Skip to main content
Addgene

Enter a sequence to perform a BLAST-based search of the Addgene plasmid sequence database.

Information

In Category

We narrowed to 587 results for: cel.2;

Showing: 201 - 250 of 587 results

  1. Advanced Uses of Cre-lox and Flp-FRT - A Neuroscientist’s View

    Type
    Blog Post
    .... “A FLEX Switch Targets Channelrhodopsin-2 to Multiple Cell Types for Imaging and Long-Range Circuit ...FLP and Cre for additional control   Figure 2: Expression of a morphological marker (eYFP) and synaptic...PMID: 18614669. PubMed Central PMCID: PMC2593125. 2. Buchholz, F, P O Angrand, and A. Francis Stewart....The FLP Recombinase of the Saccharomyces Cerevisiae 2 Micron Plasmid Attaches Covalently to DNA via a Phosphotyrosyl...specific cell types (populations) within neuronal networks in living animals. To accomplish such cell specific...individual cells. With this technique, a Neuroscientist can see the full dendritic arbor of a single cell with...Cre-mediated possibilities for site specific (and often cell type specific) control of DNA recombination and ...

  2. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    ... analogs such as 2-fluoroadenine (2-FA), organisms with functional APT will convert 2-FA to cytotoxic ...selection of plants undergoing RNAi, and LIIbeta F 1-2 RNAi, which allows assembly of intron-spliced hpRNAs...nucleotides, resulting in death. In the presence of 2-FA, potent gene silencing of APT is required for survival...genes known to be essential for cell growth. Together, this excellent work highlights the importance of...fluorescence-activated cell sorting (FACS). This is not possible in plants outside of limited, single cell protoplast...exquisitely and robustly self-organize at the cellular and multicellular scale.     References Bezanilla M, Perroud...can be found at Addgene that could serve as an excellent start (#22988, #137884). Both vectors allow the...

  3. Light Sheet Fluorescence Microscopy

    Type
    Blog Post
    ...Carballosa, et al, (2015) eNeuro, March/April 2015, 2(2) e0001-15.2015. Pubmed. Resources: http://openspim.org...orthogonal-plane fluorescence optical sectioning (OPFOS) (2). Arne Voie, David Burns and Francis Spelman focused...in LSFM. In LSFM the laser light sheet, typically 2-6  microns, illuminates only one thin plane of the...transparent are enabling microscopes to visualize the cellular architecture of whole tissues in 3D with unprecedented... Weber, M. Mickoleit, J. Huisken. (2014) Methods Cell Biol.123, 193-215. Pubmed. W. Spalteholz. (1914)...

  4. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

    Type
    Blog Post
    ...Madison lab found that H2B-GFP expressing cells exhibited a 2-4 fold increase in the number of endogenous...Denham lab made several other small changes creating 2 other versions but these did not surpass GEmCherry2...fluorescence and in-frame fluorescence.  Figure 2: The GEmCherry reporter. Image from Højland Knudsen...introduced the reporter into the cells, you’re ready to edit. These cells are then transfected with several... up, all successfully transfected cells will express RFP while cells containing the reporter gene that...using the RFP:GFP ratio, edited cells can be sorted from unedited cells. In an experiment that targeted...Activity. Frontiers in Cell and Developmental Biology 6: . https://doi.org/10.3389/fcell.2018.00054 Sander...

  5. Important Considerations in Optogenetics Behavioral Experiments

    Type
    Blog Post
    ... will repeatedly nose-poke at the active hole [1, 2].    Place preference/place aversion also models reinforcement...spend most of its times in the opposite chamber) [2]. Laser activation is usually controlled automatically...two different ways 1) your hypothesis is wrong or 2) there is some problem with the technical implementation...PMID: 21716290. Pubmed Central PMCID: PMC3775282. 2. McDevitt, Ross A., et al. "Serotonergic versus nonserotonergic...optogenetics, ICSS can now be done with neuronal cell-type and temporal precision. In ICSS, a test chamber... remote contextual memories. This study is an excellent example in which the authors started with a brain... research. Derek P. Simon received his PhD in Cellular and Molecular Biology from the University of Michigan...

