...Initiative Collection Cell Migration Consortium Center for Genome Engineering EXtracellular Plasmid RESource...Mammalian RNAi Microbiology Plant Expression Stem Cells Synthetic Biology Tet Inducible Expression Worm ...

...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 ...

...Can I directly transfect cloning grade DNA into cells? Cloning grade DNA is designed for use in cloning...cloning applications. Direct transfection into cells is not recommended. Can I order more than 2 µg of cloning...

... question about what should go into a cell, hover over the cell and instructions will pop up. When you...spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down options for you to choose from, while... into the appropriate full and partial sequence cells. *Note: If you believe you need to deposit a collection...

...Plasmids designed to express genes in a given host cell type are generally broken down into two broad categories...purpose and mechanism of polyadenylation vary across cell types, but polyadenylation generally serves to promote...additionally act as a quality control mechanism to rid the cell of mis-folded RNAs. Eukaryotic termination and polyadenylation...efficient rabbit beta-globin mRNA 3' end formation. Cell. 1987. PMID: 3568131.  Hager S, Frame FM, Collins... thought that poly(A)s are used to control the cellular concentration of regulatory RNAs and may additionally...signal by using in vitro analyses. Molecular and Cellular Biology. 1992. PMID: 1333042. PubMed Central PMCID...

...genes should be ‘read’ by the cellular machinery. This is how most cells in the body contain the same ...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,...fluorescent reporters of histone methylation in living cells." Journal of the American Chemical Society 126.19...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...

...school at MIT. “I read some of the seminal papers on cell cycle discovery from Lee Hartwell and Paul Nurse...genetic screen to identify the factors that regulate cell division. And that really got me hooked on genetics...high-resolution tracking of more than 1 million cancer cells under drug treatment.” Stegmeier especially finds...

...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,...

...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...

...fluorescent protein (GFP) was the reporter gene and green cells were observed in the kidneys at a specific developmental...genes of interest may not be expressed in the tissue/cell line you are working with. Development-specific...10.7554/eLife.67667 Springer, S. P. Gene Traps. Plant Cell. (2000) 12(7): 1007-1020. DOI: 10.1105/tpc.12.7.1007...

...two-photon microscopy. Cell, 18, 3408 - 3425.e29. (2022). https://doi.org/10.1016/j.cell.2022.07.013.   ...voltage fluctuations. Image credit: Liu Z, et al. Cell. 2022.  Find JEDI-2p viral preps here (and here...specific for human c-myc proto-oncogene product. Mol Cell Biol. Dec;5(12):3610-6.(1985). doi: 10.1128/mcb.5.12.3610...

...Mammalian cells provide scientists with the means to study biological processes on the cellular level instead...with tissue culture The cultivation of mammalian cells in the lab, or tissue culture as it is commonly ...

... edit A → Y (Y = T or C) in 67.17% of screening cells (Tong, Wang, et al., 2023). Based on this promising...Cas9 nicks the non-edited strand to encourage the cell to repair the DNA using the edited strand as a template... efficient A•T-to-C•G base editing in mammalian cells and embryos. Nature Biotechnology, 42(4), 638–650...programmable T-to-G and T-to-C base editing. Molecular Cell, 84(7), 1257-1270.e6. https://doi.org/10.1016/j....efficient T-to-G and C-to-G editing in mammalian cells. Nature Biotechnology, 42(10), 1538–1547. https:...the base editor. Black arrows indicate natural cellular processes. Created with BioRender.com. The...

...to detect dead or dying cells so that you can base your analysis on living cells only (for example, propidium...This tends to be most problematic when staining cells or tissues that are the same species as your primary...propidium iodide only stains dead cells). Compensation controls address the spectral overlap that occurs...Quantitative Fluorescence Microscopy.” Journal of Cell Biology, vol. 185, no. 7, Rockefeller University...

