We narrowed to 595 results for: cel.2

...generate a knock-out cell line to perform downstream characterization of, isolating 2 or 3 distinct clones...sites would confound your experimental results and 2) perform targeted off-target measurements at those...experiments where only localization of the Cas occurs. 2 gRNA approach Off-targets that come from mutations...millions of cells. For every individual sgRNA, you recover a minimum of 1,000 targeted cells to sequence...trying to generate a knockout cell line, with the goal of isolating single cell clones to use for multiple...generate a ssDNA break which can easily be repaired by cells with a near zero likelihood of mutation arising.... a level of, say 5%, then the majority of single cell editing events will be on target. Thus, if you isolate...

...disease, and type 2 diabetes, across four different types of human cells, including T cells and hematopoietic...(#186715) and hCtRNA_VT (#186716).   Fig. 2: Comparison of previous representative dual-gRNA architectures...to enable multiplex gene editing in a variety of cells and organisms. However, lengthy genetic payloads... over 50% averaging editing efficiency in human cells. MBE with DAP array also exhibited reduced Cas9-...hematopoietic stem and progenitor cells. Now with the DAP array, it is possible to use the state-of-art base editor... editing, and nuclease editing, in virtually any cell type or organisms of interest with only minimal ...

...Table 2: Lab strains of E. coli Strain Natural resistance Primary Use Genotype ccdB Survival 2 T1R ...resistance to tetracycline Additionally, Table 2 provides a quick reference for some of the popular...degraded, cell still can methylate DNA   hsdS(rk-,mk-) Unmethylated DNA not degraded, cell cannot methylate... gal Mutation in galactose metabolism pathway Cells cannot grow on galactose only gyrA, gyrA96 DNA...permease inactivated, lactose cannot be taken up by cell mcrA, mcrBC Inactivation of pathway that cleaves...gene that maintains the plasmids at ~250 copies per cell.  F- ∆lac169 rpoS(Am) robA1 creC510 hsdR514 endA...rpsL20 xyl-5 leu mtl-1 Stbl4   Electrocompetent cells for cloning and storage of unstable DNA and lentiviral...

...Marcotte EM, Voigt CA. Nature. 2005 Nov 24;438(7067):441-2. PubMed. Find the plasmids from this publication ...Levskaya A, Voigt CA. J Mol Biol. 2011 Jan 14;405(2):315-24. doi: 10.1016/j.jmb.2010.10.038. Epub 2010...Voigt CA, Ellington AD. Cell. 2009 Jun 26;137(7):1272-81. doi: 10.1016/j.cell.2009.04.048. PubMed. Find...with sophisticated gene expression controls. Most cell types do not respond to low-intensity light, so ...

...same parent cells that represent the preexisting resistant subpopulation 2. Resistant cells among the separate...440-448. Pubmed PMID: 25849130. 2. Hata, Aaron N., et al. "Tumor cells can follow distinct evolutionary...resistant cancer cells exist prior to drug treatment. They infected a non-small cell lung cancer cell line containing... target cell population at low multiplicity of infection (MOI) such that each individual cell receives...researchers to individually label millions of mammalian cells through lentiviral infection and to monitor their...quantify the take rate of mouse xenografts after cell implantation. Their goal was to figure out how to...ClonTracer library to determine whether or not cancer cell populations resistant to drug treatments arise during...

...parts: (1) β-strands 1-9 (β1-9) and the one ɑ-helix, (2) β-strand 10 (β10), and (3) β-strand 11 (β11). Each...activating caspase-3, and detected fluorescence within 2 to 5 hours, a time consistent with previous methods...the degradation of over 600 cellular components ultimately leading to cell fragmentation and death (Elmore...issues with tissue autofluorescence, cell heterogeneity, and/or cell position changes (To et al., 2016, ...apoptosis in HeLa cells, zebrafish embryos, and Drosophila. They treated HeLa cells expressing FlipGFP...cleavage by executioner caspases. Failure for cells to die or cells dying at the wrong time and/or place can...Apoptosis or “programmed cell death" plays a pivotal role in an array of biological processes including...