  6. Quick Guide to Working with Drosophila Part 3: Genome Engineering in Flies

    Type
    Blog Post
    ...are many docking sites available for integration. 2. How soon would you like your flies ready for use?...beautiful immunofluorescent images like in this paper (2). Metabolic assays such as Seahorse can be employed...PMID: 16547094. PubMed Central PMCID: PMC1526508. 2. H. Jiang,  et al. Cytokine/Jak/Stat signaling mediates...Drosophila cell line, I’ve studied a transcription factor important for T cell differentiation (a cell type ...regeneration and homeostasis in the Drosophila midgut. Cell. 137, 1343–55 (2009). PubMed PMID: 19563763. PubMed...et al. Digoxin and its derivatives suppress TH17 cell differentiation by antagonizing RORγt activity. ...

  7. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    ...termed fluorescence activated cell sorting or FACS (2). By analyzing single cells instead of the population...populations (Figure 2). An immunologist who studies B cells, might then gate the B cell population and create..., these cell types will separate into distinct populations on a FSC versus SSC plot (Figure 2).  A user... Figure 2: Due to differences in their size and complexity, cells from a whole blood sample will...expression. A routine flow cytometry experiment uses 2 or 3 different fluorescent colors each measuring a...plot of CD3 (T cell marker) versus CD19 (B cell marker) to separate the B cell and T cell into distinct...separate the B cell population into progenitor B cells, immature B cells, plasma cells or others.   ...

  8. TALEN Plasmids and Kits

    Type
    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...

  9. Troubleshooting Your Plasmid Cloning Experiment

    Type
    Blog Post
    ... working with is correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...colonies (5-10) and grow them in a small culture volume (2 or 3 ml) containing the corresponding antibiotic, ...product, the transformation may not result in any cells, or all the colonies screened might not contain ...verify the strain and the efficacy of your competent cells (when they get old, they lose competency). If all... look at your experimental design. Full lawn of cells. The antibiotic in your plate might not work. Check...acknowledged in hundreds of publications including Cell, Nature, and other journals.   Paolo Colombi is ...

  10. Live and Let Dye: Self-Labeling Protein Tags

    Type
    Blog Post
    ...for Multiprotein Labeling in Living Cells. Chemistry & Biology, 15(2), 128–136. https://doi.org/10.1016...super-resolution or single-molecule techniques.   Figure 2: Two-color super-resolution imaging of ER and mitochondria...stimulated emission depletion (STED) nanoscopy. Scale bar, 2 μm. Inset shows comparison with standard confocal ...enables applications like multi-color imaging (Figure 2). If you’re working with purified proteins in vitro...conjugates: A flexible chemical tag. Nature Methods, 2(4), Article 4. https://doi.org/10.1038/nmeth749 Strebinger...Bewersdorf, J. (2016). Two-colour live-cell nanoscale imaging of intracellular targets. Nature Communications,...opening up new applications.   Figure 1: Live-cell labeling strategies. The green structure represents...

  11. Plasmids 101: TOPO Cloning

    Type
    Blog Post
    ... the two DNA strands and releasing topoisomerase (2).  These days commercially available TOPO kits provide...Taq polymerase using your favorite PCR protocol.  2. Set Up TOPO Cloning Reaction: Mix together the PCR...PubMed PMID: 1658796. PubMed Central PMCID: PMC52876. 2. Novel approach to molecular cloning and polynucleotide... Transform TOPO Cloning Reaction into Competent Cells: You can use your standard lab protocol for this...

  12. Addgene’s Newest Way to Speed Your Research: The Cloning Grade DNA Service

    Type
    Blog Post
    ... be directly used in the research on the SARS-CoV-2. Nine of the overall plasmid selection will be open... cloning grade DNA beta project We will ship you  2 µg of DNA in 20 µl of Tris buffer suitable for molecular...service to further contribute to our mission to “accelerate research and discovery.” Here are the details...

  13. Hot Plasmids April 2018 - Protein Degradation, Nanoscopy, FIRE-Cas9, and Yeast Expression Tools

    Type
    Blog Post
    ...ddifferent cell types, including difficult-to-manipulate primary cells, achieving knockdown within 1-2 hr of...factor 1 controls expression of transcription factor 2, which directly controls expression of a reporter ...modulating the induction of transcription factors 1 and 2. See Aranda-Díaz et al. for more information. The ...stable cell lines overexpressing TRIM21, finding that TRIM21 overexpression did not affect cell proliferation...electroporated cells re-adhered to the culture plate within 4 hr of treatment. In some primary cell lines, endogenous...proliferation. These cells also did not contain changes in expression of TRIM21-associated genes, indicating...multiple methods for TRIM-Away delivery. For small cell populations, co-microinjection of TRIM21 protein...

