...expressing target cells. Any neutralized pseudoviruses would be unable to get into the target cells and this can...disease severity and survival. Cell 184:476-488.e11. https://doi.org/10.1016/j.cell.2020.12.015 Garcia-Beltran...vaccine-induced humoral immunity. Cell 184:2372-2383.e9. https://doi.org/10.1016/j.cell.2021.03.013 Additional resources...vaccines and allows the virus to gain entry into human cells via human ACE2 receptors. In their assay, the pseudoviruses...

...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...targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc.2016.9 Find plasmids from...for targeted cytosine demethylation in mammalian cells. This plasmid is used with pcDNA3.1-MS2-Tet1-CD ...vectors and targeted delivery into hard-to-manipulate cell types. They have also fused LSD1 to x-Cas9(3.7)....modifiers may be more frequently inherited by daughter cells. For example, KRAB induced silencing was transient...enhancers. As the effects of these tools are likely cell type- and context-dependent, it may make sense to...

... 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... our paper (1), we allowed individual bacterial cells to form colonies on a plate and then we performed..., but more population-wide (as opposed to single-cell) approach is to interrogate state with qPCR. The...sufficient for greater than 50 percent of RSM-containing cells in a population to correctly record each possible... were sufficient for greater than 90 percent of cells to correctly record each possible permuted substring...Synthetic recombinase-based state machines in living cells. Science. 353, aad8559 (2016). PubMed PMID: 27463678...

...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...biosynthesis in Drosophila melanogaster." Molecular and cellular endocrinology 215.1-2 (2004): 1-10. PubMed PMID...

...For about as long as the disciplines have existed, cell and molecular biologists have been continuously ... peer into the mysterious, microscopic world of cells. The development of antibodies as tools allowed ...monoclonals would help the field explore Myc’s role in cell proliferation and tumorigenesis.  Using two different...specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616. https://doi.org/10.1128/mcb.5.12.3610... and immunoglobulin heavy chain binding protein. Cell 46:291–300. https://doi.org/10.1016/0092-8674(86...signal prevents secretion of luminal ER proteins. Cell 48:899–907. https://doi.org/10.1016/0092-8674(87...

... Immune cells Flow cytometry (potentially others) Data repository From the Human Cell Differentiation...repository   HuBMAP Consortium Healthy human cells Imaging (IHC, ICC, IF, etc.)  Data repository...

...nuclease target multiple genes in a cell population. The resulting mutant cells are screened for phenotypes of...range. CRISPR can be difficult to deliver. Not all cells efficiently take up plasmids used to produce CRISPR...

...the fall colors are inspiring you to paint your cells in equally vivid colors, you'll want to check out...AAV9-X1.1 enables expression in brain endothelial cells via an injection into the systemic circulation. ...levels compared to AAV9. You can now target these cells with AAV-CAG-GFP packaged in AAV9-X1.1!  We also...recombinase or a fluorescent marker in the various cell types of the basal ganglia. They work via newly-...

...stable cell lines in which the plasmid is incorporated into the host DNA. For mammalian cells, you ...delivery method, it's unlikely that all of your cells will take up your plasmid. Thus, many plasmids have...them so that you can find or select for only the cells that received the plasmid. Selectable ...transduce both dividing and nondividing cells Addgene has an extensive collection of plasmids ...for viral production AdEasier®-1 cells (strain) - Bacterial strain that...or Fusion Protein Tags and fusion proteins are excellent tools for further understanding the function... as GFP, which allows you to visualize the cellular localization of your protein. Just remember...

...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. ...

...editor in mammalian cells Not all Cas13d proteins are functional in mammalian cells, but Konermann et al...make it possible to target essentially any RNA in a cell. Since catalytically dead dCasRx maintains its RNA-binding... with RNA-Targeting Type VI-D CRISPR Effectors.” Cell (2018) pii: S0092-8674(18)30207-1. PubMed PMID: ... a WYL-Domain-Containing Accessory Protein.” Mol Cell. (2018) pii: S1097-2765(18)30173-4. PubMed PMID:...

...indicator. Archon1 can embed itself into the cell membrane where cell voltage can then be measured. Archon1 ... it. Using a robotic picking approach to select cells with variant characteristics they wanted, the team...approach to control protein-protein interactions in cells because the protein binders can be customized for...TOM20 (pTriEX Tom20 mVenus BoPD). In dark adapted cells expressing these plasmids, tgRFP fluorescence localizes...offers a way to spatially and temporally control cellular processes in vivo. Many photoactive proteins exist...photo-reversible protein-protein interactions can control subcellular localization processes in vivo. They deposited...

...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...