...China, Korea, Japan, Brazil, India, and Mexico. Table 2: Addgene distributors Distributor Partnership...distribution after we partnered with them (Figure 2). In others, the distributor helped solve an issue...temperature for the duration of their journey. Figure 2: Annual distribution of items to countries with a ...we are today. As part of our 20th anniversary celebration, we thought it only fitting to share some stories...

... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 2013 ...PMID: 24086725. PubMed Central PMCID: PMC3785459. 2. Krashes MJ, Koda S, Ye C, Rogan SC, Adams AC, Cusher...expressed in cells that express cre recombinase. Typically, this feature is used to attain cell-specific ...tools so that researchers around the world can accelerate their work. After all, as we like to sayat Addgene...CRISPR applications and can be expressed from your cell line of choice after transduction with this virus...blast virus can be used to generate Cas9-expressing cell lines, which can then be used both for screens (...viral prep to create a single stable Cas9-expressing cell line and then transduce this line with lentiviruses...

...the number of plasmids per cell increases 4-5 fold (for pUC plasmids) or ~2 fold (for p15a, R6K, and ColE2...biotechnology 36.4 (2018): 352. PubMed PMID: 29553576. 2. Kittleson, Joshua T., Sherine Cheung, and JChristopher... engineer cells for specific purposes. Unfortunately, plasmid copy numbers vary within cell populations...problems. Rapidly dividing cells initiate genome replication more than once per cell division and thus, genes.... When cells are dividing rapidly, multiple rounds of replication are initiated before the cell has divided...growth rate can all impact plasmid copy number in a cell. One can attempt to eliminate fluctuations due ... pSC101 with copy numbers ranging from 3-100 per cell, a stabilized promoter buffered against the effects...

... that all look exactly alike.      Figure 2: Reagent gradient. Here, the lanes are lightly marked...guide. The gel is then removed from the membrane (2) and the membrane is cut so each lane is its own piece...in the bands, you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to ...the antibody’s dynamic range. Specifically, the 1:2, 1:1, and No dilution samples all have approximately...conditions are 4 °C overnight or room temperature for 1–2 hours, there is some evidence that antibodies may ...check its subcellular location and solubility. If it’s membrane-bound or located in a subcellular compartment...reagent. Be aware that this approach requires excellent attention to detail, since you’ll end up with ...

...models or cells lines, including stem cells, without leaving behind any accessory DNA sequences [2], [3]....leaving behind only those cells with a Footprint-Free™ genome edit.   Figure 2: Editing DNA using the...PMID: 26211958. PubMed Central PMCID: PMC4663986. 2.  A. M. Singh, V. V Adjan Steffey, T. Yeshi, and D...transgenic cell lines? One of the major uses of the piggyBac™ system is to create stable transgenic cell lines...efficiency is likely to be cell line-dependent, and should be tested in the cell line of interest. Importantly...If the cell’s chromatin structure undergoes significant rearrangement (such as when a stem cell initiates...applications, including making transgenic cell lines and genetically-modified cell lines. Furthermore, the use of...

...VP64 fusion; 2. A sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop 2; 3. The MS2...Ishitani R, Zhang F, Nureki O. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb...Kampmann M, Weissman JS. Cell. 2014 Oct 23;159(3):647-61. doi: 10.1016/j.cell.2014.09.029. Epub 2014 Oct...screening for genes that, when turned on, make melanoma cells resistant to treatment with a BRAF inhibitor. (The...Addgene, enabling systematic investigation of the cellular consequences of repressing or inducing individual...Weissman and his colleagues demonstrated in a paper in Cell that they could typically achieve 90-99% knockdown...straightforward to apply the new library for use in various cell types and in live mice. Therapeutic uses in humans...