  14. Viral Vectors 101: Calcium Sensors

    Type
    Blog Post
    ...illumination with violet light and (2) exposure to increased intracellular calcium. It has been applied to...calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) ...dynamics in vivo. Neuroscience Research. 2021 Aug 1;169:2-8. Suzuki J, Kanemaru K, Iino M. Genetically encoded... defined population of cells (e.g., a neuron subtype) and specific subcellular compartments for measurements...calcium concentrations and transients in living cells and organisms. Unlike chemical indicators, these...Localization The calcium concentrations in different subcellular compartments can vary greatly. Additionally, ...neuron-specific expression (hSyn promoter) and pan-mammalian cell expression (CAG promoter). An improved version named...

  15. 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...genes essential for West-Nile-virus-induced cell death." Cell reports 12.4 (2015): 673-683. PubMed PMID:... (and multiple) genetic perturbations in single cells. Methods to scale up multiplexed CRISPR systems ... build and constantly improve gRNA libraries, excellent resources with effective gRNAs are publicly available...perturbations and applications in functional genomics, cell reprogramming, and beyond. For further details, ...

  16. Plasmids 101: CcdB - The Toxic Key to Efficient Cloning

    Type
    Blog Post
    ...CcdB.” Biotechniques. 1996 Aug;21(2):320-3. PubMed PMID: 8862819. 2. Bahassi, EM., et al. “F plasmid CcdB... desired insert should not grow.   Figure 2: Image from http://teachline.ls.huji.ac.il/72682/tutorials... protein (CcdA) that protects the cell against the toxic CcdB. Cells that lose ccdA through the loss of...disrupted allowing cells expressing the recombinant plasmid to propagate. Any cells that contained non-recombinant...which is responsible for plasmid maintenance during cell division. ccdB codes for the toxic protein (CcdB...broken double stranded DNA and ultimately causing cell death. ccdA, another gene found in the ccd operon...Gateway takes advantage of the same principle that cells will not propagate while expressing the gene. Briefly...

  17. 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... brain, robust single-cell labeling is necessary to distinguish individual cells. The Brainbow-1.0 construct... May 5. PubMed. Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish... both of Harvard’s Department of Molecular and Cellular Biology, harnessed these attributes of Cre-lox...

  18. Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9

    Type
    Blog Post
    ... (2,3). Stable strand separation is maintained by 2 sets of interactions : 1. Interactions between the...formed by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand and the gRNA... Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal structure of ... the EMX1 locus in human embryonic kidney (HEK) cells, 11 of these initial mutants retained the on-target...off-target activity at multiple genomic locations in HEK cells. Further testing of two of the mutants, SpCas9(K855A... showed that these mutants are not toxic to HEK cells and that similar mutations can improve the specificity... researchers to make precise edits in mammalian cells and may decrease worries about off target effects...

  19. Plasmids 101: Expressing shRNAs for RNA Interference

    Type
    Blog Post
    ... References Bartel, D. P. (2004). MicroRNAs. Cell, 116(2), 281–297. https://doi.org/10.1016/s0092-8674...downstream genomic sequences (Figure 2B).   Figure 2: A comparison of an shRNA plasmid sequence and an ...module_attribute "schema_version" is_json="true" %}{% raw %}2{% endraw %}{% end_module_attribute %}{% module_attribute...miRNA-guided gene silencing. Nucleic Acids Research, 50(2), 617–634. https://doi.org/10.1093/nar/gkab1256 Lee...silencing complex. Molecular Cell, 82(1), 30–43. https://doi.org/10.1016/j.molcel.2021.11.026 Kilikevicius...miRNA and piRNA can be found endogenously in many cell types, while shRNA and siRNA are synthetic constructs...methods. shRNA processing Following delivery into cells using viral vectors or plasmids, shRNAs hijack the...

