... a template, copying the base edit. Figure 2: Cytidine deamination takes place on the free strand...SpCas9 base editors with editing windows as small as 1-2 nucleotides. To reduce off-target effects associated...formation. To further decrease indel formation 1.5-2 fold, the team fused bacteriophage protein Gam to ...that it appears “newly synthesized” to the cell. Thus, the cell repairs the DNA using the U-containing strand..., which may lead to cell death rather than NHEJ repair, thus removing these cells from the edited population...target modification in primary T cells, making them a promising tool for cell therapy applications. Dual base...relatively low frequency, especially in nondividing cells. Scientists everywhere wistfully imagined being ...

...caeomi>2.0.co;2 Uphoff CC, Drexler HG (2011) Detecting Mycoplasma Contamination in Cell Cultures by Polymerase...bringing in environmental contaminants.  Figure 2: An Addgenie works in the tissue culture hood while...mycoplasma contamination in your cell culture or you are about to begin a new cell culture project. If mycoplasmas...present in most of the cell culture facilities, tissue culture labs and every cell culturist has to deal...cost-effective (Sung, 2006). The sensor cell-based method uses specialized cells that can detect the presence of... should be made available for cell culturing and maintenance of the cell lines. Providing such an environment...Photo by Kate Harten DeMaio. Cell lines and reagents Examine your cell line stock for mycoplasma contamination...

...another. You’ll need to validate that points 1 and 2 hold true under your specific experimental conditions...when (1) other strategies may not be feasible and (2) large-scale proteomic and transcriptomic datasets...look at the subcellular localization of a protein of interest. Conveniently, you found a cell line in which...antibody binding in the transfected cells but not in control cells. But (there is always a ‘but’), you...transfect it into your favorite cell line. In parallel, you transfect cells with another plasmid not encoding...the cells transfected with your protein of interest and show just the faintest band from the cells transfected...when you start using complex samples such as whole cell lysate or tissue sections. It is important to confirm...

... a western, proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using...expensive system-specific sandwiches.   Figure 2: The western blot dry apparatus includes a stack of...readout for subcellular fractionations, a procedure that isolates proteins from different cellular compartments...interest from Addgene, transfected it into your target cells, now what? How can you tell if the protein you are... are so keen to study is expressing in your cells? Immunoblotting or simply the western blot, or western...specific protein within the complex mix derived from cells or tissues. Westerns are useful not only to detect...protein levels relative to standards, detect the cellular location of proteins, and can be a readout for...

...posts: Part 1: Genetically Engineered Mice and Part 2: Breeding and Crossing Mice. My first CRISPR mouse...is less predictable. In our best case, we saw that 2 out of 3 mice carried a 5 kb insertion in the ROSA... into a mouse embryonic stem cell (ESC) line, enrich and select for cells that had successfully incorporated... when it gets exciting – the cell sends out an “SOS” signal and cellular repair mechanisms rush in to ...Cas-Mediated Genome Engineering. Cell 154:1370–1379 . https://doi.org/10.1016/j.cell.2013.08.022 Yoon Y, Wang ...Cas-Mediated Genome Engineering. Cell 153:910–918 . https://doi.org/10.1016/j.cell.2013.04.025 Zhang X, Liang...develop into chimeric mice (with a fraction of the cells in the adult mice derived from the engineered ESCs...

...Drosophila melanogaster." Molecular and cellular endocrinology 215.1-2 (2004): 1-10. PubMed PMID: 15026169...attaches to the cell wall of green algae (Soo et al., 2015). There, it divides into two cells. At the same...Vampirovibrio). What’s left of the algae cell is just the cell wall and cytoplasmic membrane. Vampirovibrio...

...similar optical quality (magnification of 140X and 2 micron resolution). I have worked many hours in a ... to microbes by using non-invasive methods for cellular imaging with ultrasound. In his research he applies...

...access to the results of federally-funded research. 2)      In June, the White House honored 13 “Champions...Change,” including our recent guest Drew Endy, in celebration of open science. 3)      Also in June, the G8...

...you are having trouble preparing your most excellent resume. 2) Follow job boards and check them regularly...give you a chance at an interview:  1) Have an excellent resume ready to be submitted The structure and...successful application. It takes time to prepare an excellent resume…it is worth it. Read here to get help if...

...Enhanced CRISPR-based DNA demethylation Figure 2: The components of Casilio-ME1. Image from Taghbalout...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...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 ...