...while increasing the standard mutation rate by only ~2-fold over nCas9 levels. Two sets of modifications ...10-3 mutations per nucleotide per generation, and (2) the addition of the thioredoxin-binding domain (TBD...EvolvR could also be a tool for lineage tracing cells that do not tolerate double-strand DNA breaks by...researchers to trace the progeny of a single starting cell. Additionally, multiplexing of guides allows EvolvR...

...phages but to also destroy the phage upon re-exposure (2, 3). More specifically, specialized Cas proteins snip..., accessed 25 November 2013. Author: James Atmos (2). Many CRISPR/Cas moieties carry out these functions...devise new strategies to reproduce within a host cell. A few virus particles have learned to circumvent...

...one injection step! The entire protocol takes about 2-3 weeks but requires less than eight hours worth of...for C. elegans genome modification. Figure 2: Schematic from Dickinson et al. (2015) illustrating...systems may prove effective in other species or in cell culture. In addition to CRISPR/Cas9 applications...

...be decorated with proteins and remain functional, 2) break membranes outside of their natural context ...PubMed PMID: 3759909. PubMed Central PMCID: PMC213417. 2. Schrallhammer, M. et al. Tracing the role of R-bodies... can rupture E. coli spheroplasts (cells which have had their cell walls removed with lysozyme). This ...protein fusions allowed us to image R bodies inside cells, and these constructs may be useful to future scientists...applications. For example, you could use them to lyse cells (and surrounding membranes) in keeping with their...penetrate other barriers (like vasculature or extracellular matrices) in response to pH changes. Beyond...assemble and function, we might be able to endow cells with new ways to interact mechanically with their...

... systems to engineer better and faster ABEs (Fig. 2). These systems allow for rapid continuous protein...and less stringent phage selection. Figure 2: Phage-assisted evolution of base-editing activity...gives a talk at the Cell Symposia: Gene- and Cell-based Therapies: CRISPR, Stem Cells, and Beyond meeting...III product is produced and can infect bacterial cells. If a phage does not deliver an active variant, ...passage from the infected to a fresh uninfected host cell culture). This dilution step represents the major...but gets manually passaged from an infected host-cell culture to an uninfected culture which results in...

...high percentage of muscle cells throughout the body, especially stem cells 2. Remove any immunogenicity...nonsense-mediated decay. Although Nelson et al. observed only 2% genome editing in one experiment, they found the ...Science. 2015 Dec 31. doi:10.1126/science.aae0169 2. Long C, Amoasii L, Mireault AA, McAnally JR, Li H...persist over time, stem cell editing would be highly desirable. Should stem cell editing occur, the CRISPR...patient myoblasts; when injected into DMD mice, these cells expressed functional dystrophin. Editing genes in...in vivo is of course much more difficult than in cell culture, but Ran et al. have shown that CRISPR and...be delivered to both cardiac and skeletal muscle cells, where precision editing of the dystrophin gene ...

...targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc.2016.9 Find plasmids from...form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters and...pX330a dCas9-LSD1 for targeted removal of H3K4me1/2 and H3K9me2 histone modifications. Like the TALE-LSD1...mammalian cells. Co-expression markers EGFP and PuroR enable sorting and selection of transduced cells. Grant...in the Mammalian Genome. Cell 167:233–247.e17 . https://doi.org/10.1016/j.cell.2016.08.056 Find plasmids...the epigenome may make certain processes, like cellular reprogramming of iPSCs, more efficient. In general...for targeted cytosine demethylation in mammalian cells. This plasmid is used with pcDNA3.1-MS2-Tet1-CD ...

... machines in living cells. Science. 353, aad8559 (2016). PubMed PMID: 27463678. 2. Green, Alexander A....certain states of interest. For example, in Figure 2 below we present a two-input, five-state GRSM register...linking RSM inputs to endogenous gene expression (2). Our paper (1) explains how to arrange attB-attP... require minimal cellular perturbation. Also, this method requires live, intact cells; the sequencing ...Synthetic recombinase-based state machines in living cells” (1), into a more straight-forward, concise explanation...mutations, and environmental conditions drive certain cellular behaviors, but little is known about the order... may be used to better understand and engineer cellular processes that are influenced by temporally ordered...