  20. Antibodies 101: Stripping and Reprobing Western Blots

    Type
    Blog Post
    ...incubation at 37 °C. Wash the membrane for 5–10 minutes 2–3x in fresh wash buffer such as PBS or TBST. Stringent...Purpose Tris HCl 62.5 mM Buffer SDS 2% (w/v) Detergent Β-mercaptoethanol (βME) 100...minutes at 50 °C. Wash the membrane for 5–10 minutes 2–6x in fresh wash buffer such as PBS or TBST. Note ...better than a nitrocellulose membrane. If you have already run your blot using nitrocellulose, you may decide...protein of interest in a complicated solution like cell lysate. But they can be a lot of work — and what...instructions for the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed with caution...

  21. Addgene Depositors Get More Citations

    Type
    Blog Post
    ... 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010 167  Nature 3.3%  10.5%  3.2  534.1 3...Microbiology  1.6% 1.2%  0.75   125.3 In figure 2, you can see the full impact from using these weighted...plasmids deposited at Addgene still accrue on average 2 to 3 times as many citations as other papers in the... most relevant to plasmids: Genetics & Heredity, Cell Biology, Biotechnology & Applied Microbiology, Biochemistry...

  22. Using Phosphoserine to Study Protein Phosphorylation

    Type
    Blog Post
    ...phosphorylation." Nature cell biology4.5 (2002): E127-E130. PubMed PMID: 11988757. 2. Hornbeck, Peter V., ...phosphorylation events in the proteomes of higher mammals (2). How can we begin to understand the importance of... the open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...optimized Sep-OTSλ and can recommend its use (6).  2. C321, a specialized bacterial host strain for pSer-incorporation...commonly cited that approximately one-third of cellular proteins are modified through phosphorylation ...translation of many stop codons and detrimental effects on cell growth. The C321.ΔA strain was genomically recoded...is particularly interested in the intersection of cell signaling, metabolism, and the mighty mitochondrion...

  23. Fluorescent Proteins 101: Introduction to FRET

    Type
    Blog Post
    ... living plant cells." (2013). PubMed PMID: 24194740. PubMed Central PMCID: PMC3810607. 2. Bajar, Bryce... fluorophore excitation by absorption of a photon 2. Energy transfer from the excited donor directly to...PubMed PMID: 10500161. PubMed Central PMCID: PMC18018. 2. Miyawaki, Atsushi, et al. "Fluorescent indicators...fluorescent proteins available. Fortunately, several excellent reviews have collated data from various papers... alters the FRET efficiency. Detecting certain cellular changes with an intermolecular probe is often ...Miyawaki et al., 1997), was designed to measure intracellular calcium and published in 1997. Since that time...of labs to answer sophisticated questions about cellular processes. Currently, FRET experiments can probe...

  24. Chemogenetics Guide

    Type
    Guide
    ...muscarinic receptor and has not been used in vivo . Figure 2: DREADDs, their ligands, and signaling properties.... Reference Rq(R165L) Human M3 muscarinic Arrestin-2/-3 CNO* Increase Arrestin translocation Arrestin signalling...hM1Dq hM5Dq Human M3 muscarinic G αq CNO* Increase Ca 2+ Neuronal burst firing Armbruster et al., 2007 rM3D...channels, their effect, and outcome in neurons. Table 2. Descriptions of PSAMs and their activity in neurons...Forbes SL, Liggett SB (2001). Modification of the beta 2-adrenergic receptor to engineer a receptor-effector...targeted to specific tissues, cell types or even subcellular regions of a neuron. Cell-specific expression of...Human Stem Cell-Derived Neurons Repair Circuits and Restore Neural Function. Cell Stem Cell. 28:112-126...

  25. Educational Planarian Tools for Teaching Developmental Biology Techniques

    Type
    Blog Post
    ... the gene Prohormone convertase 2 (pc2) labels the central nervous system. Evaluating Gene function by... 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...stain the cells that contain this mRNA-riboprobe-antibody complex and, as a result, these cells turn blue...specific cell types or entire organs in planarians. For example, the gene Piwi labels neoblast cells and the...techniques to alter gene expression or to label specific cells, but there are too few resources to teach students... in tissue. This is useful for labeling specific cell types and for viewing how gene expression changes...