... 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...assemble and function, we might be able to endow cells with new ways to interact mechanically with their...penetrate other barriers (like vasculature or extracellular matrices) in response to pH changes. Beyond...

...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...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...Addgene, enabling systematic investigation of the cellular consequences of repressing or inducing individual...

... 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...suited for prediction of very diverse edit types and cell contexts. Figure 1: High-throughput screening...double-strand break repair pathway balance. Molecular Cell, 81(10), 2216-2230.e10. https://doi.org/10.1016/...

..., peptides, amino acids, drugs, metal ions, and cells (even pathogenic bacteria). That range is possible... combination of SELEX and fluorescence-activated cell sorting (FACS) of E. coli transformed with aptamer...and FACS, Broccoli is inherently more stable in cells. The new aptamer also has a higher melting temperature...cassettes, thereby extending their use into mammalian cells. They’ve also dimerized Broccoli to further enhance...Multicolor CRISPR labeling of chromosomal loci in human cells.” Proc. Natl. Acad. Sci. USA 112(10) (2015): 3002...Aptamers as a Tool to Study RNA-Modifying Enzymes.” Cell Chem Biol. 2016 Mar 17;23(3):415-25. PubMed PMID... over half a century; the Addgene blog has an excellent primer and tips from experts on FRET techniques...

...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...example, N-cadherin (cadherin-2) is a postsynaptic cell adhesion protein that plays a critical role in neural...Advances in fluorescent protein technology." Journal of cell science 120.24 (2007): 4247-4260. PubMed PMID: 18057027...of neuronal sub-cellular structures GFP is limited in its ability to label subcellular structures in the...

...Proteins in Living Cells Using Bimolecular Fluorescence Complementation. Molecular Cell 9:789–798. https...manner. This is straightforward in easy-to-transfect cell lines where plasmid dosage can be tightly controlled...fluorescence-based method for directly visualizing PPIs in cells. The ease with which BiFC can be visualized makes...visualization of protein interactions in living cells. Nat Protoc 1:1278–1286. https://doi.org/10.1038...(BiFC) Analysis of Protein Interactions in Live Cells. Cold Spring Harb Protoc 2013:pdb.prot076497. https...directly visualize PPIs that regulate numerous cellular functions, including ubiquitinoylation, kinase...BiFC studies used the method to interrogate the subcellular localization of interactions between basic leucine...

...measuring protein expression or localization in cells, or even single-particle tracking of the bound complex...(1-10) and GFP(11) Versatile protein tagging in cells with split fluorescent protein. Kamiyama et al. ...tracking of protein-protein interactions in living cells. Shao S, Zhang H, Zeng Y, Li Y, Sun C, Sun Y. Commun...bright enough for single-molecule tracking in live cells.  Controls  When designing experiments, it’s important... fragments themselves can sometimes interact in cells and produce background signal (especially for some...some older split-GFP constructs or certain cell environments). Also consider that a protein fusion may...) for use with GFP(11) Multiplexed labeling of cellular proteins with split fluorescent protein tags. ...

...more about. Our post on “Making Your Own Competent Cells” has been extremely popular, generating many additional... We’re happy to see scientists making their own cells to save money and create the tools that work best...Save Time and Money by Making Your Own Competent Cells By Melina Fan Plasmids 101: The Promoter Region...

...Multiplex genome engineering demonstrated in human cells They used the Golden Gate cloning method to ligate...successfully to target multiple sites in cultured human cells. While it has not yet been demonstrated, they say...Reference: Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sakuma...

...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...example, if your goal is to generate a knock-out cell line to perform downstream characterization of, ...

...Neuroscience at Harvard University. Just as computers, cell phones, and cars become more technologically advanced...gene mutation is the genetic test used for sickle cell anemia (2). Southern blotting, in conjunction with... of the mutated β globin gene that causes sickle cell anemia. The mutation happens to destroy a sequence...conducted on a sample from an individual without sickle cell anemia, two DNA fragments are observed because the... site where MstII cuts. In a patient with sickle cell anemia the enzyme does not cut the DNA so only one...Wai Kan. "A sensitive new prenatal test for sickle-cell anemia." New England Journal of Medicine 307.1 (...transgenic constructs and mice." Current Protocols in Cell Biology (2009): 19-10. PubMed PMID: 19283728. PubMed...

...pulses to the subject/cells expressing the optogenetic tool causing a change in cellular activity or gene ...positively charged ions to rapidly pass through the cell membrane and initiate an action potential - a very...such as Na+, K+, and Ca++ to passively enter the cell with the concentration gradient to depolarize it...the concentration gradient and hyperpolarize the cell (Mattis et al., 2012). Bicistronic vectors combine...different wavelength, turns it off, allowing the cell to be excited over a sustained period of time (Gong...affect how rapidly you can activate or inhibit a cell in succession and maintain fidelity with the stimulus...many downstream signaling mechanisms that are often cell-type specific. If it is something you’re considering...

...sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism...PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair ...Cas protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both...

...collection of synthetic biology plasmids related to cell-to-cell communications and environmental sensing. Sensing...

...complex cellular behaviors requires making complex manipulations to cells. For example: in a cell there ...TU, or even an individual part, will behave in a cell. For unpredictable cases, MoClo provides two answers...allow you to study natural genetic circuits in the cell. Natural and synthetic product production A major...biofuels, drug precursors, etc., microbially or in cell-free systems. These pathways typically involve multiple... yield a cellular phenotype, translational output, etc., which can support a larger cellular engineering...

...ssODN repair template in induced pluripotent stem cells made from the patient. With the first gRNA, they...homology-directed repair (HDR) taking place in 41.2% of edited cells. The results were even lower for the second gRNA...13.1% targeting efficiency with only 13% of edited cells employing HDR. Ma et al. moved forward with gRNA...babies.” The differences in editing between iPS cells and embryos make a case for conducting research ...analysis is not possible with IVF, as most of the cells are needed for proper development. Ma et al.’s experiments...as whole exome sequencing of embryo derived-ES cells.They also conducted whole genome sequencing in selected...

... Mammalian Autophagy Research. Cell,140(3), 313-326. doi:10.1016/j.cell.2010.01.028. PubMed PMID: 20144757...analyzing autophagy of cytosol and organelles in yeast cells. Autophagy (Greek for “self-eating”) is induced ...radio-labeled long-lived proteins as a proxy for autophagy. Cells are cultured with isotope-labeled amino acids for...fluorescence regardless of its localization in the cell. SEP’s pH sensitivity means Rosella will fluoresce...measurements of macroautophagy and mitophagy in mammalian cells using a single fluorescent biosensor. Scientific...

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

... 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... Cas9 in complex with guide RNA and target DNA." Cell 156.5 (2014): 935-949. PubMed PMID: 24529477. PubMed...

...Nanobody toolkit to monitor retrograde transport at the cell surface Article contributed by Marcy Patrick  Listen...nanobodies to study retrograde transport from the cell surface. Their toolkit includes a set of plasmids...new technologies to quantify proteins in tissues, cells, and biofluids. Recently, Sam Myers, a Scientist...an sgRNA expressed from a separate plasmid. When cells are treated with biotin–phenol and H2O2, APEX2 labels...

... to environmental conditions, cells must be able to detect extracellular stimuli. One way they do so is...complex converts a signal received by a GPCR on the cell’s surface into action that can result in the initiation...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...bound to the intracellular surface of the GPCR. Binding of a ligand to the extracellular surface of the...number of signaling cascades, inducing different cellular responses. One of the biggest advantages of such...

...the vacuole in yeast or the lysosome in mammalian cells. This decrease in pH quenches SEP but not DsRed,...acidity, this biosensor can’t be used with fixed cells. Find the mitochondria-targeted Keima variant at...Levine. "Methods in mammalian autophagy research." Cell 140.3 (2010): 313-326. PubMed PMID: 20144757. PubMedCentral...measurements of macroautophagy and mitophagy in mammalian cells using a single fluorescent biosensor." Scientific...

... have been extracted from a cell, instead of when it is attached to a cell membrane.  Picking the Right...epitopes that are available on the outside of the cell. The same protein may have different epitopes available...need to validate on denatured proteins or on live cells. But for others, you may need to validate that it...

...Your Favorite Fluorescent Protein Oligomerizes in Cells GFP oligomerization can lead to misleading artefacts...which specific chemical inputs are received by a cell. These so-called recombinase-based state machines...used to deliver a genes of interest to a variety of cell types in-vivo. These are widely used by the optogenetics...activation and repression of different genes in a single cell, and localization of a fluorescent signal to your... Below you’ll find brief summaries of several excellent guest posts from our depositors. Interested in...

... be stable in the cell. gDNA guides will also need to be transfected into the cells of interest. In contrast...unable to show TtAgo-mediated cleavage in mammalian cells, likely due to high temperature requirements. To...plasmid and genomic DNA in a variety of mammalian cell lines, with an efficiency similar to that of Cas9...

...R-factors could be transferred between bacteria via cell-to-cell contact; however, scientists noted that, unlike...cytoplasmic factors that could be transferred between cells. At the time, these extranuclear agents of heredity...PMCID: PMC3785787. References: Lederberg, Joshua. "Cell Genetics and Hereditary Symbiosis" Physiological...