...animal is given additional booster immunizations every 2-3 weeks to increase the antibody titer. The titer ...level of antibodies is reached - typically within 2-4 months. After the animal has the desired level of...system, antibodies are produced by B cells. Each individual B cell produces antibodies that all recognize...also all the same isotype. But together, all the B cell clones in the immune system make different isotypes...antibodies starts with selecting just one of these B cell clones for further antibody production, polyclonal... blood from the immunized animal. The red blood cells and the serum are separated, and the serum - which...

...immediate early gene expression. Genes Dev. 1988 Jun;2(6):718-29. doi: 10.1101/gad.2.6.718. PMID: 2843425...Targeted Gene Expression in Drosophila.” Genetics 186(2) (2010): 735-55. PubMed PMID: 20697123. PubMed Central... in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197-214. PubMed PMID: 22129153. Additional... by ~3,300-fold in Drosophila cells and ~24,000-fold in human HeLa cells as compared to basal expression...origins, Tet systems function well in mammalian cells, and TRE-containing promoters can be used in the...cumate switch system functions well in mammalian cells, and can be used in the repressible manner described...While originally developed for use in mammalian cells, this system has also been implemented in a variety...

...from this publication at Addgene. 2. Tang, Jonathan C.Y., et al. “Cell type-specific manipulation with ...successfully regulated expression of channelrhodopsin-2, commonly used in optogenetics, opening up the possibility...lines expressing GFP in specific cell types to do more than just label cells. If GFP could be co-opted to ...reporter output was observed in their 293 cell culture system. In cells cotransfected with GFP and the GBPs,...expression was induced in ~76% of GFP+ cells, and 100% of DsRed+ cells also expressed GFP. Using AAV constructs...fluorescent proteins as scaffolds for cell-specific gene manipulation.” Cell 154(4) (2013): 928-939. PubMed ...wanted to express a variety of proteins in single cell types in the mouse, but knew it would take years...

... pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays a higher...and a subsequent curing protocol that requires only 2-3 hours incubation. E. coli and T. citrea scarless...four major components including (1) a CRISPR RNA, (2) four proteins forming the QCascade DNA-targeting ...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...one or more genes in E. coli. and in a mammalian cell line (Qi et al., 2013). Two important features of...

...studies have shown that the conditions Sotos-type 2 and Marshall Smith Syndrome are due to mutations in...accurate diagnosis of NFIX-related syndromes (Sotos-type 2 and Marshall-Smith) and could be expanded to NFIA-...are being awarded free plasmids form Addgene to accelerate their research. Read on to learn how these researchers...the physiological and pathological roles of human cell glycans. Accordingly, she joined efforts with the...aspect of immunology, participating in events such as cell development, recognition and migration, the immune... is not surprising. However, the molecular and cellular mechanisms behind the immune defects in CDG patients...plasmids to immortalize CDG patient-derived immune cell lines and use these lines to study how immune processes...

... engineered components, like protein tags (Figure 2). Through the work of many groups, there are systems... in Addgene's repository (Table 1). Figure 2: Adding function to My Favorite Protein (MFP) through...building-blocks of living cells, rapidly forming covalent bonds to specific partners under cell-friendly conditions...using the reactive groups naturally present in a cell? By analogy to Click Chemistry, Click Biology can...the diverse kinds of proteins that one finds in cells. High yield of reaction — generating product without...reduced; in the ER and Golgi apparatus and outside the cell, most cysteines are oxidized in disulfide bonds....temperature and on ice, when we have cracked open the cells. The tools should react in all of these situations...

...linear fashion. This implies that reducing the power 2-fold does not reduce photobleaching by half. How exactly...prolonged time). Other illumination strategies, such as 2-photon-excitation, selective plane illumination, TIRF...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 ...