  26. Lentiviral Vector Guide

    Type
    Guide
    ...intact. First-generation plasmids include (Figure 2): Transfer plasmid — contains transgene and wild-type...envelope removed Envelope plasmid — contains env Figure 2: First-generation lentiviral plasmids. Created with...plasmid 1 — contains gag and pol Packaging plasmid 2 — contains rev Envelope plasmid — contains env Third-generation...The inside out of lentiviral vectors. Viruses , 3 (2), 132–159. https://doi.org/10.3390/v3020132 PMID: ...Infectivity and Integration Targeting. PLoS ONE , 2 (12), e1340. https://doi.org/10.1371/journal.pone.0001340...features of lentiviral vectors. Human Gene Therapy , 24 (2), 132–142. https://doi.org/10.1089/hum.2012.229 PMID...production cell line, typically human embryonic kidney 293T (HEK293T) cells. Standard HEK293T cells are sufficient...

  27. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    ...lately (1, 2), depends on knowledge of the structure(s) a given phage will recognize on a host cell. To make...per plate) across 20 fresh selective plates. Step 2: Express random phage DNA fragments in E. coli Now...microbiology 23.4 (2015): 185-191. PubMed PMID: 25708933. 2. Cooper, Callum J., Mohammadali Khan Mirzaei, and ... biology 34 (2015): 69-77. PubMed PMID: 26275959. 2. Weigele, Peter, and Elisabeth A. Raleigh. "Biosynthesis... cloned into E. coli cells, isolated colonies will be lifted onto nitrocellulose membranes and exposed... proteins directly onto the nitrocellulose for subsequent probing for cell-binding activity.  First, cut...with live host cells Now you can proceed with incubating this membrane with your cells of interest and...

  28. Technologies Enabled by NanoLuc® Luciferase

    Type
    Blog Post
    ...the system outlined in Schmid-Burgk, J.L., et al (2). In this post, I’ll cover two great applications ...molecularly evolved from mNeptune2 through 33 mutations and 2 deletions. CyOFP1 has a higher quantum yield, brightness...luciferin 24 hours later demonstrated a more than 2-fold brighter signal from Antares as compared to Fluc...PMID: 22894855. PubMed Central PMCID: PMC3501149. 2. Schmid, J.L., et al. (2016) CRISPaint allows modular...successfully infected/expressing cells by flow cytometry. The isolated cells were injected into mice and imaged...including monitoring multiple cellular events such as the dynamics of subcellular structures and gene expression...other cellular proteins. BRET-based biosensors utilizing NanoLuc® luciferase Many intracellular sensors...

  29. Pathways Over Time Plasmids Engage Students in Functional Genomics Research

    Type
    Blog Post
    ... DC. 2. President's Council of Advisors on Science and Technology [PCAST] (2012). Engage to Excel: Producing...research experiences to undergraduate students (1, 2), but educators face significant challenges in designing... by western blotting in other experiments (Figure 2).   Complementation reveals conservation of function...lack uracil, but contain methionine. (This allows cells carrying nonfunctional as well as functional MET... is available at our Investigations in Molecular Cell Biology Website. Many thanks to our guest blogger...right) has taught courses in biochemistry, molecular cell biology and genetics at Boston College. She is particularly...

  30. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down options for you...our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer team...Filling out and sending the deposit spreadsheet Figure 2: Example of the deposit spreadsheet showing the first... question about what should go into a cell, hover over the cell and instructions will pop up. When you... into the appropriate full and partial sequence cells. *Note: If you believe you need to deposit a collection...

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

    Type
    Blog Post
    ...neuroscience see Table 1 from the paper. 2. Manipulate your cell of interest: Cell-type specific expression of molecular...specific cell type. Find some transduction characteristics of select AAV serotypes in Table 2 from the...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...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...

  32. 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... gRNA vector that targets the gene of interest, 2) a plasmid to specify the reading frame of the insertion...and function of cellular systems. FPs have been used to track protein localization, cell structure, intracellular...associated with cellular organelles, scientists have been able to study many cellular processes, including...to fluorescently tag markers of cellular structures in mammalian cells. These plasmids use fluorescent...Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.” Cell Rep.15(1) (2016...