...By taking advantage of repair pathways within the cell — in some cases, the same repair pathways that were...adenosine (A) to inosine (I, treated as G by the cell). Surprisingly, the team noticed that this editor...cytosine editing between bacteria and mammalian cells Researchers in the Xueli Zhang lab were making similar...2021). Their GBE produced C → G edits in HEK293T cells with over 50% efficiency in 23 out of 30 target ...follow the same initial mechanism as in mammalian cells — by excising the freshly edited U bases — but E...these fusion constructs improved editing in various cell types, although efficacy varied among target sequences...CGBEs at different target sequences and in different cell types, available at www.crisprbehive.design. CGBEs...

...opsin expression in the intended brain region and/or cell type (I’ll talk more about this in detail later)...desired optogenetic construct in the brain region and cell type of interest is obviously of critical importance...wish to stimulate or inhibit neuronal firing, the cell-type specificity, the kinetics of the opsin variant...control of a generic promoter). A DIO opsin allows cell type specificity dependent on the promoter of the...expression of your opsin is no longer required to achieve cell-type specific opsin expression. However, the types...spread, and with co-localization immunofluorescence, cell-type specificity. Viruses that only express the ...experimentation.  Whether you attempt to stimulate the cell body or an axon terminal also affects the length...

... 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...Humanization (25-kbp) Created Using Embryonic Stem Cell/Blastocyst and CRISPR/Zygote Injection Approaches...

... Guide RNA and Target DNA. Cell 156:935–949. https://doi.org/10.1016/j.cell.2014.02.001  Ran FA, Cong ... Delivery by AAV Vectors. Cell 181:136–150 . https://doi.org/10.1016/j.cell.2020.03.023 Additional Resources...acids) displayed cleavage activity in mammalian cells. SaCas9 produced indels at a similar efficiency ...package each piece into an individual AAV. When a cell is transduced by both AAVs, the full length gene...SpCas9 which had modest editing rates in HEK-293T cells when compared to the full-length SpCas9. In 2016... allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes. Sci Rep 5: ....

...construct and then introduce both plasmids into the cell. The edit in the chromosome can now take place. .... After editing, pTargetF can be cured from the cell by inducing expression of the sgRNA that targets ...all the edits you want, cure pCas9 by growing the cells at 37 °C.     The new pEcCas/pEcgRNA system We ... to take place. Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image...tools for faster construction or reprogramming of cells to understand the genetic and biochemical mechanisms...phenotypes and to design a new generation of engineered cells for green chemistry and biomedical applications.... Chinese Academy of Sciences (CAS) Center for Excellence in Molecular Plant Sciences in 2019. She joined...

...interest live within a cell,” “Does this disease change the architecture of my cells,” or “How does this ...this mutation impact the types of cells found in my tissue.” It is based on the same principles as other ... visualize a target of interest within an intact cell, tissue, or, in some cases, a whole organism. But...joined to fluorescent molecules are applied to a cell or tissue sample where they bind to their targets...to visualize a target within an intact sample, a cell in the case of ICC or a tissue in the case of IHC...IF), is an immunoassay that brings to light the cellular world. The technique allows you to ask questions...

... want to make sure your cells have first reached an appropriate density. Cells in the exponential growth...collecting dead cells, and, conversely, you cannot induce too early as there are not enough cells in the culture...host protein, using up valuable resources in the cell thus leading to slowed growth. Additionally, some...such as TB or 2XYT is optimal because of the high cell-density they support; however, minimal media supplemented...

...RNA-Mediated Gene Targeting in One-Cell Embryos. Niu Y, et al. Cell. 2014 January 30. (Pubmed) Programmable...correct disease mutations, both in mice and in human cells. But that important advance was quickly followed...

...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...Silhavy, TJ., Kahne, D. & Walker S. The Bacterial Cell Envelope. Cold Spring Harb Perspect Biol. 2(5) a000414...

...target a broad range of dividing and non-dividing cell types with almost 100% efficiency. Unlike lentiviruses...recombination of adenovirus plasmids in either mammalian cells or microorganisms, including bacteria and yeast....can then be transfected into mammalian packaging cell lines to generate adenovirus particles. Read our...

...p53 are in promoting cell cycle arrest, apoptosis, or senescence in damaged cells. The p53 name arose ...or E2 Cyclin G CCNG1 CCNG2 Cyclin G1 or G2 CDC25 Cell division cycle 25C Cdk4/6 CDK4 CDK6 Cyclin-dependent...member 10b E2F-1 E2F transcription factor 1 Fas Fas cell surface death receptor Gadd45 GADD45A GADD45B GADD45G...transcription factor and tumor suppressor activated by cellular stress. Cancer...transcription factor and tumor suppressor activated by cellular stresses like DNA damage, oxidative stress, and...