...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...

...currently a Postdoctoral Fellow in the lab of Mary Jeanne Kreek at the Rockefeller University.  His interests...protein-coupled vertebrate rhodopsin in cerebellar Purkinje cells. The Journal of biological chemistry. ...Pharmacology and Optogenetics. Cell 162.3 (2015): 662-674. doi: 10.1016/j.cell.2015.06.058. Pubmed PMID: 26189679...Derek Simon! Derek P. Simon received his PhD in Cellular and Molecular Biology from the University of Michigan...PMCID: PMC3466483. 4. Kim TI, et al. Injectable, cellular-scale optoelectronics with applications for wireless...

... genetic loci within a single cell. One might imagine transfecting cells with both the nuclease-positive...Perhaps most importantly, most Cas9 orthologs are less well characterized than the conventionally used SpCas9... both to lower relative nuclease efficiencies as well as to more stringent targeting rules that result...nuclease-competent Cas9, obviating the need to generate new cell lines or transgenic animals expressing dCas9 as ...reliance on orthogonal Cas9 species. Within a single cell, full-length guides targeting one set of genetic...nanoparticles as drug delivery systems. She is also a NASA Fellow studying biosignature preservation in Mars analogue...knockin mice for genome editing and cancer modeling." Cell 159.2 (2014): 440-455. PubMed PMID: 25263330. PubMed...

...produced by a single B cell. To create a monoclonal antibody cell line, myeloma cells are fused with activated...activated B cells from the spleen of mice injected with the antigen of interest. The immortalized cells, called...distributing over 3,000 antibodies and associated hybridoma cell lines. The DSHB was created by the National Institute... individually isolated and expanded. The clonal cells are screened to identify hybridoma lines, each producing...

... infected cells to go undetected by the immune system in a novel way, by shielding host cell surface proteins... itself to escape vesicles within host cells and turn the cell into a veritable virus factory.  Their ... sets off.  The virus works its way into immune cells in ways that aren’t fully understood. Much of the... viral attachment and fusion. Once inside a host cell, new evidence in PLoS Pathogens shows that the Ebola...have information or another resource to add, please tell us about it in the comments. ...

...indicated that inntags IT5, IT6, and IT10 expressed well in cells and did not form undesirable aggregates. Using...tags. Impressively, IT6-GFP did not reduce yeast cell size or viability compared to untagged GFP overexpression...multiple other proteins. Again, little to no change in cell morphology was observed with IT5 and IT6 fusions...and HA tags had especially detrimental effects on cell size and growth rate, indicating that they compromised... replace our older epitope tags - only time will tell! References 1. Georgieva, Maya V., et al. "Inntags...correlation for protein localization in mammalian cells." Nature methods 10.4 (2013): 315-323. Pubmed PMID...

...Generating a no-fuss genetic knockout  Knock-out cells or animals are engineered by introducing Cas9-mediated... which cuts at NRN PAMs with high efficiency, as well as NYN, but with a slightly lower efficiency. Information...transcription by blocking its initiation. In mammalian cells, robust repression requires targeting dCas9 along...study essential genes whose knockout would cause cell lethality. The Krüppel associated box (KRAB) is ...repressing target genes in a variety of species and cell types can be found on Addgene’s website. Activating...activating target genes in a variety of species and cell types can be found on Addgene’s website. ...optimized for expression in different species and cell types including (but not limited to) bacteria, yeast...

...Syndrome and Related Disorders. Cell 152:984–996 . https://doi.org/10.1016/j.cell.2013.01.038 Guy J, Gan J, ...deliver the precise amount of protein to each brain cell to restore physiological levels. Much research is...repair a gene in a living animal across multiple cell types using a mouse model for a human neurological... that enough wild-type MECP2 is made inside the cells, but this is exciting progress in tackling the root...Resource Center. Researchers can also find links to cell line repositories including patient-derived iPSCs...Modifications in Human-Induced Pluripotent Stem Cells. Front Genet 10: . https://doi.org/10.3389/fgene...Neurological Disorder by Programmable RNA Editing. Cell Reports 32:107878 . https://doi.org/10.1016/j.celrep...

...bodies that will generally segregate with the cell pellet during purification procedures. Even if you produce...codons at random and expect your sequence to express well in any organism. So what are the evolutionary constraints...reasons include: Metabolic pressures - it takes cellular resources to produce tRNAs that recognize different...Tuller et al. found that translation efficiency is well correlated with codon bias in both E. coli and S...’t necessarily mean the gene is going to express well. If you do get good expression, you should also ...mismatched translation rates and potential effects on cell growth, even hosts producing alternative tRNAs may...a means to fine-tune gene expression." Molecular cell 59.2 (2015): 149-161. PubMed PMID: 26186290. PubMed...