...two-photon microscopy. Cell. 2022 Aug 16. pii: S0092-8674(22)00916-3. doi: 10.1016/j.cell.2022.07.013 pYtags...Calcium Indicator. Front Cell Dev Biol. 2022 Jun 29;10:880107. doi: 10.3389/fcell.2022.880107. KRaION potassium...sensor relies on a change in localization to the cell membrane and not a direct change in the fluorescent...of receptor tyrosine kinase signaling in living cells. bioRxiv 2022.08.13.503850; doi: 10.1101/2022.08.13.503850...better-matched to the potassium concentrations in cells than existing sensors. Using genome mining and structure-guided...fluorescence unit fused with TRansmembrane region-to-IntrAcellular loop linkers” (MTRIA for short), where a fluorescent...inserting a circularly-permuted GFP into an intracellular loop of various G-protein-coupled receptors....

...use electro-competent cells instead of the more common chemically-competent cells. You should perform, ... manufacturer’s instructions for your competent cells. For most standard cloning, you can transform 1-...1-2μl of your ligation reaction into competent cells such as DH5alpha or TOP10. If using much less total...colonies, you might want to use higher competency cells. Additionally, if your final product is going to...

...allowing a scientist to control signaling within cells expressing these engineered receptors. Chemogenetic...engineered receptor is coupled to and the specific cell type where the receptor is expressed, these receptors...

...high light doses to the cells. Thin static samples Ex: Fixed monolayers of cells or thin (<15 μm) tissue...1). Thin dynamic samples Ex: monolayers of live cells with fluorescently labelled, motile structures These... 10s of nanometers. Thick dynamic samples Ex: 3D cell cultures, small model embryos >15 μm Thsee are some...Total internal reflection fluorescence microscopy in cell biology. Methods Enzymol 361, 1-33 (2003). PubMed...Richardson DS, Lichtman JW. Clarifying Tissue Clearing. Cell 162, 246-257 (2015). PubMed PMID: 26186186.  Additional...Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University. No matter whether...Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University.     Additional ...

... make it difficult to properly gate your desired cell population. By compensating between fluorescent ... your fluorescent markers are staining for. If a cell is positive for both markers, it will show up as...Figure 3: Flow plot for a double positive sample. The cell population in the top right corner of the plot is...learning! Ashlyn has a research background in CD8+ T cells and cancer immunotherapies. She is currently a research...

... in mammalian cells Hu et al. compared xCas9 activity with wt SpCas9 in mammalian cells, finding xCas9...NG and GAT PAMs. xCas9 3.7 Activity in Mammalian Cells Construct NGG PAM NGA PAM NGC PAM NGT... percent editing efficiencies (edit frequency in cell population measured with NGS) are shown as percentages...

... acid (DAP), a component of the cell wall. Once in the recipient cell, the CRISPRi DNA (purple) flanked... is propagated during normal DNA replication and cell division. Mobile-CRISPRi does not disrupt gene function...mobilize these plasmids for transfer to recipient cells during a tri-parental mating scheme. Mobile-CRISPRi...

... 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...

...reliably detect HMOs. Fatima’s first design is a whole cell biosensor with two components - an enzyme that breaks...binding results in an increase in fluorescence of cells that actively produce the HMOs. Fatima has demonstrated...out Fatima's recent publication on her HMO work in Cell Chemical Biology! Additional Resources on the Addgene...Awardees: Xin Zhou and Fatima Enam. There were many excellent applicants but Xin and Fatima stood out for their...proposes using this sensor to detect HMOs found on cellular surfaces. After incubating the sensor with bacteria...

...to supplement your cells with biliverdin for maximal brightness. In mammalian cells, one of the improved...Imaging Center at UCSF. A common requirement for live cell imaging experiments is the ability to follow multiple...

...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...

...and see how different parts of the brain or the cells within them work together or perhaps compete against...two-photon properties, so you could activate single cells in arbitrary patterns in the brain. Addgene: Do ... different – an awake monkey is different from a cell in a dish. I always tell people, 'Be driven by your...

...abundant forms of posttranslational modifications in cells and research into its many roles in protein function...interactions can be discovered by fluorescence-activated cell sorting (FACS). The mode #2 phosphosite library ... Production of Recombinant Phosphoproteins Using Cell-Free Protein Synthesis. Oza JP, Aerni HR, Pirman...Ordureau A, Paulo JA, Rinehart J, Harper JW. Molecular Cell . 2015. Sep 9. pii: S1097-2765(15)00662-0. doi: ...