...photoconvertible fluorescent protein. Nat Methods. 2009 Feb 6(2):131-3.  PubMed. Find plasmids from this publication... are you’ve used a GFP derivative to conduct subcellular localization studies or make a reporter construct...shown to work in plant, Drosophila and mammalian cells, and it may find wide use in biological research...turnover rate. In many cases, however, a GFP-tagged cell line or organism has already been made, and it would...made by accident. After exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et al. ...spontaneous conversion did not occur. Tobacco suspension cell culture with cytosol-targeted GFP. White circles...this technique may not be appropriate for high-pH cellular compartments. Sattarzadeh et al. have shown that...

...Chinese hamster ovary (CHO) cells, as well as in cortical neurons in layer 2/3 of the mouse brain. Rubin...recombination varies for different constructs and cell types. Second, Cre may induce recombination at pseudo...standard UAS/GAL4 system is toxic to proliferating cells. A Cre-estrogen receptor ligand binding domain-fusion...recombination efficiency. When expressed in neuronal cells, each line showed broad recombinase-dependent RFP...these recombinases were also active in mammalian cells. In experiments, B3 and KD were shown to be functional... damage induced by Cre recombinase in mammalian cells. Loonstra A, Voojis M, Beverloo HB, Al Allak B, ...recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity regulation. Heidmann...

...might otherwise be hard to identify. Figure 2: Users can cross-interrogate screens from the literature... disease, cell type or drug treatment can browse through screens performed in relevant cell lines or conditions... currently houses over 890 CRISPR screens in 629 cell lines, each manually curated and annotated from ...feasibility and power of CRISPR-based library screens in cell lines from humans and other species. By accurately... screens performed in 26 different breast cancer cell lines. Screenshot updated 2024. Each screen ...screen is captured by BioGRID curators, including the cell line/tissue type, enzyme system, sgRNA library and...example phenotype screens with read-outs other than cell viability. The following video provides an in-depth...

...six different research constellations: Doench/Root (2), Zhang (3), Wu (4), Yusa (5), Wang/Lander/Sabatini...Bioinformatics 33.7 (2016): 1099-1100. PubMed PMID: 28414855. 2. Doench, John G., et al. "Optimized sgRNA design to... the single cell level), are separated by, for example, cell sorting. The separated cells are subsequently...more than 8x107 successfully targeted cells. Depending on the cells used and the readout, this could be ...My group works on immune cells in different contexts. Most of the immune cells we study are migratory, ...CRISPR modified cells are transferred into an animal and the behavior of the transferred cells is followed...with an easily measureable cellular phenotype (e.g. survival of a cancer cell as a cytotoxic drug is added...

...closer to the clinic (Tsai and Joung 2016). Table 2: Unbiased Genotyping Options Technique Detection...PMID: 23792628. PubMed Central PMCID: PMC3773023. 2. Vouillot, Léna, Aurore Thélie, and Nicolas Pollet...Digenome Seq 0.1% Cell-free (in vitro) Works across all cell types Must be verified with cell-based method ...Requires large cell population, senstive to time since cell fixing HTGTS Not reported Cell-based Identifies...method IDLV 1% Cell-based Programmable, can detect DSBs in live cells Not as senstive as other unbiased methods... use single cell clones for in vitro CRISPR experiments. The likelihood that a single cell clone derived...picture of the impact of CRISPR editing in the model cell line or organism. Several options exist for unbiased...

...Genome Editing and The CRISPR Software Matchmaker Day 2: Homology directed repair: CRISPR 101: Homology Directed...predict that we’d be able to edit RNA in mammalian cells with CRISPR? I certainly didn’t, but now we can ...updates We hope you enjoyed the #12DaysofCRISPR, a celebration of our updated blog resources. Not on social ...Improve HDR Efficiency for CRISPR Editing in Human Cells Day 3: Cpf1: Cpf1: A New Tool for CRISPR Genome ...

...fusion location, there are many choices to make. 2. Prime Editing: Adding Precision and Flexibility to...introduction of nucleic acids into bacteria or eukaryotic cells is a common technique in molecular biology and scientific...

...affinity and specificity. Trends in Immunology, 29(2), 91–97. https://doi.org/10.1016/j.it.2007.11.004 ...animation.)  Many types of immune cells, from mast cells to B cells, express Fc receptors that bind to...) antibody-dependent cellular cytotoxicity (ADCC) c) antibody-dependent cellular phagocytosis (ADCP). ...opened in the pathogen’s cell membrane (Dunkelberger & Song, 2010). As cells cannot survive when holes...die.  Antibody dependent cellular cytotoxicity (ADCC) Antibody dependent cellular cytotoxicity (ADCC) is...(NK) cells expressing Fcγ receptors binds to IgG and release cytotoxins that kill the target cell. If ...response, by binding to Fc receptors on other immune cells. (psst! Need a quick review of the different immune...