  33. How Dry Ice Affects Viral Vector Sample pH & How to Avoid It

    Type
    Blog Post
    ...pH visible at the sample interface upon thaw (Fig. 2). Before thawing, all the samples were at the control...showed that AAV infectivity was not altered by a short 2-hour exposure to pH as low as 2.5 (Potter et al., ...Methods. 2013 Apr;10(4):278-9. PubMed PMID: 23538862. 2. Potter M, Lins B, Mietzsch M, Heilbronn R, Van Vliet...measure the pH of our samples, we used a solution of cell culture medium, which is buffered and contains the...

  34. Making CRISPR Plasmids Using Fragmid

    Type
    Blog Post
    ... of components, such as your CRISPR enzyme or Pol 2 promoter. The options available in each class, or ...overhang specific to the type of module it is (Pol 2 protomer, Cas protein, ect...). Whatever individual... (KO, CRISPRa, CRISPRi, editing)   Figure 2: Selecting module fragments on the Fragmid website...Drosophilia Plasmid pRDB_051 (for stable fly cell lines; attL and attR sequences for genome integration...Drosophilia Plasmid pRDB_052 (for stable fly cell lines; Hygromycin resistance gene; for genome integration...rapid assessment of emerging CRISPR technologies. Cell Genomics, 4(3), 100519. https://doi.org/10.1016/...

  35. CRISPR Guide

    Type
    Guide
    ...Haploid Embryonic Stem Cells Carrying a Guide RNA Library. Cell Stem Cell . 17 (2), 221–232. PMID: 26165924...used to transduce your experimental cell population (Figure 8E). In a 2-vector system, you’ll need to either...of CRISPR-Cas9 with Bacteriophage Proteins. Cell , 168 (1–2), 150-158.e10. PMID: 28041849 Sakuma, T., Nishikawa... regulation of transcription in eukaryotes. Cell . 154 (2), 442–451. PMID: 23849981 Gilbert, L. A., Horlbeck...Transcriptional Programs with CRISPR RNA Scaffolds. Cell . 160 (1–2), 339–350. PMID: 25533786 Cas9 alternatives...single RNA-Guided endonuclease of a Class 2 CRISPR-CAS system. Cell , 163 (3), 759–771. PMID: 26422227 Zetsche...left free to interact with the target DNA. Figure 2: Overview of the NHEJ repair mechanism Cas9 will only...

  36. Cpf1 Update: Comparison to Cas9 and NgAgo

    Type
    Blog Post
    ... Class 2 CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674(15)01200-3. doi: 10.1016/j.cell.2015.09.038...single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227.   ... Find plasmids from this publication at Addgene. 2. Kleinstiver, Benjamin P., et al. "Genome-wide specificities...particularly helpful for targeting non-dividing cells, which are difficult to modify through homology-... Cpf1 orthologs known to be active in mammalian cells, LbCpf1 and AsCpf1. In both reports, on target editing... (RNPs) failed to induce off-target editing in a cell culture model. What we still don't know about Cpf1...reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. 2016 Jun 6. PubMed PMID: 27272384...

  37. CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems

    Type
    Blog Post
    ...base pair to another base pair at a specific site. 2. Temporal, spatial, or conditional control of CRISPR...difficult-to-engineer Sulfolobus islandicus rod-shaped virus 2 (Mayo-Muñoz et al., 2018). Only viral particles that...Editing of Sulfolobus islandicus Rod-Shaped Virus 2. Viruses 10:695 . https://doi.org/10.3390/v10120695...Inhibitor of CRISPR-Cas9. Cell 170:1224-1233.e15 . https://doi.org/10.1016/j.cell.2017.07.037 Li C, Psatha...Off-Switches for CRISPR-Cas9. Cell 167:1829-1838.e9 . https://doi.org/10.1016/j.cell.2016.11.017 Shin J, Jiang...concern is that the longer Cas remains active in a cell, the greater chances there are for off-target edits...within the first six hours. When added to human cells six hours after the introduction of Cas9 RNPs, AcrIIA4...