...editing systems by manipulating cellular determinants of editing outcomes. Cell, 184(22), 5635-5652.e29. https...Guide RNA and Target DNA. Cell, 156(5), 935–949. https://doi.org/10.1016/j.cell.2014.02.001 Yan, J., Oyler-Castrillo...type of edit, genomic context, delivery strategy, cell type, and goal of the experiment. Now, onto the ...nicking of the unedited strand and encourage the cell to use the edited strand as a template during DNA...also allows certain unintended edits to escape cellular surveillance. To limit unintended incorporation...https://doi.org/10.1016/j.cell.2021.09.018 Nelson, J. W., Randolph, P. B., Shen, S. P., Everette, K. A...

...transformation efficiency of the strain: when asking the cell to perform multiple recombination events, all linear...linear fragments need to get into the same cell. For simple cloning requiring just one or two recombination...recombination events, homemade competent cells of 107 CFU/μg are perfectly fine, yet performing multiple modifications...modifications will require ultracompetent (109 CFU/μg) cells. Primer design Using specific primer design (Figure...

...broken out of cells with a solution of salts, detergent, and ball bearings. Then a cellulose dipstick is... kits and typically follow the same principles: cells are lysed, RNAs and proteins are removed to leave... plant, microbe, blood, or mammalian cells or tissue Product: total nucleic acids While glass or silica...and RNA from plants, microbes, blood or mammalian cells in 30 seconds or less without using a pipette.  ... could be digested with restriction enzymes.  Cellulose nucleic acid purification Starting material: plant...silica are commonly used to bind and purify DNA, cellulose also efficiently binds, or at least entraps nucleic... absorb the nucleic acids. Containinants bind cellulose less tightly than nucleic acids, so placing the...

...machinery, and could thus be used in non-dividing cells. Cas13 enzymes also don’t require a PAM sequence... a maximum of 28% editing efficiency in HEK293T cells, as assessed using RNA-seq. Since the REPAIRv1 machinery...including reversibility and use in non-dividing cells. With the expanded targeting ability using both ...Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes." Mol Cell. 66(3) (2017):373-83. PubMed PMID: 28475872. Find...

... investigate how the virus interacts with human cells. Addgene’s mammalian target plasmids express these...for SARS-CoV-2 entry into human lung epithelial cells (Biering et al., 2021). In their search, they found...packaging) and contain an IRES-Puro marker for stable cell clone selection. For more information on these plasmids...Bajic G, Harrison SC, Wesemann DR (2021) Memory B cell repertoire for recognition of evolving SARS-CoV-...depends on its spike protein to bind ACE2, the host cellular receptor. To more easily study the spike variants...

...roots, as functional chloroplasts and leaf specific cell types are missing in roots. The development of transgenic...transformation” usual means that neither a stable cell line or plant is regenerated from the transformed...with the gene of interest is transferred into the cells. But for C. roseus, Agrobacterium infiltration into...Intrinsic GUS-like activities in seed plants." Plant cell reports 9.1 (1990): 1-5. PubMed PMID: 24226366. ...fused to a protein of interest to elucidate the subcellular localization of your protein of interest. Find...

...What works well in one cell or neuron type may not work well for a neighboring cell type, even within the... conditions (3,5,8), injection titers (1,2), and cell types used for packaging viral particles (6), can... vector performance in various brain regions and cell types. Power in numbers A Pubmed search for “AAV...options while AAV8 (82%) and AAV9 (84%) were both excellent at infecting our target neurons (Figure 1). However...

...using an electric charge to help the DNA enter the cells. Some samples require a reduced antibiotic concentration...concentration or need to be transformed into different cells than were originally requested. Most of our samples... Start by scanning the sample barcodes into the cells in the same order that they exist within the rack...storage process - from deposited DNA to transformed cells and finally to bacterial glycerol stocks that are...

...not generally in a cell’s best interest to overexpress a protein. Energy and cellular resources are being...being spent to make something the cell doesn’t need to make. Eukaryotes and some bacteria deploy proteosomes...proteosomes to degrade what the cell might consider junk protein. Though there are a number of chemical and...same manner as it would have in the context of a cell. FLAG tags can be cleaved by enterokinase, which...

...selection markers (for use in other cell types, e.g. eukaryotic cells). pBACKBONE-XXXXX Tip: For a catalog...