...plasmid kit consists of 20 plasmids encoding 14 alpha, 2 beta, and 4 gamma subunits of the heterotrimeric G...activation via bioluminescence resonance energy transfer 2 (BRET2). Check out our blog post on GPCR signaling...with heterotrimeric G proteins on the intracellular side of the cell membrane. Heterotrimeric G proteins...induce or inhibit intracellular signaling cascades as a response to the extracellular stimuli. Ligand dissociation...Peroxidase-Catalyzed Proximity Labeling. Cell 169:338-349.e11 . https://doi.org/10.1016/j.cell.2017.03.028 Roth BL (2016...to the seven alpha helices that transverse the cellular membrane. The receptor recognizes the appropriate...signaling is initiated when a ligand binds to the extracellular surface of the GPCR. This results in a conformational...

...increase have had that increase cancelled. Rounding out the remainder, 2% of postdocs who weren’t going...continuing with their plans, and 17 have cancelled their plans. 2 of those raising - the University of Illinois...level, in the case of some public institutions) to cancel plans to raise salaries. What are institutions ...Illinois and the University of Michigan - initially cancelled their plans, but have since reversed their position...announced that they were reversing their plans to cancel raises). A further 9 institutions, whose plans ... In addition, PIs at some of the institutions cancelling plans are going ahead with raises anyway, including...injunction, as we started to hear of institutions cancelling plans to raise salaries, the only postdocs we...

...endonuclease of a class 2 CRISPR-Cas system. Cell, 163(3): 759-771 (2015). 10.1016/j.cell.2015.09.038 Harrington...and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity. Cas7-11 does not display collateral ... Stem Cells Using Type I CRISPR-Cas. Molecular Cell, 74(5): 936-950.e5 (2019). 10.1016/j.molcel.2019.03.014...cleavage and is significantly less toxic to mammalian cells than Cas13 (likely due to the lack of collateral...

...article in the Addgene Blog Mentoring series will cover 2 of the 7 mentoring questions I set out to answer. ...101-Building Relationships for Success”. Choose 1-2 tactics to try. One of my favorites is making the ...Healthcare Businesswomen’s Association hosts an excellent group mentoring program in many of its chapters...

...PMC367857. 2. Naldini, Luigi, et al. "In vivo gene delivery and stable transduction of nondividing cells by a..., and the desired gene (transfer vector plasmid), 2) the HIV-1 genome containing all viral genes with ...protocols that have been described (protocol 1, protocol 2, protocol 3) to find the one that will give you the...-dividing cells. The ability to infect non-dividing cells is not restricted to in vitro cell culture as...target cells. If you are transducing non-adherent cells you might need to centrifuge your cells with the...favourite gene into your favourite cell line or into primary cells, this blog article will give you some...infect both dividing and non-dividing cells, including stem cells. These properties make lentiviral vectors...

...residues, which can be found throughout the antibody. 2) Endogenous cysteines, such as those that make up ...Detecting antigens by quantitative immuno-PCR. Nat Protoc 2:1918–1930. https://doi.org/10.1038/nprot.2007.267 ...and Protein Modifications in Cells and Tissues in Situ. Curr Protoc Cell Biol 89:e115. https://doi.org... like proximity ligation, immuno-PCR, and single-cell applications (Hegazy, et al., 2020; Niemeyer, et...epitope and transcriptome measurement in single cells. Nat Methods 14:865–868. https://doi.org/10.1038...

...segment on the B. subtilis libraries. Hot Plasmid 2: Transcription factor fusions modulate iPSC reprogramming...Check out additional microbiology resources Stem cell models for disease and open science: Interview with...