...bioengineering 94.2 (2006): 322-329. PubMed PMID: 16528756. 2. Oh, Steve KW, et al. "Substantial overproduction ...at bioRxiv. AAV2 interacts with the packaging cell's cell membrane When packaging adeno-associated viral...of the packaging cells (Vandenberghe, 2010). Rather than being released into the cell culture supernatant... particles associate with the cell membrane and are taken into the cell via endocytosis. In our study,...AAV2 viral particles by our packaging cells by treating the cells with the endocytosis inhibitor sucrose...target cells is blocked following sucrose treatment. Moreover, several reports have shown that cells treated... yields approximately 1.8-fold without affecting cell viability. This effect appears to be serotype specific...

...PMID: 25386923. PubMed Central PMCID: PMC4227841. 2. Young, Carissa L., Zachary T. Britton, and Anne S...doing some research, notably determining the subcellular localization and the number of disulfide bonds...

...light sensing proteins. Furthermore, during the first 2 weeks of life, larvae already exhibit distinct behaviors... light sensitive proteins (opsins) to manipulate cell activity, enables researchers to silence or incite...channels that allow charged particles to cross the cell membrane when exposed to light. Neuronal firing ...channel LiGluR both cause ion movement that results in cell depolarization. They therefore function as a neuron...in negative chloride ions which hyperpolarize the cell. It therefore functions as a neuron “off switch....They found that photostimulating the Kolmer-Agduhr cells was sufficient to trigger the symmetrical tail movement...forward swimming. Furthermore, only Kolmer-Agduhr cells on one side of the body required photostimulation...

...dissociation by bioluminescence resonance energy transfer 2, or BRET2. With binding of the drug, the alpha and... to environmental conditions, cells must be able to detect extracellular stimuli. One way they do so is...bound to the intracellular surface of the GPCR. Binding of a ligand to the extracellular surface of the...complex converts a signal received by a GPCR on the cell’s surface into action that can result in the initiation...number of signaling cascades, inducing different cellular responses. One of the biggest advantages of such...protein transducers. To use the TRUPATH system, cells are transfected with plasmids expressing the GPCR... and the appropriate Gγ-GFP2 and Gβ constructs. Cells are then treated with the luminescent enzyme substrate...

...development and therapy. Nat Rev Drug Discov (2017) 16(2):89-100. DOI: 10.1038/nrd.2016.238 Additional resources...identification of potential targets by CRISPR can accelerate the drug pipeline. High-throughput CRISPR screens...knock out, etc. – CRISPR can be used to generate cell line or animal models which have the precise disease...Since CRISPR has been adapted for use in primary cells, many tissue types, and across species, it enables... phenotype. CRISPR can also be used to generate cell line knock-ins of patient mutations which are variants...CRISPR has revolutionized the generation of knockout cell lines which has made this control step extremely...in a cancerous background and treat the parental cell line and the X knockout with your drug(s). If a ...

...remained active in up to 30% plasma. Figure 2: Human plasma longevity of SpCas9 vs GeoCas9. SpCas9...mammalian cells. Harrington et al tested GeoCas9’s editing skills by transfecting HEK293 cells with GeoCas9...choice for a number of model organisms: mammalian cells, yeast, drosophila, plants, worms, zebrafish, frogs... 45 °C 50 - 70 °C Genome Editing in Mammalian Cells? Yes Yes Longevity in Serum?* No Yes Table ...experimental results. GeoCas9 RNP editing in mammalian cells Besides being able to take some heat, GeoCas9 can...

...articles, such as: Sander JD & Joung JK, Nature Biotech, 2 March 2014. Or browse the articles related to the ...allows you to sort for GFP-positive cells and to enrich for those cells that were positively transfected....introduction of a mutation into the genome, how can cells with that mutation be selected/screened? A4: Before...can use a selection marker to select transfected cells (for example, plasmid with a puromycin resistance...paper). If you detect positive HR, isolate single-cell colonies, grow them up, then perform individual ...

... gamma (IFNγ) - and eliminate cancer cells. 2. Equilibrium - In a process analogous to the development...PMID: 25818339. 2. Curiel, Tyler J., et al. "Specific recruitment of regulatory T cells in ovarian carcinoma...activity, and consists of other cell types, especially immune cells and cells of epithelial origin. Suppressing...turns off T cell function upon interaction with APCs. Furthermore, tumor cells alter their cell-surface proteome...molecules needed for T cell effector function. Cancer cells also produce programmed cell death ligand (PD-L1... activity of CTLs and NK cells. These cells would normally kill cancer cells but are directed not to through...to use whole cell therapeutics consisting of various types of immune cells. Dendritic cells (DCs) are particularly...