...currently a Postdoctoral Fellow in the lab of Mary Jeanne Kreek at the Rockefeller University.  His interests...optogenetics, ICSS can now be done with neuronal cell-type and temporal precision. In ICSS, a test chamber...refine and optimize your stimulation parameters as well. Inhibition of neuronal activation is a little ... remote contextual memories. This study is an excellent example in which the authors started with a brain... research. Derek P. Simon received his PhD in Cellular and Molecular Biology from the University of Michigan...differential participation in reward circuitry." Cell reports 8.6 (2014): 1857-1869. Pubmed PMID: 25242321...Dynamics of retrieval strategies for remote memories. Cell. 2011;147(3):678-89. Pubmed PMID: 22179551. Pubmed...

... 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... purposes, these E. coli strains are not usually well suited for recombinant protein expression. Many ...host protein, using up valuable resources in the cell thus leading to slowed growth. Additionally, some... promoter. Although the DE3/T7 RNAP system works well for most experiments, the lac promoter can “leak...such as TB or 2XYT is optimal because of the high cell-density they support; however, minimal media supplemented...

...crystal jellyfish Aequorea victoria (Figure 1) in molecular detail. On the underside of the “umbrella” of ... chemiluminescent proteins, like luciferases, as well as fluorescent ones, like Green Fluorescent Protein...green glimmer, Shimomura collected many, many jellyfish specimens from Puget Sound in the Pacific Ocean...he was able to isolate two proteins from the jellyfish’s photoorgans; the first, which he called aequorin... could be used to measure gene transcription, as well as to track protein localization. And as it turns..., another part of the fluorescent protein puzzle fell into place, as they, taken alongside the insights...

...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 ...research field! References and Resources References Fellmann, C., et al., Cornerstones of CRISPR-Cas in drug...the Addgene blog CRISPR 101: Making a Knock-In Cell Line Using CRISPR-Cas9 to Edit Disease Out of the...

... 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...

...plasmid sharing services, as well as #happydepositors who stopped by to say hello. Needless to say we all ...booth. As well as being the Director of the Great Lakes Bioenergy Research Center and a fellow Rhodobacter... plenty of geeky fun to be had at ASM Microbe as well as the serious business of science. Two symposia... work this Broad Institute lab is doing creating well-optimized CRISPR screening technology. Addgene got...making popular screening libraries like Brie and Brunello available to the scientific community. However...

... a look! mNeonGreen: a yellow-green fluorescent protein mNeonGreen is a yellow-green fluorescent protein...protein that was derived from the tetrameric yellow fluorescent protein (LanYFP) from the marine invertebrate...was reported as the brightest monomeric green or yellow fluorescent protein at the time. It is 1.5 to 3...mNeonGreen wavelengths lie between the green and yellow fluorescent protein wavelengths, it can be imaged...with standard green fluorescent protein filters or yellow fluorescent protein filters, with no or minimal...a FRET donor to mCitrine and mOrange or for live cell imaging of fusions and FRET reporters. mTFP1 is ...

... have been extracted from a cell, instead of when it is attached to a cell membrane.  Picking the Right...even in that mythical place where antibodies are well-organized, documented, and easy to find (though ...partners, the epitopes available for binding change as well. If this change hides the epitope that the antibodies...with native proteins instead of denatured ones - well, don’t relax just yet. Proteins often change shapes...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...

...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, ...experiments on is a good way to control for off targets as well as clonal differences. If you are performing CRISPR...

...to detect dead or dying cells so that you can base your analysis on living cells only (for example, propidium...Fluorescence Microscopy.” Journal of Cell Biology, vol. 185, no. 7, Rockefeller University Press, June 2009, ... positive control group, with an alternative but well-studied kinase known to prevent tau hyperphosphorylation...interactions will be observable in preimmune serum ‌as well as with an isotype-matched control.      ...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...

... of human stem cells when students are not even allowed to manipulate prokaryotic cells? Especially, if...should be “brains-on”, but as much as I try being compelling, interactive, and application-driven to boost...where you need to apply basic molecular biology, cell biology, microbiology, genetic engineering and strategic...could be used to genetically manipulate human stem cells. Then they had 10 minutes to expose their strategy...construct and explain why they selected a certain target cell population. Who is the winner? Ultimately, society...gene therapy in the following lessons. And let me tell you more, they had brilliant ideas on how to solve...

...Awardees: Xin Zhou and Fatima Enam. There were many excellent applicants but Xin and Fatima stood out for their... is currently a graduate student in Thomas J. Mansell’s lab at Iowa State University where she is developing...reliably detect HMOs. Fatima’s first design is a whole cell biosensor with two components - an enzyme that breaks...proposes using this sensor to detect HMOs found on cellular surfaces. After incubating the sensor with bacteria...binding results in an increase in fluorescence of cells that actively produce the HMOs. Fatima has demonstrated...work at her home town in Bangladesh where it was well received. In addition to her research endeavors,...out Fatima's recent publication on her HMO work in Cell Chemical Biology! Additional Resources on the Addgene...