...somewhere else (Fig. 2). Class II TEs have LTRs on both of their ends.    Figure 2: Overview of DNA...has four major components: 1) a CRISPR RNA (crRNA), 2) four proteins (TniQ, Cas8, Cas7, Cas6) that forms... yeast, plant, insect, and mammalian cells, including human cells. piggyBac is biased to integrate at ...is active in vertebrates and integrates in human cells at rates similar to retroviral vectors. The hyperactive...piggyBac has an integration efficiency in human cells similar to retroviral vectors. The hyperactive PB...transposase (hyPB) is ~10-fold more active in mammalian cells compared to the codon-optimized wild-type piggyBac...G/C)TNA. Tol2 can deliver 10-11 kB to mammalian cells without decreased efficiency, with a maximum cargo...

...from another within a fully stained sample. Figure 2 is an example of the emission spectra for two fluorophores...another during spectral unmixing.     Figure 2: The full emission spectra for BV421 and BV711 on ...co-expression among cellular markers You’ll also need to know if markers are co-expressed in your cells when designing...immunologists, allowing for the rapid analysis of cell populations within a heterogenous tissue type, such...methods, the identification and isolation of rare cell types. In recent years, full spectrum flow cytometry...detector to detect one fluorophore and therefore one cellular marker. However, emission spectra of certain fluorophores...Spectral flow allows for autofluorescence extraction Cells produce a low level of fluorescence called autofluorescence...

...targeted DNA demethylation. Cell Discov 2, 16009. https://doi.org/10.1038/celldisc.2016.9.   ...Hrit et al. 2018, Kaas et al. 2013). Figure 2: CRISPR TET-based epigenetic editing. In reality, ...its oxidized derivatives. Cell 152, 5 P1146-1159. https://doi.org/10.1016/j.cell.2013.02.004.  Xu X, Tao...enzymatic activity cause demethylation?  Dividing cells depend on DNA methyltransferase 1 (DNMT1) to copy...methylate sites blocked by dCas9. Within a few rounds of cell division, the original methylated strand is diluted...dCas9 expression and pLM-CMV-R-Cre. Selection for cells stably and highly expressing these constructs can...can be achieved by fluorescence-activated cell sorting, and stable gRNA expression can be achieved using...

... follow the same general cyclical process (Figure 2). The differences typically come in when generating...starting point for the next cycle. Figure 2: General process of antibody directed evolution. Created...module_attribute "schema_version" is_json="true" %}{% raw %}2{% endraw %}{% end_module_attribute %}{% module_attribute...high-throughput screening techniques such as ELISA and cell surface display are common. ELISA enables quantitative...for comparing relative affinities across mutants. Cell surface display systems, such as bacterial, yeast...presentation of your mutant libraries on the surface of cells. This enables direct screening via flow cytometry...provide recombinant antibodies that target families of cell surface and secreted proteins (glypicans, integrins...

...from animals or hybridomas.   Figure 2: (Left) Figure 2: (Left) The light chain and heavy chain genes... be introduced into host cells, such as bacterial, yeast, or mammalian cells, for antibody production ...Nanobodies and recombinant binders in cell biology. Journal of Cell Biology 209:633–644 . https://doi.org...animals and bleed dates and even clonal hybridoma cell lines can produce more than one mAb (Bradbury et...plasmid or from an integrated sequence in a stable cell line. The genes encode the heavy chain and light...antibody protein binds an antigen. Image from Fvasconcellos.     How are Recombinant Antibodies Made...

...FRET signal back again. The Ting lab constructed 2 versions of this reporter to target the K9 (pcDNA3... 525.7570 (2015): 533-537. PubMed PMID: 26352475. 2. Bygren, Lars O., et al. "Change in paternal grandmothers...genes should be ‘read’ by the cellular machinery. This is how most cells in the body contain the same ...complex of DNA and histone proteins that compacts cellular DNA into chromosomes and reduces the risk of dangerous...the more compacted it becomes. This prevents the cellular machinery from physically interacting with the...visualise changes in histone methylation in living cells. Essentially this reporter construct consists of...recognise lysine 27 methylation. When tested in living cells where methylation of these sites was controlled,...