...Almo, Institute for Protein Innovation  1:18:45 - 2:01:18 (end) - Panel Alex Tucker (Ginkgo Bioworks)...their curation of citable, linkable figures. Cause #2: Hidden “negative” data We all want to be able to ... characterised antibodies against all human extracellular proteins with community-based validation. They...

...teaching and mentoring others (see more in Figure 2). Starting early demonstrates not only your interest...application process so you can learn more! Figure 2: The many ways to prepare for a science policy career...example proposals and consider applying for their accelerator for some hands-on guidance.   Check out my webinar...

...establishing the dynamic range of your FRET assay (Figure 2). These labeling methods are freely interchangeable...PMID: 20161833. PubMed Central PMCID: PMC2802762. 2. Dumas, Anaëlle, et al. "Designing logical codon reassignment–Expanding...Mammalian Cells pCMV-DnpK dinitrophenyl hapten Mammalian Cells pANAP AnapRS Mammalian Cells Note:... Qi, and Marcel P. Bruchez. "Advances in chemical labeling of proteins in living cells." Cell and tissue...endogenous biomolecules in cells nonspecifically, and they cannot cross cell membranes, so they are best... ion channels, or cell surface receptors) or with experiments on permeabilized cells. Biarsenical labeling...these compounds cross cell membranes, thus enabling FRET measurements in intact cells. The Gradia Lab has...

...majority of deposited materials to study SARS-CoV-2 would be fully open and available to industry requestors...they grasped the crucial role Addgene plays in accelerating research. Yet this was still not enough to ensure...type of biomedical science research. Our work accelerates and enhances productive collaborations as well...distribution of plasmids for COVID-19 research is accelerating essential research in laboratories all around...scientists worldwide with Addgene’s traditional excellence through whatever lies ahead this year. We can...

... ligands can elicit varying degrees of β-arrestin-2 recruitment to receptors, indicating their agonistic...fluorescent labels. In Nature Methods (Vol. 19, Issue 2, pp. 149–158). Nature Research. https://doi.org/10.1038... is technically cell-permeable, its use in live cells can sometimes result in cellular toxicity due to... Dyes that can pass through cell membranes can prove useful for live-cell imaging. Experimental context...Additionally, cell permeability is a critical factor, as it influences the dye's ability to enter cells without...enabled the visualization of both organic tissues and cell cultures, opening biological interiors to many inquisitive...fluorophores to enable highly specific visualization of cellular components, typically a protein of interest. Fluorescent...

...developed fluorescent-protein based DA sensors (Figure 2). The dLight1 dopamine sensor In 2018, Lin Tian’s ...the brain has been in vivo microdialysis of extracellular fluid (Darvesh et al., 2011). This technique... across the semipermeable membrane from the extracellular fluid into the perfused liquid. The probe then...detection of dopamine in flies, fish, and mice." Cell 174.2 (2018): 481-496.PubMed PMID: 30007419. PubMed...

...method relies on the fluorescence ratio between the 2 fluorophores, rather than on the fluorescence intensity...Fluorescence Activated Cell Sorting (FACS), allowing for enrichment of successfully targeted cells that can be used...investigating genetic mechanisms related to morphogenesis, cell fate specification, and human disease.     Sun,...

...make it easier to study these mutants.   Fig. 2: (A) Betacyanin is produced from tyrosine by the activity...graduate student studying plant development and cell-cell communication in the Nimchuk Lab in the Biology...untransformed seedlings or callus (undifferentiated plant cell culture), which is great for a wide variety of applications...

...Pringle’s former lab at UNC Chapel Hill. Bähler et al.(2) describe a modular collection of plasmids for a wide...insertion. The linear PCR product is introduced into the cell by direct transformation. A given insert will typically...

...E. coli: acoustic gene 1 (arg1) and acoustic gene 2 (arg2). The key difference between arg1 and arg2 vesicles...limit of detection for arg1 expressing ECN is 109 cells/ml, which is within the range of  the ~1011 commensal...

... last ~2-7 days at moderate levels. With regard to target cells or organism, some primary cells and differentiated...packaging plasmids into a viral production cell line, usually HEK-293 cells. When all of these viral plasmids ...secreted into the cellular media, making viral collection fairly easy. Producer cell media can be collected... rogue HEK-293 cell). This virus-containing media can then be placed on whatever cells you would like ...just zapping your cells? That really depends on two things. First, what type of cell or organism context...process of transferring foreign nucleic acid into a cell by a viral agent. Transductions are similar to transfections...processes which introduce foreign nucleic acid into cells by any means, but are unique in that they require...

