscopy) Visually compelling. Easy to convey a large number of details without...

  14. Save Time with Transient Plant Leaf Transformations

    Type
    Blog Post
    Published
    July 25, 2019, 12:03 p.m.
    ...into the cells. But for C. roseus, Agrobacterium infiltration into leaves does not work well, as the leaves...tumefaciens strain GV3101(pMP90), which works well for us. Use a well working vector system This allows you to...roots, as functional chloroplasts and leaf specific cell types are missing in roots. The development of transgenic...transformation” usual means that neither a stable cell line or plant is regenerated from the transformed...interest (Unver and Budak, 2009). This method works well in C. roseus. A common method for overexpressing...transient transformation systems for other species as well. Things to consider in developing a transient leaf...fused to a protein of interest to elucidate the subcellular localization of your protein of interest. Find...

  15. Beyond the Blood Brain Barrier: PHP.B Enters the Central Nervous System Via the LY6A Receptor

    Type
    Blog Post
    Published
    July 23, 2019, 12:46 p.m.
    ...relied on for transducing cells: galactose and the AAV receptor (AAVR). Cell lines lacking galactose were...Concise review: stem cell antigen‐1: expression, function, and enigma." Stem cells 25.6 (2007): 1339-1347... Wilson labs determined LY6A, also known as stem cell antigen-1 (SCA-1), is the receptor used by PHP.B...commonly used to enrich adult mouse hematopoietic stem cells. Its ligand(s) has not been identified. While LY6A...out of Ly6a in primary C57BL/6J brain endothelial cells decreased transduction rates while overexpression...overexpression of the C57BL/6J version of Ly6a in 293T cells enhanced PHP.B transduction rates. The group also determined...PHP.B, virus still reached the brain endothelial cells of these mice, although at lower levels than in ...

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

    Type
    Blog Post
    Published
    May 2, 2019, 12:41 p.m.
    ...Xenopus frog cell lines to develop robust methods for frog gene editing. They edit in a cell line, then ...is the smallest photosynthetic cell in existence, but most abundant cell on earth (twice the human biomass...Mormyridae (Jason Gallant) Weakly electric fish are excellent systems to study for convergent evolution because...Faithful in 60 °C soils. Dendrobatidae (Lauren O’Connell)  Poison frogs are great for studying parenting... currently for terrestrial frogs. Cnidarian-Dinoflagellate symbionts (Virginia Weis) Sea anemone Aiptasia...killing frogs all over the world. These are less well known fungi (they are not yeast or mushrooms) but...Hawaiian bobtail squid has an extreme number of nerve cells and an unusually high level of RNA recoding in the...

  17. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    Published
    April 13, 2021, 1:15 p.m.
    ... into a pEcCas/pEcgRNA system. This system works well for engineering single genome edits but the double...construct and then introduce both plasmids into the cell. The edit in the chromosome can now take place. .... After editing, pTargetF can be cured from the cell by inducing expression of the sgRNA that targets ...all the edits you want, cure pCas9 by growing the cells at 37 °C.     The new pEcCas/pEcgRNA system We ... to take place. Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image... E. coli B strains, 1 E. coli W strain, and 1 Tatumella citrea, with a total of 70 sites edited. We optimized...crRNA. The spacers of the crRNA can be customized as well, including concatenations into crRNA arrays targeting...

  18. Antibodies 101: The Four ELISAs and When to Use Them

    Type
    Blog Post
    Published
    July 25, 2024, 1:15 p.m.
    ... the same overall process. First users coat a multiwell plate with a capture reagent. The capture reagent...conjugated antibody binds to antigen coated on the multiwell plate. In an indirect ELISA a primary antibody...the primary antibody. In a sandwich ELISA the multiwell plate is first coated with a capture antibody ...antibody can be used. In a competitive ELISA a multiwell plate is coated with a competing antigen (green...enzyme labeled antibody and then added to the multiwell plate. The more antigen in the sample, the less...method, users immobilize the target antigen on the multiwell dish and incubate it with a reporter conjugated...ELISA, the sandwich ELISA, users first coat a multiwell plate with a capture antibody and then add their...

  19. Plasmids 101: Stringent Regulation of Replication

    Type
    Blog Post
    Published
    June 20, 2024, 1:15 p.m.
    ...replicating too quickly can slow cell growth or even kill the host cell. For stringently controlled plasmids...All plasmids rely on their host cell's replication machinery in order to replicate—but not always to ...be synchronized with the replication of the host cell's chromosomal DNA.  Plasmids are said to be under... stringent control when they depend on the host cell’s initiation proteins to start their own replication... occurs at the appropriate time—for example, the cell must have enough nutrients available to complete...the amount of active DnaA available is one of a cell’s best tools to regulate DNA replication. DnaA negatively...available to bind DnaA. Fun fact! As far as we can tell, DnaA doesn’t need to hydrolyze ATP to do its job...

  20. Hot Plasmids Spring 2024

    Type
    Blog Post
    Published
    May 23, 2024, 1:15 p.m.
    ...with enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene offers vectors...reverse transcriptase enzymes, inhibition of host cell proteins, and modifications to the pegRNA. The Britt...shows enhanced editing efficiencies in a number of cell types at different genomic loci — in some cases,...stabilizing the 3’ end of the pegRNA, which is not well protected by Cas9. This is similar to the proposed...exciting to see future studies enabled by this tool as well as additional potential improvements to the editor... editing efficiencies at different loci in U2OS cells. Image adapted from Yan et al. 2024 under a CC-BY...western blots (Figure 3).      Figure 3: HeLa cells transduced with SARS-CoV-2 nucleocapsid protein ...
Showing: 261 - 280 of 845 results