  38. Mapping the 4D nucleome with CRISPR/Cas9

    Type
    Blog Post
    ...resolved loci with physical map distances of 75 and 2 Mbp, with the calculated fluorescent distances correlating...established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that they could evaluate the...be applicable to studies of cell cycle progression, epigenetics and cellular reactions to external stimuli...Weissman JS, Qi LS, Huang B. Cell. 2013 Dec 19;155(7):1479-91. doi: 10.1016/j.cell.2013.12.001. Pubmed. Find...LS, Weissman JS, Vale RD. Cell. 2014 Oct 23;159(3):635-46. doi: 10.1016/j.cell.2014.09.039. Epub 2014 Oct...labeled Cas9 to label multiple genomic loci in live cells. While other systems can be used to label loci, ...CRISPR/Cas9’s ease of use and ability to label live cells make this system truly advantageous. This new technique...

  39. Tetracycline Inducible Expression

    Type
    Collection
    ...expression tTA Off Verma 26803 pEnt L1L3 EF1a-tTA-2 Contains Gateway L1L3 sites and EF1α promoter driving...systems were tested for functionality in mammalian cells. The lac system with the inducer isopropylthio-β-galactoside... and Bujard tested the tet system in a mammalian cell system (HeLa) and found that the tet system was ... interest rtTA or TetR expression plasmid Stable cell lines can be made that continuously express a system...derivatives can be viewed in Krueger et al, 2004. Some cell culture sera may contain tetracycline or its derivatives...of gene expression at a single copy in mammalian cells; Expresses firefly luciferase hairpin and GFP under...vector lentiviral Gateway RNAi system for conditional cell line generation; contains expression cassette for...

  40. AAV Vector Quality Control: Going the Extra Mile with NGS

    Type
    Blog Post
    ...the mixed sample in our pipeline (see Figure 2, sample 2). When we blindly analyzed AAV-68544, we were...look for potential contaminants. The analysis is a 2-step process using Geneious software: First, the individual...our viral service to continue our mission of accelerating scientific research. To make sure that adeno-associated... check viral transgene expression in transduced cells whenever possible. This post will describe our workflow...fragments of DNA from the helper plasmids or the cell genome can be packaged inside the vectors during...earlier, it is common to find DNA from the packaging cell genome, bacterial genomes, cloning vectors, and ...

  41. Antibodies 101: Flow Compensation

    Type
    Blog Post
    ...Figure 2, following compensation. Figure 3: Flow plot for a double positive sample. The cell population.... Let's look at an experimental example in Figure 2. Figure 2A shows the sample prior to compensation.... line with the other populations.   Figure 2: Compensation example based on experimental sample... make it difficult to properly gate your desired cell population. By compensating between fluorescent ... your fluorescent markers are staining for. If a cell is positive for both markers, it will show up as...learning! Ashlyn has a research background in CD8+ T cells and cancer immunotherapies. She is currently a research...

  42. Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

    Type
    Blog Post
    ...PMC2875081. 2. Snapp, Erik Lee. "Fluorescent proteins: a cell biologist's user guide." Trends in cell biology...imaging in a variety of cellular compartments including the secretory pathway (Figure 2), the inner membrane...properly exiting the ER and fluorescing (see Figure 2) [5].  When using FPs containing two cysteine residues...numerous cell biology assays. However, there has been a pervasive view that most FPs in a cell will fold...fluorescent fusion proteins in cell biology."Current Protocols in Cell Biology (2005): 21-4. Pubmed PMID...Fluorescent proteins in cellular organelles: serious pitfalls and some solutions." DNA and cell biology 32.11 (...-fusions on quantitative imaging experiments and cell physiology in general. Pitfalls of Fluorescent Protein...

  43. Plasmids 101: Control Plasmids

    Type
    Blog Post
    ...the result in Figure 1 could look like this: Figure 2: Expression of Plasmid B (as internal control) This...down expression of Gene X but, as shown in Figure 2, this was likely due to the original transfection ...PMID: 20074371. PubMed Central PMCID: PMC2817684. 2. Replicates and repeats—what is the difference and...untreated cells—but can we be certain? Do we know whether the plasmid delivered into the cells successfully...amount of DNA transfected into the cells and the ability of the cells to express the protein.   In analysis...Gene X in backbone Y) was transfected into human cells. From this result one could simply conclude that...didn’t work, as the expression level of Gene X in cells treated with Plasmid A is similar to the expression...

  44. The AAVantages of AAV in CRISPR Screens

    Type
    Blog Post
    ...AAV8 for kidney cells — can help improve infection rates in your experiment. Figure 2: Visual representation...handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires a less involved setup when working..., 10(1). https://doi.org/10.1038/s41467-019-12449-2 Pupo, A., Fernández, A., Low, S. H., François, A.,...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...

