...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...

... to share information about the emerging SARS-CoV-2 virus quickly and openly. By sharing results faster...Open Scientific Communication Ecosystem Fig. 2: The scientific ecosystem, enriched by open access...from peer-reviewed publishing in a journal.  Accelerating Scientific Communication Preprints have been...Fig. 1: Stand aside, CERN - it's a new age of accelerating science! Photo courtesy of under a CC license...Lacy was also employed by the American Society for Cell Biology in a position working with preprints in ...

...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...

...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...