... used HEK293T cells in our first study, but we're planning to soon expand to other cell types, including...26 of the 29 SARS-CoV-2 viral proteins in human cells and used these viral “bait” proteins to fish for...human proteins that the virus interacts with in cells. Because viruses rely on the help of human proteins...monkeywrench into the virus’ hijacking of human cells. Even better, more than half the drugs hitting these...they have an IRES-Puro marker to facilitate stable cell clone selection. Expression is driven by a fairly... as the EU, Russia, India and Saudi Arabia -- as well as closer to home in the US and Canada. In fact,...

... glutamate release from neurons and other brain cells in vivo. Glutamate plays a variety of roles in synaptic...(SF-Azurite-iGluSnFR), green (SF-iGluSnFR), and yellow (SF-Venus-iGluSnFR). iGluSnFR3: Improved Glutamate...photostability, quantum yield, and expression. Create yellow (v82) and lime green (v857) variants differing ... versatility through two different variants The yellow (v82) and lime green (v857) versions can be summarized...sensor to the membrane and displays it on the extracellular side. In addition to the original PDGFR TMD,...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 ...

...’re saving trees! The Earth thanks you. 2. Gibco Cell Culture by Gibco Whenever I start to learn a new...spectrometry app. I digress. When I started learning proper cell culture technique, I tried to find an app that could...calculations. This is how I came across the Gibco Cell Culture app. Gibco organizes its app into four categories...tools are a lifesaver when your research requires cell culture and include a counter, lab timer, dilution...educational section. This section holds the Gibco Cell culture manual and basic instructional videos. The... Overall, it’s a great tool for someone starting cell culture work. 3. NEB Tools by New England BioLabs...alternative, similar apps: Findings Lab Notebook Cell Culture Split Calculator by DRDP Restriction Enzyme...

...Marcotte EM, Voigt CA, Ellington AD. Cell. 2009 Jun 26;137(7):1272-81. doi: 10.1016/j.cell.2009.04.048. PubMed...with sophisticated gene expression controls. Most cell types do not respond to low-intensity light, so ...bacterial camera that prints a chemical image, as well as a genetic method for the computational problem... are compatible with many strains of E. coli, as well as with other plasmid-based systems, which will ...ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM, Voigt CA. Nature. 2005 Nov 24...

...carefully select first hires who are well-suited, productive, and fit well with your goals. These first hires...and foster an effective working environment? Tom Ellis was offered a start-up package that included a postdoc... most surprising thing about becoming a PI? Tom Ellis has a one word answer: “Email. Nothing prepared ...differently or the same as your adviser did? Tom Ellis merges the influence from his two PhD advisers who...our latest results as soon as the gels ran.” Dr. Ellis tries to lead his team as a scientists and a friend... lab!”   Acknowledgments Thank you to Dr. Tom Ellis (Imperial College London), Dr. Connie Cepko (Harvard...

...editor in mammalian cells Not all Cas13d proteins are functional in mammalian cells, but Konermann et al... Ruminococcus flavefaciens (nicknamed CasRx), as well as EsCas13d. Find the Yan et al. plasmids here ...make it possible to target essentially any RNA in a cell. Since catalytically dead dCasRx maintains its RNA-binding...natural diversity of CRISPR systems, only time will tell how large the genome and transcriptome engineering... 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:...

...Drosophila cell line, I’ve studied a transcription factor important for T cell differentiation (a cell type ...integration will have eyes that are anywhere from light yellow to red. The variation in color depends on how transcriptionally...vermillion (another gene controlling eye color) and yellow (a gene affecting the body color of the adult fly...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. ...

...expression and needing to work with a transfectable cell line. 2) They require larger expression vectors... of 4 crRNAs in vitro and when expressed in 293 cells. This allows for a single promoter to drive expression... were used.  With transfection, 6.4% of HEK293T cells had edits at 4 of 4 targets when an array of 4 crRNAs...transfected on a single plasmid. This is versus 2.4% of cells transfected with a pool of plasmids that contained...processing of the crRNA array when expressed in cells, but notice how Zetsche et al reverses the orientation...mouse neurons and in vivo. For these experiments, cells were infected with a 1:1 ratio of two AAVs,  one...RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell,163(3), 759-771. PubMed PMID: 26422227. PubMed Central...

...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... update, courtesy of Addgene depositor Stephen Elledge. His new method, named sequence- and ligation-independent...assembled in a unique order with no cloning scars. The Elledge Lab showed that a five-piece assembly reaction ...vitro to generate recombinant DNA via SLIC. Li MZ, Elledge SJ. Nat Methods. 2007 Mar;4(3):251-6. Epub 2007...sequence- and ligation-independent cloning. Li MZ, Elledge SJ. Methods Mol Biol. 2012;852:51-9. doi: 10.1007...