...access this tool, set up an IDT account. Figure 2: Addgene's Analyze Sequence tool.   DNA sequence... repository of CRISPR screens Cellosaurus: A knowledge resource for cell lines IBEX Knowledge Base: A ...

... temporal activation of a transgene. Figure 2: FLEX Cre-Lox system.  WPRE The Woodchuck Hepatitis...about synthetic promoter AAVs for cell-type expression in retinal cells Resources on Addgene.org Find ...

... stabilization of unknown bacteria. Figure 2: Christina Mork collecting samples at the hot spring... life that remained unexplored. Using new single-cell sequencing approaches, the lab was able to eliminate...sample was separated into smaller samples of 5-10 cells each for sequencing, allowing for lower complexity...

...PMID: 30083608. PubMed Central PMCID: PMC6070312. 2. Stark, Jessica C., et al. "BioBits™ Bright: A fluorescent...on freeze-dried, cell-free (FD-CF) components. Making proteins outside of a cell - cell free extracts Typically... into a host cell (through a process called transformation), essentially giving the cell all the instructions...to dispose of the live cells. The alternative is to express the protein in a cell-free manner. This is ...biology, especially those at the molecular and cellular level. The reason for this is cost and complexity...electrophoresis to separate DNA or transforming cells with green fluorescent protein, these activities...transcription and translation. Unfortunately, having live cells in a classroom presents equipment needs, such as...

...structure-based framework engineering. Methods, 34(2), 184–199. PubMed PMID: 15312672. Inbar, D., Hochman...Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable...systems, and often do not fold properly once inside cells.  Developing the HA frankenbody To combat the issues... detects the widely used HA epitope in diverse cellular environments. They called this probe the HA frankenbody...previously proven to fold and function in living cells. To make these scaffolds specific to different targets...the tagged protein upon probe binding in living cells.  In their proof of concept experiment the Stasevich...mCherry, 4xHA tagged histone protein (H2B) in U2OS cells. They then showed that the HA frankenbody probe ...

...MagFRET: The First Genetically Encoded Fluorescent Mg(2+) Sensor. PLoS One. 2013 Dec 2;8(12):e82009. doi: ... there is one problem: how to get that into the cells of interest,” Griesbeck said. Because they are chemical...understand the connection between molecular- and cellular-level events and behavior. It’s a problem that...advantage for observing events taking place inside cells that are on the move, such as T lymphocytes. Griesbeck...-binding protein, troponin C (TnC), from muscle cells. They identified a toadfish TnC variant which had...cAMP-responsive element-binding protein (CREB) in living cells. “Now we are making this really good,” Griesbeck...FRET-based sensor proteins, particularly for the intracellular imaging of transition metal ions, such as zinc...

... & Walker S. The Bacterial Cell Envelope. Cold Spring Harb Perspect Biol. 2(5) a000414 (2010). Cleveland...negative bacteria is due to differences in cell wall composition. Cell wall structure Gram positive bacteria...complex within the peptidoglycan layer, staining the cell purple. When the alcohol is added, it dehydrates...

... and whether I would be able to carry out a yeast-2-hybrid library screen; instead I thought they would...would continue on with my academic dream of studying cell division. However, after 6 years in the business...Cambridge University where he studied mechanisms of cell division. Recently Seán got the entrepreneurial ...information on ELISA troubleshooting, ELISA kits and cell death assays. You can find Seán on twitter @Macfhearraigh...

...created for the Brunello library is shown in Figure 2. You can use a small PCR product because the plasmids... Typically, Addgene uses electrocompetent Stbl4 cells when performing amplifications, and we use electroporation...DNA. This bias can be counteracted by growing the cells on solid plates, where each transformant forms its...own colony, uninfluenced by the growth of other cells. For libraries where the plasmids are all very similar...

...project my team & I have poured our hearts into for 2 yrs) has 200K+ figures by 95K+ scientists 👨🏽‍🔬👩‍...four main categories: anatomy and the human body, cellular biology, medical specialities, and general items...

... master's degree prepared them for their career.  2. Are you looking for career advancement and growth... or so I thought. So I entered a molecular and cellular biology PhD program in 2011, which I left four...

...motion videos is very time consuming; producing a 1-2 minute video takes an entire week of our free time... to mold it all into a full laboratory. It is priceless to see our “toys” gaining life and working as ...

...much has been done with CRISPR technology in just 2 years and excitement about how much more there is ...alone were worth the trip. Dr. Dana Carroll's excellent keynote address was the first of 19 talks given...