...desired mutation in mouse ES cells, select for the mutation and inject the ES cells into a mouse embryo to ...Cas-mediated genome engineering." Cell 154 (2013):1370-9. DOI: 10.1016/j.cell.2013.08.022 PubMed: PMID: 23992847...then electroporate it into mouse embryonic stem cells. After selecting for clones with the recombined ... your chimeric mice did not contain edited germ cells, your experiment would be unsuccessful. CRISPR made...

...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...context (Pillai-Kastoori et al., 2019). Tissues or cell lines other than those used by the vendor for validation...negative controls. Positive controls typically include cell lines or tissues that are known to express the protein...readout for subcellular fractionations, a procedure that isolates proteins from different cellular compartments...protein levels relative to standards, detect the cellular location of proteins, and can be a readout for...

...immunocytochemistry (ICC) when the sample is cultured cells or immunohistochemistry (IHC) with tissues), where...know if they co-localize at the same place in the cell? Or you know that the presence of several biomarkers...tissue sections, methanol- or paraformaldehyde-fixed cell samples, or other preparations. Sequential IF For...hippocampus, or counting the number of puncta in each cell. When making figures for a paper or presentation...approach for deep phenotyping and spatial analysis of cells in complex tissues. Proceedings of the National ...

...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...

...co-delivering different AAV particles into the same cells in vivo? A: This will be highly dependent on vector...particularly one that can target the epithelial cells in the blood-brain barrier?   A: For liver, we recommend...worth investigating for both Q6 & Q7.For epithelial cells of the blood brain barrier we would currently recommend...and to all our participants for their time and excellent questions.    Additional Resources on the Addgene...

... glutamate release from neurons and other brain cells in vivo. Glutamate plays a variety of roles in synaptic...promoter, FLEXed for expression in Cre-expressing cells hSynap: Human Synapsin-1 promoter. Good for neuronal...promoter, FLEXed for expression in Cre-expressing cells. hSynap-FLP: Synapsin-1 promoter, FLP-dependent ...sensor to the membrane and displays it on the extracellular side. In addition to the original PDGFR TMD,...

...they either trace downstream from the soma (neuron cell body) toward the axon (anterograde tracer) or upstream...particles require receptor-mediated entry into the cell. This means that not all neurons and pathways will...chemogenetic vector directly into the region where the cell bodies of the projection neurons reside.   Impact...

...electrocompetent cells in which the phage recombination machinery is induced using L-arabinose. These cells can be...Enzymes Required No No Yes Yes Lab Made Competent Cells Required Yes No No No Comparison of REPLACR-mutagenesis...

... in therapeutic settings. As a tool in mammalian cell systems, silencing is achieved through the delivery...). Origins and mechanisms of miRNAs and siRNAs . Cell, 136 (4), 642–655. PubMed (Link opens in a new window...

...patterning of cells and tissues is fundamental to development. I'm interested in how cells find their way...competitive CV You don't necessarily need a Nature/Cell/Science paper. You do need multiple first/corresponding... optimization of fluorescent proteins,  and live cell imaging approaches. Erik is also an avid long distance...

...plasmids is associated with the "Barcoding cells using cell-surface programmable DNA-binding domains" ...

...bacteria, so it can be transformed as-is into cells. The cells then get to work fixing up the “damage” and...nicked plasmid together long enough to get it into cells and let them do the repair work. The key is T4 DNA...

...Mutations. Berger AH, Brooks AN, Wu X, et al. Cancer Cell, 2016. PubMed PMID: 27478040. The lung cancer mutant... profiling of human gene and allele function via Cell Painting. Rohban MH, Elife, 2017. PubMed PMID: 28315521... of MAPK1/ERK2 Missense Mutants. Brenan L et al. Cell Reports, 2016. PubMed PMID: 27760319. PubMed Central...poorly classified function and a well established cellular signaling gene, thus providing a starting point...reference cDNA clones mapping to well-annotated cellular pathways). It wasn’t clear whether gene-agnostic...sequence-confirmed series of clones corresponding to known cellular signaling pathways as part of our Genetic Pathway...functional assays for these mutations and map other cellular functions and phenotypes. Since time is of the...