  45. Neuronal labeling with Spaghetti Monster

    Type
    Blog Post
    ...and standard neuronal tracers. 2. Visualization of neuronal sub-cellular structures GFP is limited in its...proteins. For example, N-cadherin (cadherin-2) is a postsynaptic cell adhesion protein that plays a critical...PMID: 25915120. PubMed Central PMCID: PMC4573404. 2. Shepherd, G.M. (2004). In "The synaptic organization...ability to interact with protein partners within the cell. However, antibodies can have low affinity for these... generally bright, stable and well tolerated by cells. They can be used for protein localization, isolation...trigger protein aggregation which is toxic for the cells and affects the labeling of small structures like...team, an ideal probe should combine the solubility, cell tolerance, and optional fluorescence of FPs with...

  46. The Future of Research Symposium Boston 2015

    Type
    Blog Post
    ...Quotes taken directly from participants (summarized in 2) touched upon many problems in this area: “ [The] ...PMID: 24733905. Pubmed Central PMCID: PMC4000813. 2. McDowell, Gary S., et al. "Shaping the Future of ...A fair deal for PhD students and postdocs." Elife 2 (2013): e01139. Pubmed PMID: 24137543. Pubmed Central...following panel members: Jonathan Dinman, Chair of the Cell Biology and Molecular Genetics Department at University...

  47. PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs

    Type
    Blog Post
    ... flanking sequences to their input string (Figure 2). This string can then be used as input for the prediction...top 5) and test them in their labs. Figure 2: Guidelines to predict pegRNA efficiencies with pridict.it...Biotechnology. https://doi.org/10.1038/s41587-024-02268-2 Schep, R., Brinkman, E. K., Leemans, C., Vergara, ... systems in multiple cell types. Cell, 186, 1–17. https://doi.org/10.1016/j.cell.2023.03.034 Resources...Integrated in Parallel. Cell, 154(4), 914–927. https://doi.org/10.1016/J.CELL.2013.07.018 Anzalone, A....epigenetic manipulation of prime editing. Cell. https://doi.org/10.1016/j.cell.2024.03.020 Mathis, N., Allam, A... balance. Molecular Cell, 81(10), 2216-2230.e10. https://doi.org/10.1016/J.MOLCEL.2021.03.032 Yu, G., ...

  48. FLEx Technology and Optogenetics: Flipping the switch on gene expression with high spatial and temporal resolution

    Type
    Blog Post
    ...channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry) to easily detect opsin-expressing cells. The resulting.... "A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit ...the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable strong... Abdallah, Khaled, et al. "Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents... specific cell-type promoter. Once injected into the brain, the viruses infect all the cells with an inactive...energy conversion or the induction of intra- or intercellular signaling pathways (Ernst et al., 2014). Microbial...proteins control the flow of electric charges across cell membranes and maintain membrane potential in response...

  49. Hot Plasmids: Winter 2025

    Type
    Blog Post
    ...resistance to the herbicide chlorsulfuron (Homo line 2) or wild type (WT), two weeks after herbicide was ... CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed a cost-effective expression... when associated with microtubules. Figure 2: CLIP-SNAP fusion proteins for single-molecule FRET... primary cells. The authors used this platform to assay dozens of GPCRs across many cell types in...biosensor platform. Cell, 187(6), 1527–1546.e25. doi: https://doi.org/10.1016/j.cell.2024.01.028. New...Prime editing (PE), initially developed in human cells, has been uniquely challenging to apply in dicotyledonous...labeling approach using these constructs in HEK 293 cells (Smith et al., 2024). They then used single-molecule...

  50. "What Makes a Good Mentor?" and 6 More FAQs About Science Mentoring

    Type
    Blog Post
    ...portion of our careers. In that time we may have only 2-3 formal supervisors that will provide guidance and...be challenging – is advice and guidance from only 2 people enough?  For many years I’ve been organizing...Apparently, a good Jedi does not necessarily an excellent mentor make. Are your current mentors/advisors...What Kind of Mentor Do I Want To Be?” In this excellent essay Acclimatrix, an assistant professor at a...
Showing: 201 - 250 of 587 results